**The Next Challenge in the Treatment of Renal Cell Carcinoma: Overcoming the Resistance Mechanisms to Antiangiogenic Agents**

Michele Guida and Giuseppe Colucci *Department of Medical Oncology National Cancer Institute Viale Orazio Flacco Bari Italy* 

#### **1. Introduction**

82 Emerging Research and Treatments in Renal Cell Carcinoma

Zhou, G.X., Ireland, J., Rayman, P., Finke, J. & Zhou, M. (2010). Quantification of carbonic

*Urology* Vol.75, No.2, (Feb), pp.257-61

anhydrase IX expression in serum and tissue of renal cell carcinoma patients using enzyme-linked immunosorbent assay: prognostic and diagnostic potentials.

> In recent years, important advances have been made in the medical therapy of metastatic renal cell carcinoma (mRCC). These advances are due on the one hand to the availability of many new molecules directed at specific biomolecular targets, and on the other hand to the understanding of both the pathogenetic mechanisms which have led to the identification of the key role of some gene mutations and angiogenesis, fundamental mechanisms in the process of tumour proliferation (1,2). In particular, there have been great developments in molecules capable of inhibiting the activity of the pro-angiogenesis receptors of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) such as tyrosine-kinase inhibitors (TKI) sunitinib, sorafenib, pazopanib, and monoclonal antibodies bevacizumab. Also inhibitors of specific pathways correlated with tumour growth such as the mTOR inhibitors temsirolinmus and everolimus have become crucial drugs in the management of mRCC (3).

> In the last few years, these drugs have radically changed the course of medical therapy of mRCC and other molecules currently in an advanced stage of clinical development will soon further enrich the therapeutic options of mRCC: axitinib (new, powerful anti-tyrosine kinases inhibitor), dovitinib (multi-target inhibitor particularly active against Beta Fibroblast Growth Factor Receptor (FGFR)), volociximab (new chimeric antibody with powerful antiangiogenic activity directed towards the α5β1integrin), regorafenib, cediranib etc.

> As is known, RCC is a highly vascularized neoplasm which is dependent on VEGFmediated angiogenesis. In fact, mRCC is among neoplasms showing the highest level of circulating VEGF. The importance of VEGF signaling for tumoral growth is also supported by the high frequency of von Hippel-Lindau *(*VHL*)* gene mutations found in about 70% of clear cell RCC. The VHL gene product regulates VEGF expression through suppression of the HIF transcription factor. Loss of function mutations in VHL lead to unregulated activation of HIF and overexpression of VEGF and other proangiogenic factors. For these reasons, anti-angiogenic drugs are particularly active in clear cell RCC and these drugs are currently considered the standard of care for first-line treatment. They include the monoclonal antibody bevacizumab which binds to the soluble ligand of VEGF, and the inhibitors of multiple receptor TK for vascular endothelial growth factor receptors (VEGFR-

The Next Challenge in the Treatment of Renal Cell Carcinoma:

**2.1 Definition of resistance and its clinical implications** 

patients with mRCC.

are not available.

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 85

Resistance is divided into primary (also "refractoriness" or "intrinsic responsiveness"), which is characterized by a lack of efficacy to anti-angiogenic agents from the start of therapy, and secondary (also "acquired" or "adaptive" or "evasive" or "angiogenesis escape"), which begins after an initial response to TKI lasting for a period of time of variable length. Notably, early treatment failure involves all anti-angiogenic agents and all type of

Nevertheless, primary resistance to TKI in mRCC is heavily influenced by the patient risk score (low-intermediate *vs* poor) and by the type of first line therapy used. Primary refractory patients are about 20% in good-intermediate risk patients treated with different TKI, and it arises over 30% in poor risk patients (6, 8, 10, 11). In addition, the mTORi everolimus generally utilized as second line therapy is characterized by a resistance involving about 20% of patients (12). It is not clear if the patients who present primary resistance are the same as those who also present secondary resistance as data on this topic

The influence of prior therapies on the risk of primary resistance in patients with mRCC treated with sunitinib as first line has recently been reported in a systemic review and metaanalysis of 10 clinical studies including a total of 4,320 (13). The overall incidence of primary resistance to sunitinib was 22.4%. Moreover, the risk of developing primary resistance was significantly lower in patients with clear-cell cancer compared with non-clear-cell cancer. Notably, patients with prior cytokine therapy exhibited a significantly higher risk of primary progressive disease with sunitinib compared with those who had no prior treatment (RR, 1.18, 95% CI, 1.05-1.34, p=0.007). Although not statistically significant, there was a trend supporting that prior treatment with another mTKI sorafenib increased the risk of resistance to sunitinib

The conclusions of the Authors are that the risk of primary resistance to sunitinib may vary with tumor histology and prior therapies. In particular, previous exposure to cytokines significantly increased the risk of primary resistance suggesting that an immune mechanism

A similar meta-analysis was done in patients treated with sorafenib as first line therapy (14). A total of 3,269 patients from 20 studies were included for the analysis. The overall incidence of primary resistance was 22.6% without significant difference between clear cell and non-clear cell nor between prior cytokine therapies and no prior treatment. Notably, patients with prior exposure to sunitinib had a significantly higher incidence of resistance when treated with sorafenib (52.2%). The conclusions of the Authors are that prior exposure to sunitinib but not cytokines significantly increased the risk of resistance with sorafenib in mRCC patients, suggesting that initial therapy with angiogenesis inhibitors may promote

The conclusive considerations regarding the primary resistance to anti-angiogenic agents in mRCC are that about 30% of mRCC have an innate resistance to all available treatments and the resistance to angiogenic drugs seems to be independent from the type of TKI used.

In second line treatment, resistance to mTORi everolimus occurs in about 20% of patients (12), but when a second TKI was used, the risk of resistance increased to about 50%.

in comparison with no prior treatment (RR 1.33, 95% CI: 0.98-1.80, p=0.069).

may underlie the resistance to this drug.

the development of resistance to sorafenib.

1, VEGFR-2, and VEGFR-3), PDGFR-α and PDGFR-β, FLT3, the stem cell growth factor receptor KIT, and RET (4).

Despite the efficacy of TKI and bevacizumab therapy, the development of resistance is of major clinical concern; in fact, almost all patients with mRCC develop resistance and the disease inexorably progress.

Conventionally, patients are categorised as "early progressors" when they develop resistance within approximately 6 months of the beginning of first-line therapy, and "late progressors" when they develop resistance later. About 30% of patients present a primary resistance to these drugs with a rapid spreading of disease and a very poor survival (primary refractory). Another 40% of patients, after an initial positive response, exhibit disease progression after about 1 year of treatment (5).

Consequently, the number of patients who receive a second line therapy after antiangiogenic agents is only about half of the total. In the registrative phase III trial which compared sunitinib to interferon alpha, of 375 patients treated in the sunitinib arm, only 182 patients, corresponding to 56% of the total, received a second line therapy with an antimTOR or with a second anti-angiogenic drug (6). Similarly, in the AVOREN study with bevacizumab plus interferon *vs* interferon alone, of 325 patients in the bevacizumab plus interferon arm only 180 patients corresponding to 55% received a second line therapy (7). These data have been confirmed in the similar CALGB 90206 study (8). Notably, outside large controlled studies the percentage of patients receiving a second line treatment after anti-angiogenic agents is much lower. In a recent retrospective analysis of 645 patients from 7 centers and recruited in various studies, only 216 (30%) underwent second line therapy with anti-VEGF/anti-mammalian target of rapamycin (mTOR) drugs (9). Of interest, basal performance status resulted the only significant independent predictor of receiving secondline targeted therapy. Moreover, patients who received a second-line anti-VEGF drug appeared to have a similar overall survival to those who receive a second-line anti-mTOR drug (9).

The adoption of alternative angiogenic signaling pathways to compensate for inhibition of VEGF/VEGFR-mediated signaling seems to be the main, but not the only, common mechanism for the development of cancer resistance to VEGF pathway inhibitors. Nevertheless, to date very few data are available in literature about which alternative pathways are involved in resistant disease. Therefore, understanding the escape mechanisms of resistance to anti-angiogenic agents could improve clinical outcomes and the number of responsive patients.

#### **2. Mechanisms of resistance in MRC**

Resistance is generally defined as the capability of tumors to evade the antineoplastic effects of various treatments. About 30% of mRCC have an innate resistance to all available treatments independently from the type of anti-angiogenic agent used. Furthermore, treatment with mTORi as second line therapy results in primary resistance in about 20% of patients.

In this chapter we will attempt to give some partial responses to the numerous questions regarding the significance of resistance in mRCC: what is the definition of resistance? Which mechanisms sustain it? How can we overcome the resistance mechanisms?

1, VEGFR-2, and VEGFR-3), PDGFR-α and PDGFR-β, FLT3, the stem cell growth factor

Despite the efficacy of TKI and bevacizumab therapy, the development of resistance is of major clinical concern; in fact, almost all patients with mRCC develop resistance and the

Conventionally, patients are categorised as "early progressors" when they develop resistance within approximately 6 months of the beginning of first-line therapy, and "late progressors" when they develop resistance later. About 30% of patients present a primary resistance to these drugs with a rapid spreading of disease and a very poor survival (primary refractory). Another 40% of patients, after an initial positive response, exhibit

Consequently, the number of patients who receive a second line therapy after antiangiogenic agents is only about half of the total. In the registrative phase III trial which compared sunitinib to interferon alpha, of 375 patients treated in the sunitinib arm, only 182 patients, corresponding to 56% of the total, received a second line therapy with an antimTOR or with a second anti-angiogenic drug (6). Similarly, in the AVOREN study with bevacizumab plus interferon *vs* interferon alone, of 325 patients in the bevacizumab plus interferon arm only 180 patients corresponding to 55% received a second line therapy (7). These data have been confirmed in the similar CALGB 90206 study (8). Notably, outside large controlled studies the percentage of patients receiving a second line treatment after anti-angiogenic agents is much lower. In a recent retrospective analysis of 645 patients from 7 centers and recruited in various studies, only 216 (30%) underwent second line therapy with anti-VEGF/anti-mammalian target of rapamycin (mTOR) drugs (9). Of interest, basal performance status resulted the only significant independent predictor of receiving secondline targeted therapy. Moreover, patients who received a second-line anti-VEGF drug appeared to have a similar overall survival to those who receive a second-line anti-mTOR

The adoption of alternative angiogenic signaling pathways to compensate for inhibition of VEGF/VEGFR-mediated signaling seems to be the main, but not the only, common mechanism for the development of cancer resistance to VEGF pathway inhibitors. Nevertheless, to date very few data are available in literature about which alternative pathways are involved in resistant disease. Therefore, understanding the escape mechanisms of resistance to anti-angiogenic agents could improve clinical outcomes and the

Resistance is generally defined as the capability of tumors to evade the antineoplastic effects of various treatments. About 30% of mRCC have an innate resistance to all available treatments independently from the type of anti-angiogenic agent used. Furthermore, treatment with

In this chapter we will attempt to give some partial responses to the numerous questions regarding the significance of resistance in mRCC: what is the definition of resistance? Which

mTORi as second line therapy results in primary resistance in about 20% of patients.

mechanisms sustain it? How can we overcome the resistance mechanisms?

receptor KIT, and RET (4).

disease inexorably progress.

drug (9).

number of responsive patients.

**2. Mechanisms of resistance in MRC** 

disease progression after about 1 year of treatment (5).

#### **2.1 Definition of resistance and its clinical implications**

Resistance is divided into primary (also "refractoriness" or "intrinsic responsiveness"), which is characterized by a lack of efficacy to anti-angiogenic agents from the start of therapy, and secondary (also "acquired" or "adaptive" or "evasive" or "angiogenesis escape"), which begins after an initial response to TKI lasting for a period of time of variable length. Notably, early treatment failure involves all anti-angiogenic agents and all type of patients with mRCC.

Nevertheless, primary resistance to TKI in mRCC is heavily influenced by the patient risk score (low-intermediate *vs* poor) and by the type of first line therapy used. Primary refractory patients are about 20% in good-intermediate risk patients treated with different TKI, and it arises over 30% in poor risk patients (6, 8, 10, 11). In addition, the mTORi everolimus generally utilized as second line therapy is characterized by a resistance involving about 20% of patients (12). It is not clear if the patients who present primary resistance are the same as those who also present secondary resistance as data on this topic are not available.

The influence of prior therapies on the risk of primary resistance in patients with mRCC treated with sunitinib as first line has recently been reported in a systemic review and metaanalysis of 10 clinical studies including a total of 4,320 (13). The overall incidence of primary resistance to sunitinib was 22.4%. Moreover, the risk of developing primary resistance was significantly lower in patients with clear-cell cancer compared with non-clear-cell cancer. Notably, patients with prior cytokine therapy exhibited a significantly higher risk of primary progressive disease with sunitinib compared with those who had no prior treatment (RR, 1.18, 95% CI, 1.05-1.34, p=0.007). Although not statistically significant, there was a trend supporting that prior treatment with another mTKI sorafenib increased the risk of resistance to sunitinib in comparison with no prior treatment (RR 1.33, 95% CI: 0.98-1.80, p=0.069).

The conclusions of the Authors are that the risk of primary resistance to sunitinib may vary with tumor histology and prior therapies. In particular, previous exposure to cytokines significantly increased the risk of primary resistance suggesting that an immune mechanism may underlie the resistance to this drug.

A similar meta-analysis was done in patients treated with sorafenib as first line therapy (14). A total of 3,269 patients from 20 studies were included for the analysis. The overall incidence of primary resistance was 22.6% without significant difference between clear cell and non-clear cell nor between prior cytokine therapies and no prior treatment. Notably, patients with prior exposure to sunitinib had a significantly higher incidence of resistance when treated with sorafenib (52.2%). The conclusions of the Authors are that prior exposure to sunitinib but not cytokines significantly increased the risk of resistance with sorafenib in mRCC patients, suggesting that initial therapy with angiogenesis inhibitors may promote the development of resistance to sorafenib.

The conclusive considerations regarding the primary resistance to anti-angiogenic agents in mRCC are that about 30% of mRCC have an innate resistance to all available treatments and the resistance to angiogenic drugs seems to be independent from the type of TKI used.

In second line treatment, resistance to mTORi everolimus occurs in about 20% of patients (12), but when a second TKI was used, the risk of resistance increased to about 50%.

The Next Challenge in the Treatment of Renal Cell Carcinoma:

inhibition (15).

tumours express FGF2.

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 87

Function-blocking antibodies to VEGF receptors R1 and R2 were used to probe their roles in controlling angiogenesis in a mouse model of pancreatic islet carcinogenesis. Inhibition of VEGFR2 but not VEGFR1 markedly disrupted angiogenic switching, persistent angiogenesis, and initial tumor growth. In late-stage tumors, phenotypic resistance to VEGFR2 blockade emerged, as tumors regrew during treatment after an initial period of growth suppression. This resistance to VEGF blockade involves reactivation of tumor angiogenesis, independent of VEGF and associated with hypoxia-mediated induction of other proangiogenic factors, including members of the FGF family. These other proangiogenic signals are functionally implicated in the revascularization and regrowth of tumors in the evasion phase, as FGF blockade impairs progression in the face of VEGF

Recently, it has been demonstrated that the FGF pathway is important in patients who develop resistance to sunitinib. Welti and collegues (21) reported that FGF2 supports endothelial proliferation and de novo tubule formation in the presence of sunitinib and that FGF2 can suppress sunitinib-induced retraction of tubules. Importantly, these effects of FGF2 were ablated by PD173074, a small molecule inhibitor of FGF receptor signalling. They also showed that FGF2 can stimulate pro-angiogenic signalling pathways in endothelial cells despite the presence of sunitinib. Finally, analysis of clinical renal-cancer samples demonstrated that a large proportion of renal cancers strongly express FGF2. In conclusion, they suggest that therapeutic strategies designed to simultaneously target both VEGF and FGF2 signalling may prove more efficacious than sunitinib in renal cancer patients whose

Interestingly, it has been demonstrated that FGFR is highly expressed in RCC. Tsimafeyeu and collegues analyzed the expression of FGFR1 in 140 patients with mRCC. Expression of FGFR1 was observed in 98% of primary tumors and in 82.5% of lymph node metastases. Moreover, a significant rise in plasma bFGF levels was reported in patients with disease progression but a non-significant fall in patients with response or stable disease. Plasma VEGF-A level increased in patients with response whereas no detectable changes in plasma VEGF-A level was found in patients with progressive disease. The conclusions of the Authors are that plasma levels of bFGF and VEGF-A are altered in MRCC patients receiving sunitinib, and the increases in bFGF levels may represent biomarker of resistance to targeted therapy (22). Recently it has confirmed that the subset of clear cell RCC tumors with increased expression of FGFR1 is associated with a shorter progression free survival (23).

Also the role of IL-8 in resistance mechanisms seems to be determinant. In xenograft models, sunitinib resistance/refractoriness has been reported associated to higher levels of IL-8 (16). Moreover, the resistance to sunitinib was associated with a higher microvessel density, indicating an escape from anti-angiogenesis mechanisms. Finally, the addition of monoclonal antibody anti-IL-8 resensitized the tumor to sunitinib activity. The conclusions of the Authors are that IL-8 mediates resistance to sunitinib and could represent a candidate

Higher levels of IL-8 were associated with shorter progression free survival in mRCC

Some Authors also demonstrated in pre-clinical models that antiangiogenic drugs could elicit malignant progression of tumors with an increase of local invasion and distant

target to reverse acquired or intrinsic resistance to sunitinib.

patients treated in phase III trials of pazopanib (24).

Therefore, considering that only 30%-50% of patients receive second line therapy, the rechallenge with a second TKI is an option available for very few and selected patients.

#### **2.2 The resistance mechanisms**

Resistance has yet to be thoroughly understood in kidney cancer. The "angiogenic escape" to anti-VEGF treatment may be dependent both on cancer cell phenomena or endothelial cell phenomena. It is believed that multiple factors affect resistance including factors that decrease angiogenesis and factors that increase angiogenesis. Often these mechanisms are present contemporarily in a single patient. Several of these factors need to be accounted for when developing a comprehensive treatment approach and in understanding why a patient may be resistant to any one approach.

Hypoxia is a known inducer of angiogenic response in a wide variety of tumors. Nevertheless, it is strongly believed that hypoxia is also the key mechanism of angiogenic escape. It involves induction of gene expression via HIF transcription factor of various proangiogenic factors including VEGF, FGFs and ephrins. When angiogenesis is inhibited, tumors are in a hypoxic state and develop new alternative pathways to guarantee their further growth (15).

#### **2.3 Primary resistance mechanisms**

It is thought that patients with primary resistance to TKI have already activated one or more alternative mechanisms of resistance in response to the selective pressure of their microenvironment. Probably these cases are not, or not only, sustained by angiogenesis mechanisms. Moreover, in patients with primary resistance there is frequently an upregulation of alternative pro-angiogenic pathways mediated by FGFR, interleukin-8 (IL-8), insulin-like GFR, ephrins, and angiopoietins. In particular, FGF/FGFR system has been reported as one of the most important escape pathways of anti-VEGFR therapies.

Other possible mechanisms include the pre-existing inflammatory cell-mediated vascular protection (myeloid cell); an hypovascularity status with consequent indifference toward angiogenesis inhibitors (desmoplastic stroma); the co-option of normal vessels without requisite angiogenesis (4, 16-18).

#### **2.4 Secondary resistance mechanisms**

Regarding secondary resistance, many Authors believe that it is precisely the state of **hypoxia** determined by anti-angiogenic drugs which is at the root of the onset of the *escape* mechanisms sustained by new HIF, FGF, IL-8, ephrine etc transcript factors, which lead to the activation of alternative pathways which support a "*new angiogenic wave*" (15). It is notable that during therapy with anti-VEGF the expression of new and ever-increasing proangiogenic factors is observed. It is known that the early phase of angiogenesis is generally characterized by a response to anti-VEGF treatment. On the contrary, the late phase of angiogenesis is characterized by the escape to anti-VEGF treatment. This late phase is sustained by FGF, IL-8 and other factors. It has been reported that in the presence of sunitinib the tumor is able to produce until 19 pro-angiogenic factors to rescue endothelia cell proliferation (19,20).

Therefore, considering that only 30%-50% of patients receive second line therapy, the rechallenge with a second TKI is an option available for very few and selected patients.

Resistance has yet to be thoroughly understood in kidney cancer. The "angiogenic escape" to anti-VEGF treatment may be dependent both on cancer cell phenomena or endothelial cell phenomena. It is believed that multiple factors affect resistance including factors that decrease angiogenesis and factors that increase angiogenesis. Often these mechanisms are present contemporarily in a single patient. Several of these factors need to be accounted for when developing a comprehensive treatment approach and in understanding why a patient

Hypoxia is a known inducer of angiogenic response in a wide variety of tumors. Nevertheless, it is strongly believed that hypoxia is also the key mechanism of angiogenic escape. It involves induction of gene expression via HIF transcription factor of various proangiogenic factors including VEGF, FGFs and ephrins. When angiogenesis is inhibited, tumors are in a hypoxic state and develop new alternative pathways to guarantee their

It is thought that patients with primary resistance to TKI have already activated one or more alternative mechanisms of resistance in response to the selective pressure of their microenvironment. Probably these cases are not, or not only, sustained by angiogenesis mechanisms. Moreover, in patients with primary resistance there is frequently an upregulation of alternative pro-angiogenic pathways mediated by FGFR, interleukin-8 (IL-8), insulin-like GFR, ephrins, and angiopoietins. In particular, FGF/FGFR system has been

Other possible mechanisms include the pre-existing inflammatory cell-mediated vascular protection (myeloid cell); an hypovascularity status with consequent indifference toward angiogenesis inhibitors (desmoplastic stroma); the co-option of normal vessels without

Regarding secondary resistance, many Authors believe that it is precisely the state of **hypoxia** determined by anti-angiogenic drugs which is at the root of the onset of the *escape* mechanisms sustained by new HIF, FGF, IL-8, ephrine etc transcript factors, which lead to the activation of alternative pathways which support a "*new angiogenic wave*" (15). It is notable that during therapy with anti-VEGF the expression of new and ever-increasing proangiogenic factors is observed. It is known that the early phase of angiogenesis is generally characterized by a response to anti-VEGF treatment. On the contrary, the late phase of angiogenesis is characterized by the escape to anti-VEGF treatment. This late phase is sustained by FGF, IL-8 and other factors. It has been reported that in the presence of sunitinib the tumor is able to produce until 19 pro-angiogenic factors to rescue endothelia

reported as one of the most important escape pathways of anti-VEGFR therapies.

**2.2 The resistance mechanisms** 

may be resistant to any one approach.

**2.3 Primary resistance mechanisms** 

requisite angiogenesis (4, 16-18).

cell proliferation (19,20).

**2.4 Secondary resistance mechanisms** 

further growth (15).

Function-blocking antibodies to VEGF receptors R1 and R2 were used to probe their roles in controlling angiogenesis in a mouse model of pancreatic islet carcinogenesis. Inhibition of VEGFR2 but not VEGFR1 markedly disrupted angiogenic switching, persistent angiogenesis, and initial tumor growth. In late-stage tumors, phenotypic resistance to VEGFR2 blockade emerged, as tumors regrew during treatment after an initial period of growth suppression. This resistance to VEGF blockade involves reactivation of tumor angiogenesis, independent of VEGF and associated with hypoxia-mediated induction of other proangiogenic factors, including members of the FGF family. These other proangiogenic signals are functionally implicated in the revascularization and regrowth of tumors in the evasion phase, as FGF blockade impairs progression in the face of VEGF inhibition (15).

Recently, it has been demonstrated that the FGF pathway is important in patients who develop resistance to sunitinib. Welti and collegues (21) reported that FGF2 supports endothelial proliferation and de novo tubule formation in the presence of sunitinib and that FGF2 can suppress sunitinib-induced retraction of tubules. Importantly, these effects of FGF2 were ablated by PD173074, a small molecule inhibitor of FGF receptor signalling. They also showed that FGF2 can stimulate pro-angiogenic signalling pathways in endothelial cells despite the presence of sunitinib. Finally, analysis of clinical renal-cancer samples demonstrated that a large proportion of renal cancers strongly express FGF2. In conclusion, they suggest that therapeutic strategies designed to simultaneously target both VEGF and FGF2 signalling may prove more efficacious than sunitinib in renal cancer patients whose tumours express FGF2.

Interestingly, it has been demonstrated that FGFR is highly expressed in RCC. Tsimafeyeu and collegues analyzed the expression of FGFR1 in 140 patients with mRCC. Expression of FGFR1 was observed in 98% of primary tumors and in 82.5% of lymph node metastases. Moreover, a significant rise in plasma bFGF levels was reported in patients with disease progression but a non-significant fall in patients with response or stable disease. Plasma VEGF-A level increased in patients with response whereas no detectable changes in plasma VEGF-A level was found in patients with progressive disease. The conclusions of the Authors are that plasma levels of bFGF and VEGF-A are altered in MRCC patients receiving sunitinib, and the increases in bFGF levels may represent biomarker of resistance to targeted therapy (22). Recently it has confirmed that the subset of clear cell RCC tumors with increased expression of FGFR1 is associated with a shorter progression free survival (23).

Also the role of IL-8 in resistance mechanisms seems to be determinant. In xenograft models, sunitinib resistance/refractoriness has been reported associated to higher levels of IL-8 (16). Moreover, the resistance to sunitinib was associated with a higher microvessel density, indicating an escape from anti-angiogenesis mechanisms. Finally, the addition of monoclonal antibody anti-IL-8 resensitized the tumor to sunitinib activity. The conclusions of the Authors are that IL-8 mediates resistance to sunitinib and could represent a candidate target to reverse acquired or intrinsic resistance to sunitinib.

Higher levels of IL-8 were associated with shorter progression free survival in mRCC patients treated in phase III trials of pazopanib (24).

Some Authors also demonstrated in pre-clinical models that antiangiogenic drugs could elicit malignant progression of tumors with an increase of local invasion and distant

The Next Challenge in the Treatment of Renal Cell Carcinoma:

**Drug/Author N. Pts Type** 

Motzer, 2007

of 23 mRCC patients re-treated with sunitinib (36, 37).

reacquired drug-sensitivity by clones become resistant to TKI drugs.

375 (sunitinib arm)

325 (bevacizumab-IFN arm)

Sunitinib *Motzer et al, JCO 2009*

Beva + IFN *Escudier et al, JCO* 

\* Multi-institutional studies

1a line therapy Good-intermediate

2a line therapy

\*Meta-analysis

mRCC

prognosis

1a line

Abbreviations: PS: Performance status

*2010*

TKI\* *Vikers et al, Urology 2010*

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 89

<sup>645</sup>Anti-VEGF/

Table 1. Percentage of patients who access to a second line treatment after TKi in mRCC

Poor prognosis Hudes 2007 Temsirolimus 33

Ranpura, 2010\* Sorafenib after

Su, 2010\* Sunitinib after

**Setting Author Drug % of incidence resistance** 

Table 2. Percentage of patients with resistance according to the risk score and treatments in

Due to this genomic instability, it is strongly believed that resistance is a dynamic mechanism changing in different conditions (treatment pressure, hypoxia pressure, etc) and during the tumor growth. This aspect could explain the response obtained in some patients re-challenged with sunitinib. It thought that during treatment interruption, the selective pressure from drugs is removed and drug-sensitive clones re-growth. Recently, Zama and colleagues reported the results of a retrospective study describing 5 partial response (22%)

Also a "holiday" period from anti-VEGF therapies it is thought able to determine a

Various genes associated with resistance have been identified which could become a target for future treatments. Recently, Sanjmyatas and colleagues also reported a specific gene expression signature able to characterize the different metastatic potential in ccRCC (38).

Ranpura, 2010\* Sunitinib 22.4 Su, 2010\* Sorafenib 22.6

Motzer, 2008 Everolimus after TKI 20

**First line Second line Predictive** 

TKI <sup>180</sup>

Sunitinib <sup>33</sup>

Sorafenib 52.2

**N. of Pts (%)**

182

216

(56) -

(55) -

(30) Basal PS

**of therapy**

Anti-VEGF/ anti-mTOR

anti-mTOR

**factors** 

metastasis. In particular, it has been reported that short-term treatment with a potent inhibitor of tumor angiogenesis is able to induce an acceleration of metastasis formation (25). Moreover, other Authors reported that angiogenesis inhibitors targeting the VEGF pathway had antitumor effects in mouse models of pancreatic neuroendocrine carcinoma and glioblastoma, but concomitantly these drugs elicit tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and in some cases increased lymphatic and distant metastasis (26). Increased invasiveness is also seen by genetic ablation of the VEGF-A gene in both models, substantiating the results of the pharmacological inhibitors. The realization that potent angiogenesis inhibition can alter the natural history of tumors by increasing invasion and metastasis warrants clinical investigation, as the prospect has important implications for the development of enduring antiangiogenic therapies (26).

Other two main mechanisms that could partially explain the ability of the tumor to become resistant to treatment are their capability to epithelial-mesenchimal transformation and the intra-tumoral heterogeneity.

The **epithelial to mesenchymal transition** (EMT) process has been described in different neoplasms and associated with metastatic disease, drug resistance, and develop of angiogenesis (27-30). Treatment-associated tumor hypoxia has been reported to induce an EMT in several tumor models (31). How EMT as a mechanism of acquired resistance occurs in human tumors is unknown and deserves further investigation. In RCC, sarcomatoid phenotype is observed across all histological subtypes, and associated with a poorer prognosis and an increased resistance to VEGF inhibitors. A growing number of interdependent pathways have been linked to the induction of EMT, which, by definition, is a potentially transient/reversible phenotype of epithelial cancers. The reverted histologic phenotype observed in the xenografts also suggests that this escape mechanisms against anti-VEGF therapies may be transient (30, 32, 33).

According to this hypothesis, patients who have initially received clinical benefit from treatment with TKIs and then developed resistant disease may respond again to TKIs following a break from anti-VEGF therapies. The "holiday" period from anti-VEGF therapies may lead to "reset" the tumor microenvironment and reestablish a primarily EGF driven tumor growth. This hypothesis is supported by anecdotic reports of patients who were treated with sunitinib with initial response and subsequent progression who responded again to sunitinib following different targeted therapies such as mTOR inhibitors. The apparent transient/reversible mechanism of resistance to anti-VEGF therapies may also explain why clinical benefit has been reported by sequencing different anti-VEGF therapies despite the fact that these agents target the same VEGF pathway.

Regarding **intratumoral heterogeneity**, it has been demonstrated that mRCC, like other cancer, is characterized by a significant chromosomal instability that creates a selection of multiple clonal tumor subpopulations with an intrinsic multidrug resistance. Multiple intermixed cell subpopulations within one tumour differ by large genomic events as focal amplifications and deletions. For this reason, it is thought that single biopsy is often not representative of mutational landscape of the tumor (34). Recently have been developed methods able to study multiple subpopulations from different anatomic locations of neoplastic tissue (35).


\* Multi-institutional studies

88 Emerging Research and Treatments in Renal Cell Carcinoma

metastasis. In particular, it has been reported that short-term treatment with a potent inhibitor of tumor angiogenesis is able to induce an acceleration of metastasis formation (25). Moreover, other Authors reported that angiogenesis inhibitors targeting the VEGF pathway had antitumor effects in mouse models of pancreatic neuroendocrine carcinoma and glioblastoma, but concomitantly these drugs elicit tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and in some cases increased lymphatic and distant metastasis (26). Increased invasiveness is also seen by genetic ablation of the VEGF-A gene in both models, substantiating the results of the pharmacological inhibitors. The realization that potent angiogenesis inhibition can alter the natural history of tumors by increasing invasion and metastasis warrants clinical investigation, as the prospect has important implications for the development of enduring antiangiogenic therapies (26). Other two main mechanisms that could partially explain the ability of the tumor to become resistant to treatment are their capability to epithelial-mesenchimal transformation and the

The **epithelial to mesenchymal transition** (EMT) process has been described in different neoplasms and associated with metastatic disease, drug resistance, and develop of angiogenesis (27-30). Treatment-associated tumor hypoxia has been reported to induce an EMT in several tumor models (31). How EMT as a mechanism of acquired resistance occurs in human tumors is unknown and deserves further investigation. In RCC, sarcomatoid phenotype is observed across all histological subtypes, and associated with a poorer prognosis and an increased resistance to VEGF inhibitors. A growing number of interdependent pathways have been linked to the induction of EMT, which, by definition, is a potentially transient/reversible phenotype of epithelial cancers. The reverted histologic phenotype observed in the xenografts also suggests that this escape mechanisms against

According to this hypothesis, patients who have initially received clinical benefit from treatment with TKIs and then developed resistant disease may respond again to TKIs following a break from anti-VEGF therapies. The "holiday" period from anti-VEGF therapies may lead to "reset" the tumor microenvironment and reestablish a primarily EGF driven tumor growth. This hypothesis is supported by anecdotic reports of patients who were treated with sunitinib with initial response and subsequent progression who responded again to sunitinib following different targeted therapies such as mTOR inhibitors. The apparent transient/reversible mechanism of resistance to anti-VEGF therapies may also explain why clinical benefit has been reported by sequencing different anti-VEGF therapies despite the fact that these agents target the same VEGF pathway.

Regarding **intratumoral heterogeneity**, it has been demonstrated that mRCC, like other cancer, is characterized by a significant chromosomal instability that creates a selection of multiple clonal tumor subpopulations with an intrinsic multidrug resistance. Multiple intermixed cell subpopulations within one tumour differ by large genomic events as focal amplifications and deletions. For this reason, it is thought that single biopsy is often not representative of mutational landscape of the tumor (34). Recently have been developed methods able to study multiple subpopulations from different anatomic locations of

intra-tumoral heterogeneity.

neoplastic tissue (35).

anti-VEGF therapies may be transient (30, 32, 33).

Abbreviations: PS: Performance status

Table 1. Percentage of patients who access to a second line treatment after TKi in mRCC


\*Meta-analysis

Table 2. Percentage of patients with resistance according to the risk score and treatments in mRCC

Due to this genomic instability, it is strongly believed that resistance is a dynamic mechanism changing in different conditions (treatment pressure, hypoxia pressure, etc) and during the tumor growth. This aspect could explain the response obtained in some patients re-challenged with sunitinib. It thought that during treatment interruption, the selective pressure from drugs is removed and drug-sensitive clones re-growth. Recently, Zama and colleagues reported the results of a retrospective study describing 5 partial response (22%) of 23 mRCC patients re-treated with sunitinib (36, 37).

Also a "holiday" period from anti-VEGF therapies it is thought able to determine a reacquired drug-sensitivity by clones become resistant to TKI drugs.

Various genes associated with resistance have been identified which could become a target for future treatments. Recently, Sanjmyatas and colleagues also reported a specific gene expression signature able to characterize the different metastatic potential in ccRCC (38).

The Next Challenge in the Treatment of Renal Cell Carcinoma:

angiopoietins;





necessity of VEGF signalling;

VEGF-mediated survival;

neovascularisation

Table 3. Main mechanisms of primary and secondary resistance in mRCC


To overcome secondary resistance, various strategies are being explored: increasing the dose of the current drug, the use of non cross-resistant drugs (for example changing to a mTOR inhibitor such as everolimus after a anti-angiogenic drug), changing to another VEGF inhibitor (for example sunitinib after bevacizumab, or sorafenib after sunitinib, or axitinib after sorafenib), the use of a "drug holiday" (12, 42, 43). However, results obtained so far have been modest, above all because in general the choice of strategy has been empirical rather than determined by a strong biological rationale. It is therefore desirable that new

As previously mentioned, several studies using combinations of drugs targeted to different biomolecular targets have been started with the aim of increasing clinical activity. Many attempts have been made to verify if the combination of drugs with different mechanisms of action was able to improve the results of single agent therapy. Unfortunately, so far this

Some combinations have proved to be very toxic and relatively inactive and therefore they were quickly abandoned, as was the case of the combination of TKI and bevacizumab (44, 45).

strategy has given disappointing or negative results with a heavier profile of toxicity.

Figure 1 shows the possible drug combination strategies in mCRC therapy.


angiogenic drugs

(myeloid cell);

**Primary resistance** 

**Secondary resistance** 

**3.1 Drug combinations** 

Note: to bibliographic references see the text

studies are founded on convincing preclinical data.

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 91








angiogenesis inhibitors (desmoplastic stroma); -Co-option of normal vessels without requisite angiogenesis

It has been demonstrated that some genes are hyperexpressed when there is resistance, for example the gene which encodes sphingosine kinase, calvasculin, chemokine receptor 4 (CXCR4), NNP1, arginase II, hypoxia-inducible protein-2 (HIG2) and VEGF. Other antiangiogenic genes, however, show reduced expression in resistant tumors, such as the genes which encode cytokines associated with interferon-gamma, in particular IP10 (CXCL10) and Mig (CXCL9) (39). Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid derived from sphingosine through sphingosine kinase (SphK) action, is dysregulated in a variety of disease conditions including cancer. S1P is a tumorigenic and angiogenic growth factor produced normally by blood platelets, mast cells and possibly fibroblasts in the tumour microenvironment. It is capable of determining proliferation and migration of endothelial cells, favouring angiogenesis and tumour proliferation. Notably, several tumors up regulate the expression of SPHK1, which may greatly contribute to the putative increased levels of S1P. In experimental models it has been demonstrated that SphK and S1P expression was increased during sunitinib resistance (39).

In xenografts models Bhatt and colleagues provided evidence that resistance to VEGF receptor therapy is due at least in part to resumption of angiogenesis in association with reduction of IFNγ-related angiostatic chemokines, and that this resistance can be delayed by restoration of angiostatic signalling with the concomitant administration of CXCL9 (40).

An emerging area of drug discovery called lipidomic-based therapeutics is in rapid develop. It directly targets pleiotropic bioactive lipids involved in cancer as well as other disorders. It has been postulated that S1P antibodies could represented a potential therapeutic strategies in the treatment of renal cancer (41).

Other mechanisms, not completely known, sustaining secondary resistance in mRCC include: **secondary mutations in tyrosine kinase receptors** (analogous to EGFR TKI); **recruitment of bone marrow-derived pro-angiogenic cells** which can obviate the necessity of VEGF signalling, thereby affecting re-initiaton and continuance of tumour angiogenesis; **increasing of pericyte** coverage of the tumour vasculature, serving to support its integrity and attenuate the necessity for VEGF-mediated survival signalling has been described; activation and enhancement of invasion and metastasis to provide **access to normal tissue vasculature** without obligate neovascularisation (4).

In table 3 are reported the main mechanisms of primary and secondary resistance in mRCC.

#### **3. How can we overcome resistance to anti-angiogenic agents?**

Many attempts have been made in the effort to overcome resistance to anti-VEGF treatments, but so far the results are disappointing. They include the use of non crossresistant drugs, integrating or combining current treatment, optimization of sequential therapies and TKI re-challenge. Finally, several ongoing studies are trying to clarify the optimal sequence of the different drugs and the significance of the rechallenge with TKI in the treatment strategies.

As regards primary resistance, other than new experimental molecules the main route taken up until now has been to the combination of drugs for different biomolecular targets.

It has been demonstrated that some genes are hyperexpressed when there is resistance, for example the gene which encodes sphingosine kinase, calvasculin, chemokine receptor 4 (CXCR4), NNP1, arginase II, hypoxia-inducible protein-2 (HIG2) and VEGF. Other antiangiogenic genes, however, show reduced expression in resistant tumors, such as the genes which encode cytokines associated with interferon-gamma, in particular IP10 (CXCL10) and Mig (CXCL9) (39). Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid derived from sphingosine through sphingosine kinase (SphK) action, is dysregulated in a variety of disease conditions including cancer. S1P is a tumorigenic and angiogenic growth factor produced normally by blood platelets, mast cells and possibly fibroblasts in the tumour microenvironment. It is capable of determining proliferation and migration of endothelial cells, favouring angiogenesis and tumour proliferation. Notably, several tumors up regulate the expression of SPHK1, which may greatly contribute to the putative increased levels of S1P. In experimental models it has been demonstrated that SphK and S1P expression was

In xenografts models Bhatt and colleagues provided evidence that resistance to VEGF receptor therapy is due at least in part to resumption of angiogenesis in association with reduction of IFNγ-related angiostatic chemokines, and that this resistance can be delayed by restoration of angiostatic signalling with the concomitant administration of CXCL9

An emerging area of drug discovery called lipidomic-based therapeutics is in rapid develop. It directly targets pleiotropic bioactive lipids involved in cancer as well as other disorders. It has been postulated that S1P antibodies could represented a potential therapeutic strategies

Other mechanisms, not completely known, sustaining secondary resistance in mRCC include: **secondary mutations in tyrosine kinase receptors** (analogous to EGFR TKI); **recruitment of bone marrow-derived pro-angiogenic cells** which can obviate the necessity of VEGF signalling, thereby affecting re-initiaton and continuance of tumour angiogenesis; **increasing of pericyte** coverage of the tumour vasculature, serving to support its integrity and attenuate the necessity for VEGF-mediated survival signalling has been described; activation and enhancement of invasion and metastasis to provide **access to normal tissue** 

In table 3 are reported the main mechanisms of primary and secondary resistance in mRCC.

Many attempts have been made in the effort to overcome resistance to anti-VEGF treatments, but so far the results are disappointing. They include the use of non crossresistant drugs, integrating or combining current treatment, optimization of sequential therapies and TKI re-challenge. Finally, several ongoing studies are trying to clarify the optimal sequence of the different drugs and the significance of the rechallenge with TKI in

As regards primary resistance, other than new experimental molecules the main route taken

up until now has been to the combination of drugs for different biomolecular targets.

**3. How can we overcome resistance to anti-angiogenic agents?** 

increased during sunitinib resistance (39).

in the treatment of renal cancer (41).

the treatment strategies.

**vasculature** without obligate neovascularisation (4).

(40).


Note: to bibliographic references see the text

Table 3. Main mechanisms of primary and secondary resistance in mRCC

To overcome secondary resistance, various strategies are being explored: increasing the dose of the current drug, the use of non cross-resistant drugs (for example changing to a mTOR inhibitor such as everolimus after a anti-angiogenic drug), changing to another VEGF inhibitor (for example sunitinib after bevacizumab, or sorafenib after sunitinib, or axitinib after sorafenib), the use of a "drug holiday" (12, 42, 43). However, results obtained so far have been modest, above all because in general the choice of strategy has been empirical rather than determined by a strong biological rationale. It is therefore desirable that new studies are founded on convincing preclinical data.

#### **3.1 Drug combinations**

As previously mentioned, several studies using combinations of drugs targeted to different biomolecular targets have been started with the aim of increasing clinical activity. Many attempts have been made to verify if the combination of drugs with different mechanisms of action was able to improve the results of single agent therapy. Unfortunately, so far this strategy has given disappointing or negative results with a heavier profile of toxicity.

Figure 1 shows the possible drug combination strategies in mCRC therapy.

Some combinations have proved to be very toxic and relatively inactive and therefore they were quickly abandoned, as was the case of the combination of TKI and bevacizumab (44, 45).

The Next Challenge in the Treatment of Renal Cell Carcinoma:

phase II (1a e 2a line)

phase II 1a e 2a lines

phase II R Beva + Tem

Beva +

Abbreviations: PFS: progression free survival.

**3.2 New drugs and sequences** 

phase I <sup>31</sup>

**Drugs combinations/ Authors** 

*Gollob et al, JCO 2007* 

**Beva + Sunitinib\*** 

**Beva + Sorafenib** 

*Garcia et al, ASCO 2008* 

*Sosman et al, ASCO 2008*

**Sunitinib + Gefitinib** 

*Motzer et al, AJCO 2010* 

**Beva + Everolimus** 

*Whorf et al, ASCO '08 Hainsworth, Whorf, JCO* 

**Beva + Temsirolimus** 

*Merchan et al, JCO 2009* 

**Beva + Temsirolimus**  *vs* **Sunitinib**  *vs* **IFN + Beva** 

*Escudier et al, ASCO* 

*(TORAVA Trial)*

*2010* 

*2010* 

**Sorafenib + Interferone** 

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 93

Generally speaking, even if it is necessary to wait for definitive results of ongoing phase III

40 10 33% PR


**(Mo) OR/SD Toxicity Notes** 

50% pts in 2a line

Good activity

A negative experience

Activity similar to that of sunitinib alone

Higher toxicity for experimental arm

No confirmed results of phase II studies

moderate Good activity

moderate Good activity

high (hypertensi on, stomatite, hand-foot sindrome)

acceptable (diarrea G3-4 in 14% of pts)

High (41% of pts stop therapy)

> As aspected

> As aspected

29% SD moderate

3/7 mRCC 1 /3 melanoma 1 surrene

18% SD

37% OR 34% SD

30/23% OR 50/64% SD (1a/2a linea)

phae I-II 45 18/5.3 14,5% acceptable Long PFS

50% SD

34% SD

8,2 27,3% OR 47,7% SD

Table 4 shows the most significant experiences of the different drug combinations used.

trials, the results reported so far do not encourage this therapeutic strategy.

**Setting N. of pts PFS** 

(varius histology)

phase II 42 11

phase I-II 48 14 25% PR

<sup>80</sup>9,1

Sunitinib 8,2 23,8% OR

alpha IFN 16,8 39% OR

Clearly, another approach to overcoming resistance mechanisms is the use of new molecules which have a more powerful anti-angiogenic activity or which are more directly aimed at the targets involved in resistance mechanisms. Axitinib and dovitinib are of particular

Table 4. Most significant experiences with drug combinations in mRCC

7,1

Fig. 1. Possible strategies of drugs association in mRCC

The high expression of EGFR in renal tumours from 50 to 90% (46), has also encouraged the use of anti-EGFR drugs in combination with anti-angiogenic agents. A study has recently been published by Motzer and colleagues at the Memorial Sloan-Kettering Cancer Center in New York in which gefitinib was combined with sunitinib in order to realise a double target. However, the reported results were similar to those obtained with sunitinib alone, but with an increase in toxicity. The Authors therefore discourage further studies on this combination (47).

Another route which seems more promising is the combination of bevacizumab and m-TOR inhibitors. However, after some encouraging early experiences (48-50), more recent studies are re-dimensioning the preliminary results. Of particular note are the results of a randomized phase II trial which compared temsirolimus and bevacizumab *vs* sunitinib *vs* interferon alfa and bevacizumab (TORAVA study). Unfortunately, in view of clearly higher toxicity in the temsirolimus plus bevacizumab arm, superiority of this combination compared to other arms was not reported (51). The conclusions of Authors are that the toxicity of the temsirolimus and bevacizumab combination was much higher than anticipated and limited treatment continuation over time, whereas clinical activity was low compared with the benefit expected from sequential use of each targeted therapy. Thus, this combination cannot be recommended for first-line treatment in patients with mRCC.

The combination of targeted drugs with immunological molecules such as interferon is proving to be more interesting. In particular, encouraging results have been reported on the combination of sorafenib and interferon alpha (52).

**IFN** 

**Tumoral proliferation** 

**Inhibitors of EGFR**  - **erlotinib**  - **gefitinib**  - **lapatinib**  - **cetuximab**

**Inhibitors of mTOR/akt**  - **temsirolimus**  - **everolimus**  - **perifosine** 

The high expression of EGFR in renal tumours from 50 to 90% (46), has also encouraged the use of anti-EGFR drugs in combination with anti-angiogenic agents. A study has recently been published by Motzer and colleagues at the Memorial Sloan-Kettering Cancer Center in New York in which gefitinib was combined with sunitinib in order to realise a double target. However, the reported results were similar to those obtained with sunitinib alone, but with an increase in toxicity. The Authors therefore discourage further studies on this combination (47). Another route which seems more promising is the combination of bevacizumab and m-TOR inhibitors. However, after some encouraging early experiences (48-50), more recent studies are re-dimensioning the preliminary results. Of particular note are the results of a randomized phase II trial which compared temsirolimus and bevacizumab *vs* sunitinib *vs* interferon alfa and bevacizumab (TORAVA study). Unfortunately, in view of clearly higher toxicity in the temsirolimus plus bevacizumab arm, superiority of this combination compared to other arms was not reported (51). The conclusions of Authors are that the toxicity of the temsirolimus and bevacizumab combination was much higher than anticipated and limited treatment continuation over time, whereas clinical activity was low compared with the benefit expected from sequential use of each targeted therapy. Thus, this

combination cannot be recommended for first-line treatment in patients with mRCC.

The combination of targeted drugs with immunological molecules such as interferon is proving to be more interesting. In particular, encouraging results have been reported on the

Fig. 1. Possible strategies of drugs association in mRCC

**Angiogenesis** 

**Inhibitors of VEGF**  - **sunitinib**  - **sorafenib**  - **axitinib**  - **pazopanib**  - **bevacizumab** 

**Inhibitors of escape** 

**mecanisms**  - **dovitinib**  - **AMG 386**  - **M200** 

combination of sorafenib and interferon alpha (52).

Generally speaking, even if it is necessary to wait for definitive results of ongoing phase III trials, the results reported so far do not encourage this therapeutic strategy.

Table 4 shows the most significant experiences of the different drug combinations used.


Abbreviations: PFS: progression free survival.

Table 4. Most significant experiences with drug combinations in mRCC

#### **3.2 New drugs and sequences**

Clearly, another approach to overcoming resistance mechanisms is the use of new molecules which have a more powerful anti-angiogenic activity or which are more directly aimed at the targets involved in resistance mechanisms. Axitinib and dovitinib are of particular

The Next Challenge in the Treatment of Renal Cell Carcinoma:

retrospective studies regarding selected patients (36,37,56-61).

planned trials are evaluating what are the best sequences and timing.

**4. Conclusions** 

the treatment of renal cell carcinoma.

with the linguistic revision of this review.

Oncol 2006; 24:1-3.

2009; 10:992-1000.

Reviews 2008; 8(8):592-603.

**5. Acknowledgements** 

**6. References** 

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 95

the basis of some preliminary experiences. Also the rechallenge with the same drug has been proposed, especially when a "holiday" period from anti-VEGF therapies is given to the patient. This break could be able to determine a reacquired drug-sensitivity by clones become resistant to TKI. Nevertheless, at present the majority of data are from small and

Recently, the results of the phase III study with Axitinib as second line therapy have been published (62). Axitinib resulted in significantly longer PFS compared with sorafenib. Nevertheless, in the subgroup of the patients treated previously with the TKI inhibitor the PFS was similar to what has been reported for the mTOR inhibitor everolimus (4.8 vs. 4.9 months). Of course, further controlled studies are needed to determine the real effect of prior antiangiogenesis therapy on the development of resistance to further therapies. A series of

The adoption of alternative angiogenic signaling pathways to compensate for inhibition of VEGF/VEGFR-mediated signaling seems to be the common mechanism for the development of cancer resistance to VEGF pathway inhibitors. Nevertheless, until now very few data are known about which alternative pathways are involved in resistant disease.

Many attempts have been proposed to overcome resistance. These include the use of non cross-resistant drugs, the optimization of sequential therapies, and the use of combined therapies. Unfortunately, all these approaches have given only modest results. Therefore, the overcome resistance mechanisms to antiangiogenic agents remains the next challenge in

The author would like to thank Caroline Oakley and Silvana Valerio for their assistance

[1] Rini BI, Small EJ: Biology and clinical development of vascular endothelial growth factor-targeted therapy in renal cell carcinoma. J Clin Oncol 2005; 23:1028-1043. [2] Motzer RJ, Michaelson MD, Redman BG et al: Activity of SUI 1248, a multitargeted

[3] Schmidinger M, Bellmunt J: Plethora of agents, plethora of targets, plethora of side effects in metastatic renal cell carcinoma. Cancer Treat Rev 2010; 36:416-424. [4] Bergers G and Hanahan D: Modes of resistance to anti-angiogenic therapy. Nature

[5] Rini B, Atkins MB: Resistance to targeted therapy in renal cell carcinoma. Lancet Oncol

inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin

interest here. In preclinical trials, axitinib has shown much more powerful antiangiogenic activity than other TKIs (53, 54). Furthermore, interesting results have been reported in phase II studies as a second line therapy after sorafenib with an overall response of 23% and a stable disease of 55%; interestingly, the progression free survival was 7.4 months, one of the longest ever reported (42).

Recently, beta FGFR has been identified as a new target for anti-angiogenic therapy. The system FGF/FGF receptor (FGFR) has been frequently reported as one of the most important escape pathways of anti-VEGFR therapies. It is involved in primary and secondary resistance mechanisms. The activation of FGFR3 is associated with cell proliferation and survival in certain cancer cell types. Thus, beta FGFR is proving to be a new interesting target for anti-angiogenic therapy.

Dovitinib, a new small multi-target molecule, is able to strongly binds to FGFR3 and inhibits its phosphorylation, which may result in the inhibition of tumor cell proliferation and the induction of tumor cell death. In addition, this agent may inhibit other members of the TK receptors superfamily, including the VEGFR; FGFR1; PDGFR3; FMS-like tyrosine kinase 3; stem cell factor receptor (c-KIT); and colony-stimulating factor receptor 1; this may result in an additional reduction in cellular proliferation and angiogenesis, and the induction of tumor cell apoptosis. A phase I/II has been recently concluded (55) and a large phase III clinical trial is ongoing to evaluate the efficacy of this drug as third line therapy in mRCC.

Other drugs of great interest are the monoclonal antibody anti-S1P, a molecule directly involved in resistance mechanisms already being developed clinically, and the anti- IL-8 and anti-IL-12 antibodies, which are still being studied in preclinical trials.

Regarding **sequences**, factors that could drive the choice of a more appropriate second line therapy are the response to primary treatment with TKI, the side effects reported in first line therapy, the patient risk score, and the histology of the tumor.

At present, the use of non cross-resistant mTOR inhibitor everolimus is the only registered agent available as second line therapy for mRCC resistant to anti-angiogenic drugs. In fact, the registrative trial showed a significant benefit in terms of PFS of 4.9 months for everolimus *vs* 1.9 months for placebo (12).

A second TKi as second line therapy is another option to consider for patients resistant to antiangiogenetic agents. Nevertheless, it is thought that this treatment must be propose only in carefully selected patients who did not show a rapid progression at the first line TKi. At present, this choice has a weaker recommendation because no definitive data from phase III studies are available yet.

Notably, in the AVOREN study it has been reported a median overall survival of 23.3 months for the sequence alfa interferone/bevacizumab followed by a second antiangiogenetic agent (TKI), with respect to only 21.3 months for the sequence alfa interferone/placebo followed by a TKI (7).

Some Authors believe that better clinical outcomes are correlated with a higher number of lines of treatments used rather than with the sequences utilized. Consequently, they have hypothesize specific sequences with the aim to utilize the maximum of therapeutic options available. Others suppose that the sequence TKI-TKI is to prefer to that with TKI-mTORi on the basis of some preliminary experiences. Also the rechallenge with the same drug has been proposed, especially when a "holiday" period from anti-VEGF therapies is given to the patient. This break could be able to determine a reacquired drug-sensitivity by clones become resistant to TKI. Nevertheless, at present the majority of data are from small and retrospective studies regarding selected patients (36,37,56-61).

Recently, the results of the phase III study with Axitinib as second line therapy have been published (62). Axitinib resulted in significantly longer PFS compared with sorafenib. Nevertheless, in the subgroup of the patients treated previously with the TKI inhibitor the PFS was similar to what has been reported for the mTOR inhibitor everolimus (4.8 vs. 4.9 months).

Of course, further controlled studies are needed to determine the real effect of prior antiangiogenesis therapy on the development of resistance to further therapies. A series of planned trials are evaluating what are the best sequences and timing.

#### **4. Conclusions**

94 Emerging Research and Treatments in Renal Cell Carcinoma

interest here. In preclinical trials, axitinib has shown much more powerful antiangiogenic activity than other TKIs (53, 54). Furthermore, interesting results have been reported in phase II studies as a second line therapy after sorafenib with an overall response of 23% and a stable disease of 55%; interestingly, the progression free survival was 7.4 months, one of

Recently, beta FGFR has been identified as a new target for anti-angiogenic therapy. The system FGF/FGF receptor (FGFR) has been frequently reported as one of the most important escape pathways of anti-VEGFR therapies. It is involved in primary and secondary resistance mechanisms. The activation of FGFR3 is associated with cell proliferation and survival in certain cancer cell types. Thus, beta FGFR is proving to be a

Dovitinib, a new small multi-target molecule, is able to strongly binds to FGFR3 and inhibits its phosphorylation, which may result in the inhibition of tumor cell proliferation and the induction of tumor cell death. In addition, this agent may inhibit other members of the TK receptors superfamily, including the VEGFR; FGFR1; PDGFR3; FMS-like tyrosine kinase 3; stem cell factor receptor (c-KIT); and colony-stimulating factor receptor 1; this may result in an additional reduction in cellular proliferation and angiogenesis, and the induction of tumor cell apoptosis. A phase I/II has been recently concluded (55) and a large phase III clinical trial is ongoing to evaluate the efficacy of this drug as third line therapy in mRCC. Other drugs of great interest are the monoclonal antibody anti-S1P, a molecule directly involved in resistance mechanisms already being developed clinically, and the anti- IL-8 and

Regarding **sequences**, factors that could drive the choice of a more appropriate second line therapy are the response to primary treatment with TKI, the side effects reported in first line

At present, the use of non cross-resistant mTOR inhibitor everolimus is the only registered agent available as second line therapy for mRCC resistant to anti-angiogenic drugs. In fact, the registrative trial showed a significant benefit in terms of PFS of 4.9 months for

A second TKi as second line therapy is another option to consider for patients resistant to antiangiogenetic agents. Nevertheless, it is thought that this treatment must be propose only in carefully selected patients who did not show a rapid progression at the first line TKi. At present, this choice has a weaker recommendation because no definitive data from phase III

Notably, in the AVOREN study it has been reported a median overall survival of 23.3 months for the sequence alfa interferone/bevacizumab followed by a second antiangiogenetic agent (TKI), with respect to only 21.3 months for the sequence alfa

Some Authors believe that better clinical outcomes are correlated with a higher number of lines of treatments used rather than with the sequences utilized. Consequently, they have hypothesize specific sequences with the aim to utilize the maximum of therapeutic options available. Others suppose that the sequence TKI-TKI is to prefer to that with TKI-mTORi on

the longest ever reported (42).

new interesting target for anti-angiogenic therapy.

anti-IL-12 antibodies, which are still being studied in preclinical trials.

therapy, the patient risk score, and the histology of the tumor.

everolimus *vs* 1.9 months for placebo (12).

interferone/placebo followed by a TKI (7).

studies are available yet.

The adoption of alternative angiogenic signaling pathways to compensate for inhibition of VEGF/VEGFR-mediated signaling seems to be the common mechanism for the development of cancer resistance to VEGF pathway inhibitors. Nevertheless, until now very few data are known about which alternative pathways are involved in resistant disease.

Many attempts have been proposed to overcome resistance. These include the use of non cross-resistant drugs, the optimization of sequential therapies, and the use of combined therapies. Unfortunately, all these approaches have given only modest results. Therefore, the overcome resistance mechanisms to antiangiogenic agents remains the next challenge in the treatment of renal cell carcinoma.

#### **5. Acknowledgements**

The author would like to thank Caroline Oakley and Silvana Valerio for their assistance with the linguistic revision of this review.

#### **6. References**


The Next Challenge in the Treatment of Renal Cell Carcinoma:

Cell 2009; 3;15(3):220-31. Review.

Nature 2011; 472(7341):90-94.

186(1):289-294.

95:1131-1135.

15(3):232-239.

Overcoming the Resistance Mechanisms to Antiangiogenic Agents 97

[25] Ebos JM, Lee CR, Cruz-Munoz W, et al: Accelerated metastasis after short-term

[26] Paez-Ribes M, Allen E, Hudock J, et al: Antiangiogenic therapy elicits malignant

[27] Frederick BA, Helfrich BA, Coldren CD, et al: Epithelial to mesenchymal transition

[30] Ghersi G: Roles of molecules involved in epithelial/mesenchymal transition during

[31] Hugo H, Ackland ML, Blick T, et al: Epithelial-mesenchymal and mesenchymalepithelial transitions in carcinoma progression. J Cell Physiol 2007; 213:374–383. [32] Hammers HJ, Verheul HM, Salumbides B, et al: Reversible epithelial to mesenchymal

[35] Navin N, Kendall J, Troge J, et al: Tumour evolution inferred by single-cell sequencing.

[36] Zama IN, Hutson TE, Elson P, et al: Sunitinib rechallenge in metastatic renal cell

[37] Rini BI, Hutson HE, Elson P et al: Clinical activity of sunitinib rechallenge in metastatic

[38] Sanjmyatas J, Steiner T, Wunderlich H, et al: A specific gene expression signature

[39] Sabbadini RA: Targeting sphingosine-1 -phosphate for cancer therapy. Br J Cancer 2006;

[40] Bhatt RS, Wang X, Zhang L, et al: Renal cancer resistance to antiangiogenic therapy is

[41] Sabbadini RA. Sphingosine-1-phosphate antibodies as potential agents in the treatment of

[42] Rini BI, Wilding G, Hudes G et al: Phase II study of axitinib in sorafenib-refractory

[43] Rini BI, Hutson HE, Elson P et al: Clinical activity of sunitinib rechallenge in metastatic renal cell carcinoma. GU ASCO meeting 2010, J Clin Oncol 2010, Abst 396. [44] Rini BI, Garcia JA, Cooney MM, et al: Toxicity of sunitinib plus bevacizumab in renal

metastatic renal cell carcinoma. J Clin Oncol 2009; 27:4462-4468.

cell carcinoma. J Clin Oncol. 2010; 10;28(17):284-285.

characterizes metastatic potential in clear cell renal cell carcinoma. J Urol 2011;

delayed by restoration of angiostatic signaling. Mol Cancer Ther. 2010; 9(10):2793-802.

cancer and age-related macular degeneration. Br J Pharmacol. 2011; 162(6):1225-1238.

evidence from a xenograft study. Mol Cancer Ther 2010; 9:1525-1535. [33] Klymkowsky MW, Savagner P: Epithelial-mesenchymal transition: A cancer researcher's conceptual friend and foe. Am J Pathol 2009; 174:1588–1593. [34] Lee AJ, Endesfelder D, Rowan AJ, Walther A, et al: Chromosomal instability confers

intrinsic multidrug resistance. Cancer Res. 2011; 71(5):1858-70.

renal cell carcinoma. GU ASCO 2010, J Clin Oncol 2010, Abst 396.

carcinoma patients. Cancer 2010; 116(23):5400-5406.

and non-small cell lung carcinoma. Mol Cancer Ther 2007; 6:1683–1691. [28] Shah AN, Summy JM, Zhang J, et al: Development and characterization of gemcitabineresistant pancreatic tumor cells. Ann Surg Oncol 2007; 14:3629–3637. [29] Kajiyama H, Shibata K, Terauchi M, et al: Chemoresistance to paclitaxel induces

ovarian carcinoma cells. Int J Oncol 2007; 31:277–283.

angiogenesis. Front Biosci 2008; 13:2335–2355.

treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 2009;

progression of tumors to increased local invasion and distant metastasis. Cancer

predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma

epithelial-mesenchymal transition and enhances metastatic potential for epithelial

transition and acquired resistance to sunitinib in patients with renal cell carcinoma:


[6] Motzer RJ, Hutson TE, Tomczak P, et al: Overall survival and updated results for

[7] Escudier B, Bellmunt J, Négrier S et al: Phase III trial of bevacizumab plus interferon alfa-

[8] Rini, BI Halabi S, Rosenberg JE, et al. Phase III trial of bevacizumab plus interferon alfa

[9] Vickers MM, Choueiri TK, Rogers M, et al: Clinical outcome in metastatic renal cell

[10] Hutson, TE, Davis ID, Machiels JP, et al. Efficacy and safety of pazopanib in patients with metastatic renal cell carcinoma.J Clin Oncol 2010 ; 28(3):475-480. [11] Hudes G, Carducci M, Tomczak P et al: Temsirolimus, interferon alfa, or both for

[12] Motzer RJ, Escudier B, Oudard S et al: Efficacy of everolimus in advanced renal cell

[13] RanpuraV, Su X, Wu S: Influence of prior therapies on the risk of primary progressive

[15] Casanovas O, Hicklin DJ, Bergers G, Hanahan D: Drug resistance by evasion of

[16] Sleijfer S, Wiemer E, Seynaeve C, Verweij J: Improved insight into resistance

[18] Huang D, Ding Y, Zhou M, et al: Interleukin-8 mediates resistance to antiangiogenic agent sunitinib in renal cell carcinoma.. Cancer Res 2010; 70(3):1063-1071.

[20] Faivre S, Demetri G, Sargent W, Raymond E: Molecular basis for sunitinib efficacy and future clinical development. Nat Rev Drug Discov. 2007; 6(9):734-745. Review. [21] Welti JC, Gourlaouen M, Powles T, et al: Fibroblast growth factor 2 regulates endothelial cell sensitivity to sunitinib. Oncogene 2011; 30(10):1183-1193. [22] Tsimafeyeu I, Demidov L, Ta H, et al: Fibroblast growth factor pathway in renal cell

[23] Ho TH, Wang F, Hoang A, et al: FGFR1) expression and activation in clear cell renal cell carcinoma (ccRCC). ASCO meeting 2011. J Clin Oncol 29: 2011 (suppl; abstr e15015). [24] Liu Y, Tran HT, Lin Y, et al: Plasma cytokine and angiogenic factors (CAFs) predictive

of clinical benefit and prognosis in patients (Pts) with advanced or metastatic renal cell cancer (mRCC) treated in phase III trials of pazopanib (PAZO). ASCO meeting

advanced renal-cell carcinoma. N Engl J Med 2007; 356: 2271-2281.

meta-analysis. GU ASCO meeting 2010. J Clin Oncol 2011, Abst 347. [14] Su X, Wu S: Treatment failure secondary to primary progressive disease in patients

final results of CALGB 90206. J Clin Oncol 2010; 28:2137-2143.

carcinoma. J Clin Oncol. 2009; 27(22):3584-3590.

overall survival. J Clin Oncol 2010; 28:2144-2150.

targeted therapy. Urology 2010; 76(2):430-434.

2008; 372:449-456.

ASCO 2010, Abst 391.

Cancer Cell. 2005; 8(4):299-309.

Rep 2008;10(4):344-349. Review.

[19] Folkman J: Principles and practice in Oncology Cancer 2005.

2011. J Clin Oncol 29: 2011 (suppl 7; abstr 334).

carcinoma. J Clin Oncol 28:15s, 2010 (suppl; abstr 4621).

sunitinib compared with interferon alfa in patients with metastatic renal cell

2a in patients with metastatic renai celi carcinoma (AVOREN): final analysis of

versus interferon alfa monotherapy in patients with metastatic renal cell carcinoma:

carcinoma patients after failure of initial vascular endothelial growth factor-

carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet

disease in patients with metastatic renal cell carcinoma treated with sunitinib: A

with metastatic renal cell carcinoma treated with sorafenib: A meta-analysis. GU

antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors.

mechanisms to imatinib in gastrointestinal stromal tumors: a basis for novel approaches and individualization of treatment. Oncologist. 2007; 12(6):719-726. [17] Reichardt P: Novel approaches to imatinib- and sunitinib-resistant GIST. Curr Oncol


**5** 

*USA* 

**Steroid Receptors in Renal Cell Carcinoma** 

*Department of Pathology and Laboratory Medicine, Rhode Island Hospital and* 

Renal cell carcinomas (RCCs) are the most common epithelial neoplasms of adult kidney. It has been estimated that there will be about 60,920 new cases of kidney cancer in the United States in 2011 and about 13,120 people will die from this disease (Siegel et al. 2011). Currently, surgery remains the only effective treatment for RCC, since metastatic disease is highly resistant to radiotherapy and chemotherapy. Approximately 20 to 30% of patients with RCCs present with non-resectable metastatic disease and 20 to 40% of patients undergoing nephrectomy for clinically localized RCC will develop metastatic disease. In the past two decades significant advances in the diagnosis and treatment of patients with RCC have resulted in improved survival of a select group of patients. Prior to the availability of targeted therapies, Interferon-(IFN) was the standard of care but was associated with a low response rate and significant toxicity (Interferon-alpha and survival in metastatic renal carcinoma: early results of a randomized controlled trial. Medical Research Council Renal Cancer Collaborators 1999). High dose interleukin-2 (IL-2) has a similar response rate as IFN, but can cure approximately 3-5% of patients (Yang, Sherry, et al. 2003). Targeted molecular therapies include inhibitors of angiogenesis (Yang, Haworth, et al. 2003), inhibitors of receptor tyrosine kinases with promiscuous targets including VEGFR1 and VEGFR2, PDGFR, C-Kit, Raf kinase, mammalian target of rapamycin (mTOR) (Atkins et al. 2004; Motzer et al. 2006; Porta et al. 2011) and combination treatment modalities (Escudier et al. 2007; Hudes et al. 2007; Motzer et al. 2007). These novel therapies have demonstrated improved outcomes and have become the first line of therapy in patients with advanced metastatic disease or second line of therapy in patients who have failed prior cytokine immunotherapy (Leveridge & Jewett 2011). As new treatment modalities become standard of care, clinical practices in diagnosis and treatment of the primary tumor will undergo revision. For instance, the role of cytoreductive surgery in patients selected for targeted therapy has not yet been established. This could increase the number of cases diagnosed and treated based on core needle biopsies alone, presenting new challenges to surgical pathologists who will likely have to use smaller amounts of tissue to accurately classify the

tumor and provide molecular information aimed to personalize clinical care.

RCC is a heterogeneous neoplasm, which includes distinct histological subtypes (Table 1). Among the adult population, clear cell RCC constitutes the most prevalent subtype (70-80%)

**1. Introduction** 

Evgeny Yakirevich, Andres Matoso, David J. Morris and Murray B. Resnick

*Alpert Medical School of Brown University,* 


## **Steroid Receptors in Renal Cell Carcinoma**

Evgeny Yakirevich, Andres Matoso,

David J. Morris and Murray B. Resnick *Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Alpert Medical School of Brown University, USA* 

#### **1. Introduction**

98 Emerging Research and Treatments in Renal Cell Carcinoma

[45] Sosman JA, Flaherty KT, Atkins MB et al: Updated results of phase I trial of sorafenib

[46] Yoshida K, Hosoya Y, Sumi S, et al: Studies of the expression of epidermal growth factor

[47] Motzer RJ, Hudes GR, Ginsberg MS, et al: Phase I/II trial of sunitinib plus gefitinib in patients with metastatic renal cell carcinoma. Am J Clin Oncol 2010; 33(6):614-618. [48] Whorf RC, Hainsworth JD, Spigel DR, et al: Phase II study of bevacizumab and

[49] Hainsworth JD, Spigel DR, Burris HA, et al: Phase II trial of bevacizumab and

[50] Merchan J R, Pitot HC, Qin R, et al: Phase I/II trial of CCI 779 and bevacizumab in

[51] Négrier S, Gravis G, Pérol D, et al: Temsirolimus and bevacizumab, or sunitinib, or

(TORAVA): a randomised phase 2 trial. Lancet Oncol. 2011; 12(7):673-680. [52] Gollob JA, Rathmell WK, Richmond TM., et al: Phase II trial of sorafenib plus interferon

[53] Larkin JM, Chowdhury S, Gore ME: Drug insight: advances in renal cell carcinoma and the role of targeted therapies. Nat Clin Pract Oncol. 2007; 4(8):470-479. [54] Kelly RJ, Rixe O: Axitinib (AG-013736). Recent Results Cancer Res 2010;184:33-44. [55] Angevin E, Lopez JA, Pande A, et al: TKI258 (dovitinib lactate) in metastatic renal cell

[56] Dudek AZ, Zolnierek J, Dham A et al: Sequential therapy with sorafenib and sunitinib

[57] Di Lorenzo G, Cartenì G, Autorino R et al: Phase II study of sorafenib in patients with sunitinib-refractory metastatic renai celi cancer. J Clin Oncol 2009; 27:4469-4474. [58] Tamaskar I, Garcia JA, Elson P et al: Antitumor effects of sunitinib or sorafenib in

[59] Merseburger AS, Simon A, Waalkes S, Kuczyk MA: Sorafenib reveals efficacy in

[60] Sablin MP, Négrier S, Ravaud A et al: Sequential sorafenib and sunitinib for renai celi

[61] Zimmermann K, Schmittel A, Steiner U et al: Sunitinib treatment for patients with

[62] Rini BI, Escudier B, Tomczak P, et al: Comparative effectiveness of axitinib versus

method versus ligand binding assay. Oncology. 1997; 54(3):220-225.

ASCO meeting 2008, J Clin Oncol 2008, abst 5010.

in renal cell carcinoma. Cancer 2009; 115: 61-67

patients. ASCO meeting 2009, J Clin Oncol 2011, Abst 5039.

ASCO meeting 2008, abst. 5011.

Clin Oncol 2007; 25:3288-3295.

therapy. J Urol 2008; 179:81-86.

carcinoma. J Urol 2009; 182:29-34.

Lancet. 2011 [Epub ahead of print].

2009; 9:1429-1434.

2009; 76:350-354.

28:2131-2136.

(S) and bevacizumab (B) in patients with metastatic renal cell cancer (mRCC).

receptor in human renal cell carcinoma: a comparison of immunohistochemical

everolimus (RAD001) in the treatment of advanced renal cell carcinoma (RCC).

everolimus in patients with advanced renal cell carcinoma. J Clin Oncol 2010;

advanced renal cell carcinoma (RCC): Safety and activity in RTKI refractory RCC

interferon alfa and bevacizumab for patients with advanced renal cell carcinoma

alfa-2b as first- or second-line therapy in patients with metastatic renal cell cancer. J

carcinoma (mRCC) patients refractory to approved targeted therapies: A phase I/II dose finding and biomarker study. ASCO meeting 2009, J Clin Oncol 2009, Abst 3563.

patients with metastatic renal cell carcinoma who received prior antiangiogenic

sequential treatment of metastatic renal cell cancer. Expert Rev Anticancer Ther

advanced clear-cell renal-cell carcinoma after progression on sorafenib. Oncology

sorafenib in advanced renal cell carcinoma (AXIS): a randomised phase 3 trial.

Renal cell carcinomas (RCCs) are the most common epithelial neoplasms of adult kidney. It has been estimated that there will be about 60,920 new cases of kidney cancer in the United States in 2011 and about 13,120 people will die from this disease (Siegel et al. 2011). Currently, surgery remains the only effective treatment for RCC, since metastatic disease is highly resistant to radiotherapy and chemotherapy. Approximately 20 to 30% of patients with RCCs present with non-resectable metastatic disease and 20 to 40% of patients undergoing nephrectomy for clinically localized RCC will develop metastatic disease. In the past two decades significant advances in the diagnosis and treatment of patients with RCC have resulted in improved survival of a select group of patients. Prior to the availability of targeted therapies, Interferon-(IFN) was the standard of care but was associated with a low response rate and significant toxicity (Interferon-alpha and survival in metastatic renal carcinoma: early results of a randomized controlled trial. Medical Research Council Renal Cancer Collaborators 1999). High dose interleukin-2 (IL-2) has a similar response rate as IFN, but can cure approximately 3-5% of patients (Yang, Sherry, et al. 2003). Targeted molecular therapies include inhibitors of angiogenesis (Yang, Haworth, et al. 2003), inhibitors of receptor tyrosine kinases with promiscuous targets including VEGFR1 and VEGFR2, PDGFR, C-Kit, Raf kinase, mammalian target of rapamycin (mTOR) (Atkins et al. 2004; Motzer et al. 2006; Porta et al. 2011) and combination treatment modalities (Escudier et al. 2007; Hudes et al. 2007; Motzer et al. 2007). These novel therapies have demonstrated improved outcomes and have become the first line of therapy in patients with advanced metastatic disease or second line of therapy in patients who have failed prior cytokine immunotherapy (Leveridge & Jewett 2011). As new treatment modalities become standard of care, clinical practices in diagnosis and treatment of the primary tumor will undergo revision. For instance, the role of cytoreductive surgery in patients selected for targeted therapy has not yet been established. This could increase the number of cases diagnosed and treated based on core needle biopsies alone, presenting new challenges to surgical pathologists who will likely have to use smaller amounts of tissue to accurately classify the tumor and provide molecular information aimed to personalize clinical care.

RCC is a heterogeneous neoplasm, which includes distinct histological subtypes (Table 1). Among the adult population, clear cell RCC constitutes the most prevalent subtype (70-80%)

Steroid Receptors in Renal Cell Carcinoma 101

Clear cell RCC

Oncocytoma and chromophobe RCC

Cystic nephroma, mixed epithelial and stromal tumor, angiomyolipoma with epithelial cysts

Cystic nephroma, mixed epithelial and stromal tumor, angiomyolipoma with epithelial cysts, chromophobe

Clear cell, papillary, and chromophobe

Papillary RCC, chromophobe RCC, oncocytoma, collecting duct carcinoma

Clear cell RCC, chromophobe RCC

(RAR-)

RCC and oncocytoma

RCC

Tumor Type Clinical

Relevance

Increased expression is a favorable marker

Diagnostic marker

Hormonal mechanism of pathogenesis

Hormonal mechanism of pathogenesis Diagnostic marker

Increased expression is a favorable marker

Diagnostic marker

Increased expression of RXR- is a favorable marker

References

Yakirevich et al.

Yakirevich et al.

Adsay et al. 2000

Adsay et al. 2000 Tickoo et al.

Mai et al. 2008

Kimura et al

Langner et al,

Obara, Konda et

Goelden et al.

Obara, Konda et

1993

2004

al. 2007 Liu et al. 2006

2005

al. 2007

2011

2008

2008

Steroid Receptor

GR

MR

ER

PR

AR

VDR

RAR and RXR

Expression in Normal Kidney

Proximal tubules,

Distal tubules, loops of Henle and collecting

glomeruli

ducts

Interstitial stromal cells

Interstitial stromal cells

Proximal and distal tubules

Distal tubules and collecting

Proximal tubules, interstitial cells

Table 2. Steroid Receptors in Renal Cell Neoplasms

ducts


Table 1. Major Histological Subtypes of Renal Cell Neoplasms with Corresponding Incidence, Survival, and Cell of Origin

and has a relatively unfavorable prognosis (Amin et al. 2002; Eble et al. 2004). Papillary and chromophobe RCCs are less common, comprising 10-15% and 5%, respectively, and have a better prognosis compared to clear cell RCC (Amin et al. 2002). Oncocytoma is a benign renal cell tumor characterized by an extremely favorable prognosis. Renal epithelial tumors are thought to originate in cells of different compartments along the nephron. Clear cell RCC is believed to arise from the proximal tubules. Tumors that originate in the collecting tubules and ducts include chromophobe RCC, oncocytomas, and the more rare collecting duct and medullary carcinomas. The histogenesis of papillary carcinoma is controversial with some studies suggesting a proximal tubule origin while phenotyping by immunohistochemistry supports a distal nephron origin. Renal tumors with papillary growth include papillary RCC types 1 and 2, clear cell RCC with papillary features and the recently described clear cell papillary RCC (CPRCC) (Gobbo et al. 2008). CPRCC is a subtype of renal cell carcinoma characterized by cells with clear cytoplasm arranged in papillary structures which was first described in patients with end stage renal disease, but later also identified in kidneys unaffected by end stage renal disease (Fuzesi et al. 1999; Gobbo et al. 2008).

Immunohistochemistry is useful in distinguishing the different subtypes of renal neoplasms. Clear cell RCCs are frequently CD10 positive but AMACR and CK7 negative; papillary carcinoma, on the other hand, is positive for CK7 and AMACR and usually negative for CD10. Recent studies in CPRCC demonstrate positive immunoreactivity for CK7 but negative AMACR and CD10. These and other immunohistochemical markers are currently used routinely in diagnostic histopathology to help classify tumors. However, this is a constantly evolving field and new immunohistochemical and molecular markers are being investigated to address new clinical needs.

Steroid receptors are a family of ligand dependent transcription factors, which have important roles in control of growth and differentiation in many non-neoplastic and neoplastic cell types. The steroid receptor family is characterized by a unique modular structure, with receptors classically divided into three main domains and several

70-80%

10-15%

5%

5%

unaffected by end stage renal disease (Fuzesi et al. 1999; Gobbo et al. 2008).

Table 1. Major Histological Subtypes of Renal Cell Neoplasms with Corresponding

and has a relatively unfavorable prognosis (Amin et al. 2002; Eble et al. 2004). Papillary and chromophobe RCCs are less common, comprising 10-15% and 5%, respectively, and have a better prognosis compared to clear cell RCC (Amin et al. 2002). Oncocytoma is a benign renal cell tumor characterized by an extremely favorable prognosis. Renal epithelial tumors are thought to originate in cells of different compartments along the nephron. Clear cell RCC is believed to arise from the proximal tubules. Tumors that originate in the collecting tubules and ducts include chromophobe RCC, oncocytomas, and the more rare collecting duct and medullary carcinomas. The histogenesis of papillary carcinoma is controversial with some studies suggesting a proximal tubule origin while phenotyping by immunohistochemistry supports a distal nephron origin. Renal tumors with papillary growth include papillary RCC types 1 and 2, clear cell RCC with papillary features and the recently described clear cell papillary RCC (CPRCC) (Gobbo et al. 2008). CPRCC is a subtype of renal cell carcinoma characterized by cells with clear cytoplasm arranged in papillary structures which was first described in patients with end stage renal disease, but later also identified in kidneys

Immunohistochemistry is useful in distinguishing the different subtypes of renal neoplasms. Clear cell RCCs are frequently CD10 positive but AMACR and CK7 negative; papillary carcinoma, on the other hand, is positive for CK7 and AMACR and usually negative for CD10. Recent studies in CPRCC demonstrate positive immunoreactivity for CK7 but negative AMACR and CD10. These and other immunohistochemical markers are currently used routinely in diagnostic histopathology to help classify tumors. However, this is a constantly evolving field and new immunohistochemical and molecular markers are being

Steroid receptors are a family of ligand dependent transcription factors, which have important roles in control of growth and differentiation in many non-neoplastic and neoplastic cell types. The steroid receptor family is characterized by a unique modular structure, with receptors classically divided into three main domains and several

Incidence 5-Year Survival Cell of Origin

45-76%

Proximal

and ducts

and ducts

convoluted tubules

Shared phenotype of proximal and distal tubules

Intercalated cells of collecting tubules

Intercalated cells of collecting tubules

82-90%

78-92%

100%

Histological Subtype

Clear Cell RCC

Papillary RCC

Oncocytoma

Chromophobe RCC

Incidence, Survival, and Cell of Origin

investigated to address new clinical needs.


Table 2. Steroid Receptors in Renal Cell Neoplasms

Steroid Receptors in Renal Cell Carcinoma 103

along the kidney nephron. *In vitro* studies have implicated GRs in the regulation of ammoniagenesis, gluconeogenesis, GFR, Na-H exchange and Na-phosphate co-transport, all of which are proximal renal tubule processes (Baylis et al. 1990; Boross et al. 1986; Campen et al. 1983; Freiberg et al. 1982). Measurement of GRs in normal rat kidney cortical tubules enriched in proximal tubules yielded three to six fold higher GR content as compared to the distal tubules (Mishina et al. 1981). Predominant proximal tubule localization of GR was demonstrated by quantitation of GR mRNA levels in microdissected nephron segments from the rat kidney by a competitive polymerase chain reaction (PCR) technique (Todd-Turla et al. 1993). GR mRNA was twofold more abundant in glomeruli, proximal tubule, and thick ascending limb segments than in the collecting duct segments (Todd-Turla et al. 1993). In an additional study GR mRNA was localized by in-situ hybridization predominantly to renal proximal tubules and cortical collecting tubules with lower levels in distal collecting tubules of the rat kidney (Roland et al. 1995). In a recent study we provided immunohistochemical evidence of GR expression in the proximal tubular epithelium of normal human kidneys and in the epithelial cells of normal renal glomeruli (Yakirevich et al. 2011). However, several *in vitro* and *in vivo* studies have demonstrated that glucocorticoids can exert mineralocorticoid-like effects, such as Na+ reabsorption and K+ secretion, in the distal nephron (Morris & Souness 1992; Naray-Fejes-Toth & Fejes-Toth 1990;

Initial studies of GR expression in RCCs based on ligand-binding assays in the early 1980's demonstrated the presence of GRs in kidney tumors (Bojar et al. 1979; Chen et al. 1980; Hemstreet et al. 1980; Liu et al. 1980). In these pioneer studies renal tumors were not subdivided into different histologic subtypes and were all designated as RCCs. Bojar et al. demonstrated GRs in 10 of 15 tumors studied (Bojar et al. 1979). The average dexamethasone binding capacity was calculated and found to be 7.1 fmol/mg of cytosol protein. The ligand specificity experiments clearly indicated that binding to GRs is not restricted to glucocorticoids alone. Progesterone and aldosterone turned out to be moderate competitors for dexamethasone binding. Medroxyprogesterone acetate, the compound widely used in hormone therapy of advanced renal cancer in man, was demonstrated to be one of the strongest inhibitors of [3H] dexamethasone. The binding of medroxyprogesterone acetate to GRs may represent the primary mechanism of action of the compound in causing tumor regression. Hemstreet et al. identified and measured the levels of GRs in 47 autologous pairs of normal and neoplastic renal tissue (Hemstreet et al. 1980). Glucocorticoid receptors were demonstrated in this study in normal and neoplastic tissues of both sexes. The levels of GRs were higher in the tumors (mean 31.3 fmol/mg) than in the normal tissue (18.5 fmol/mg). In an additional study conducted at the same time, Liu et al. reported high concentrations of GRs in four of seven RCC cases (Liu et al. 1980). The levels of GRs in RCCs were comparable to those in the glucocorticoid-responsive rat liver. Furthermore, the GR levels in RCCs were comparable to human acute lymphocytic leukemia cells sensitive (0.03 pmol/mg cytosol protein), in contrast to those that have become resistant (0.015 pmol/mg cytosol protein) to glucocorticoids. Chen et al. detected GRs in cytosol of RCCs (Chen et al. 1980). Competition experiments demonstrated that progestin competed for the GR sites in all renal tumors

tested, whereas diethylstilbestrol and testosterone were weak or not competitive.

Thomas et al. 2006).

**2.2 Expression of GR in kidney tumors** 

subdomains or regions. In general, the receptor members share a variable amino-terminal transactivation domain, a central and well-conserved DNA-binding domain (DBD), and a moderately conserved carboxy-terminal domain responsible for ligand binding. The latter domain also contains activating functions. The well known members of the steroid receptor family includes glucocorticoid (GR), mineralocorticoid (MR), progesterone (PR), androgen (AR), estrogen (ER), vitamin D (VDR), thyroid, and retinoic acid (RAR)/retinoid X receptors (RXR) (Fuller 1991).

There is emerging evidence that steroid receptors can induce gene expression through both ligand-dependent and ligand-independent pathways, and distinct families of genes are likely to be regulated depending on the mechanism of nuclear receptor signaling. Until recently, the study of steroid receptors in renal cell neoplasm's (RCNs) has been limited to ER and PR. The employment of novel techniques for studying steroid receptors in RCCs, such as immunohistochemistry, tissue microarray technology, and quantitative real-time PCR has revealed the presence and biologic importance of several steroid receptors in RCNs, including GR, MR, VDR, and others (Table 2). This review will focus on histogenetic, diagnostic, and prognostic implications of steroid receptor expression in RCNs.

#### **2. Glucocorticoid receptor**

Glucocorticoids mediate their effects via their intracellular glucocorticoid receptors. Studies of GRs have revealed that there is only one GR gene, but several GR receptor isoforms resulting from alternative splicing or alternative translation initiation (Pujols et al. 2002; Revollo & Cidlowski 2009). Two main human isoforms, GR- and GR-, have a different distribution pattern and biologic activity in healthy and diseased human cells and tissues. It has been demonstrated that GR- is the predominant isoform expressed in a large number of healthy human tissues including brain, liver, kidney, skeletal muscle, lung, and other organs. The GR- isoform possesses steroid binding activity. In contrast, GR- expression level is lower than that of the GR- isoform and is relatively abundant in inflammatory blood cells (Pujols et al. 2002). In non-activated cells, the GR resides in the cytoplasm as a part of a large complex consisting of chaperone and cochaperone proteins including heat shock proteins hsp90, hsp70, immunophilins FKBP51 and FKBP52, and others (De Bosscher et al. 2003). Upon ligand binding, GR undergoes phosphorylation and activation and translocates from the cytoplasm to the nucleus where it converts to a DNA-binding form. Transcriptional responses triggered by activated GR include both positive and negative gene regulation. The direct positive transcriptional regulation of genes (transactivation) requires binding of the GR homodimer to glucocorticoid-response elements (GRE) in gene promoters. The indirect negative regulation (transrepression) is mediated through negative cross-talk with other transcription factors including AP-1, NF-kB and p53 (Beato et al. 1995). As a result, glucocorticoids modulate a variety of physiologic and pathologic processes, including among others cellular differentiation, growth, inflammation, immune response, and carbohydrate metabolism.

#### **2.1 Expression of GR in the normal kidney**

In normal human kidneys GRs contribute to the regulation of renal fluid and electrolyte homeostasis. Keeping with their physiologic function, GRs are differentially distributed

subdomains or regions. In general, the receptor members share a variable amino-terminal transactivation domain, a central and well-conserved DNA-binding domain (DBD), and a moderately conserved carboxy-terminal domain responsible for ligand binding. The latter domain also contains activating functions. The well known members of the steroid receptor family includes glucocorticoid (GR), mineralocorticoid (MR), progesterone (PR), androgen (AR), estrogen (ER), vitamin D (VDR), thyroid, and retinoic acid (RAR)/retinoid X receptors

There is emerging evidence that steroid receptors can induce gene expression through both ligand-dependent and ligand-independent pathways, and distinct families of genes are likely to be regulated depending on the mechanism of nuclear receptor signaling. Until recently, the study of steroid receptors in renal cell neoplasm's (RCNs) has been limited to ER and PR. The employment of novel techniques for studying steroid receptors in RCCs, such as immunohistochemistry, tissue microarray technology, and quantitative real-time PCR has revealed the presence and biologic importance of several steroid receptors in RCNs, including GR, MR, VDR, and others (Table 2). This review will focus on histogenetic,

Glucocorticoids mediate their effects via their intracellular glucocorticoid receptors. Studies of GRs have revealed that there is only one GR gene, but several GR receptor isoforms resulting from alternative splicing or alternative translation initiation (Pujols et al. 2002; Revollo & Cidlowski 2009). Two main human isoforms, GR- and GR-, have a different distribution pattern and biologic activity in healthy and diseased human cells and tissues. It has been demonstrated that GR- is the predominant isoform expressed in a large number of healthy human tissues including brain, liver, kidney, skeletal muscle, lung, and other organs. The GR- isoform possesses steroid binding activity. In contrast, GR- expression level is lower than that of the GR- isoform and is relatively abundant in inflammatory blood cells (Pujols et al. 2002). In non-activated cells, the GR resides in the cytoplasm as a part of a large complex consisting of chaperone and cochaperone proteins including heat shock proteins hsp90, hsp70, immunophilins FKBP51 and FKBP52, and others (De Bosscher et al. 2003). Upon ligand binding, GR undergoes phosphorylation and activation and translocates from the cytoplasm to the nucleus where it converts to a DNA-binding form. Transcriptional responses triggered by activated GR include both positive and negative gene regulation. The direct positive transcriptional regulation of genes (transactivation) requires binding of the GR homodimer to glucocorticoid-response elements (GRE) in gene promoters. The indirect negative regulation (transrepression) is mediated through negative cross-talk with other transcription factors including AP-1, NF-kB and p53 (Beato et al. 1995). As a result, glucocorticoids modulate a variety of physiologic and pathologic processes, including among others cellular differentiation, growth, inflammation, immune response,

In normal human kidneys GRs contribute to the regulation of renal fluid and electrolyte homeostasis. Keeping with their physiologic function, GRs are differentially distributed

diagnostic, and prognostic implications of steroid receptor expression in RCNs.

(RXR) (Fuller 1991).

**2. Glucocorticoid receptor** 

and carbohydrate metabolism.

**2.1 Expression of GR in the normal kidney** 

along the kidney nephron. *In vitro* studies have implicated GRs in the regulation of ammoniagenesis, gluconeogenesis, GFR, Na-H exchange and Na-phosphate co-transport, all of which are proximal renal tubule processes (Baylis et al. 1990; Boross et al. 1986; Campen et al. 1983; Freiberg et al. 1982). Measurement of GRs in normal rat kidney cortical tubules enriched in proximal tubules yielded three to six fold higher GR content as compared to the distal tubules (Mishina et al. 1981). Predominant proximal tubule localization of GR was demonstrated by quantitation of GR mRNA levels in microdissected nephron segments from the rat kidney by a competitive polymerase chain reaction (PCR) technique (Todd-Turla et al. 1993). GR mRNA was twofold more abundant in glomeruli, proximal tubule, and thick ascending limb segments than in the collecting duct segments (Todd-Turla et al. 1993). In an additional study GR mRNA was localized by in-situ hybridization predominantly to renal proximal tubules and cortical collecting tubules with lower levels in distal collecting tubules of the rat kidney (Roland et al. 1995). In a recent study we provided immunohistochemical evidence of GR expression in the proximal tubular epithelium of normal human kidneys and in the epithelial cells of normal renal glomeruli (Yakirevich et al. 2011). However, several *in vitro* and *in vivo* studies have demonstrated that glucocorticoids can exert mineralocorticoid-like effects, such as Na+ reabsorption and K+ secretion, in the distal nephron (Morris & Souness 1992; Naray-Fejes-Toth & Fejes-Toth 1990; Thomas et al. 2006).

#### **2.2 Expression of GR in kidney tumors**

Initial studies of GR expression in RCCs based on ligand-binding assays in the early 1980's demonstrated the presence of GRs in kidney tumors (Bojar et al. 1979; Chen et al. 1980; Hemstreet et al. 1980; Liu et al. 1980). In these pioneer studies renal tumors were not subdivided into different histologic subtypes and were all designated as RCCs. Bojar et al. demonstrated GRs in 10 of 15 tumors studied (Bojar et al. 1979). The average dexamethasone binding capacity was calculated and found to be 7.1 fmol/mg of cytosol protein. The ligand specificity experiments clearly indicated that binding to GRs is not restricted to glucocorticoids alone. Progesterone and aldosterone turned out to be moderate competitors for dexamethasone binding. Medroxyprogesterone acetate, the compound widely used in hormone therapy of advanced renal cancer in man, was demonstrated to be one of the strongest inhibitors of [3H] dexamethasone. The binding of medroxyprogesterone acetate to GRs may represent the primary mechanism of action of the compound in causing tumor regression. Hemstreet et al. identified and measured the levels of GRs in 47 autologous pairs of normal and neoplastic renal tissue (Hemstreet et al. 1980). Glucocorticoid receptors were demonstrated in this study in normal and neoplastic tissues of both sexes. The levels of GRs were higher in the tumors (mean 31.3 fmol/mg) than in the normal tissue (18.5 fmol/mg). In an additional study conducted at the same time, Liu et al. reported high concentrations of GRs in four of seven RCC cases (Liu et al. 1980). The levels of GRs in RCCs were comparable to those in the glucocorticoid-responsive rat liver. Furthermore, the GR levels in RCCs were comparable to human acute lymphocytic leukemia cells sensitive (0.03 pmol/mg cytosol protein), in contrast to those that have become resistant (0.015 pmol/mg cytosol protein) to glucocorticoids. Chen et al. detected GRs in cytosol of RCCs (Chen et al. 1980). Competition experiments demonstrated that progestin competed for the GR sites in all renal tumors tested, whereas diethylstilbestrol and testosterone were weak or not competitive.

Steroid Receptors in Renal Cell Carcinoma 105

regressed after palliative treatment with betamethasone (Tanaka et al. 2003). A 10 year complete remission of metastatic RCC to the liver and retroperitoneal lymph nodes was described in a patient who received palliative cortisone therapy (Christophersen et al. 2006). The mechanism of metastases regression in these cases is unknown and is not likely to be immune related, because glucocorticoids are known to suppress the immune system. These observations suggest that GR and its agonists may have a potential role in novel anti-cancer

The mineralocorticoid receptor (MR) has long been considered as a secondary glucocorticoid receptor, even though specific roles of its natural ligand, aldosterone, have been well established since the purification of electrocortin more than 50 years ago. Aldosterone was initially restricted to the control of sodium reabsorption in the kidney, thereby being recognized as a major regulator of volume status and blood pressure. The cloning of a specific receptor for aldosterone (Arriza et al. 1987) definitively moved MR out of the shadow of GR and opened a new era of exciting biological, biochemical, and genetic studies that have provided important insights into the complexity of MR action. The MR is closely related to GR and is 94% homologous in the DNA binding domain and 57% homologous in the ligand binding domain, but only 15% homologous in the N-terminal region (Evans 1988). The MR has a similar affinity for the mineralocorticoid aldosterone and the glucocorticoids corticosterone and cortisol (Krozowski & Funder 1983). Although rats and mice synthesize only corticosterone, cortisol is the predominant glucocorticoid in humans and many other mammals, including rodents. Since the circulating levels of glucocorticoids are several orders of magnitude higher than those of aldosterone, the primary mineralocorticoid, glucocorticoid activation of MR may be functionally significant. Specificity is conferred by the enzyme 11-hydroxysteroid dehydrogenase type II (11- HSD2) which converts the cortisol to the less active compound cortisone, thus allowing aldosterone binding to MR. In the absence of ligand, MRs are located in both the cytosol and nucleus bound by a variety of chaperone proteins, including hsp90. Upon exposure to either aldosterone or corticosterone, most MRs are found in the nucleus, where they bind to hormone-response elements and mediate gene expression of signaling proteins regulating water and electrolyte transport including K-ras, serine-threonine kinase Sgk1, and corticosteroid hormone-induced factor (Connell & Davies 2005). The most recent role of aldosterone in renal and cardiac fibrosis has indicated a pro-fibrotic role for MR and the product of 11-HSD2, cortisone or 11-dehydro-corticosterone in the regulation of this

In contrast to GRs which are expressed in a broad variety of cells, expression of MRs is restricted to fewer cell types. The MR is expressed in so-called "classical" aldosterone target tissues, which are sodium-transporting epithelia (kidney, colon, pancreas, salivary, and sweat glands) and in a variety of non-epithelial target tissues such as the central nervous system, mononuclear lymphocytes, large blood vessels, and the heart (Arriza et al. 1987; Sasano et al. 1992). A general agreement exists that the distal nephron is an aldosteronespecific target site. Specific nuclear binding sites for aldosterone exist from the thick

hormonal therapies in clear cell RCC.

**3. Mineralocorticoid receptor** 

process (Brem et al.).

**3.1 Expression of MR in the normal kidney** 

Development of antibodies against human GR enabled immunohistochemical and Western blot assessment of GR protein expression. In addition, molecular studies utilizing reverse transcriptase polymerase chain reaction (RT-PCR) revealed that most commonly used RCC cell lines express high levels of GR. In a study by Arai et al., two RCC cell lines OUR-10 and NC65 expressed high levels of GR, whereas Caki-1 cell exhibited low levels of GR expression by Western blot (Arai et al. 2008). Iwai et al. demonstrated GR mRNA expression in the A498, RCC270, Caki1, and ACHN renal carcinoma cells. A498 and RCC270 expressed especially high levels of the GR gene (Iwai et al. 2004). Recently, using tissue microarray technology and real-time RT-PCR we described the immunohistochemical and mRNA expression of GRs in different histologic subtypes of RCNs including clear cell RCC, papillary RCC, chromophobe RCC, and oncocytoma (Yakirevich et al. 2011). We found that GRs are strongly expressed in the majority of clear cell RCCs (66%), in 26% of papillary RCCs, and in only 6% of chromophobe RCC and 14% of oncocytomas. Within the clear cell carcinoma group, most positive cases (87%) demonstrated strong expression, whereas only 1 papillary RCC, 1 chromophobe RCC and none of the oncocytomas demonstrated strong immunoreactivity. In this study we used commercially available rabbit-antihuman GR polyclonal antibody PA1-511A from Affinity Bioreagents (Golden, CO) which recognizes both the - and - isoforms of GR. In order to recognize specific isoform expressed in RCC, we measured both isoforms by quantitative real-time PCR and demonstrated that RCCs express GR- isoform. We found that GR expression is associated with tumors of low nuclear grade (Fuhrman grade 1 and 2) and low stage (stage 1 and 2). Although GR expression was demonstrated predominantly in clear cell RCC group, the loss of GR expression in high-grade tumors and overlap with other histologic subtypes of RCCs limit the diagnostic utility of this marker. GR appears to be a marker of less aggressive behavior in RCC as there is significant correlation between GR expression and overall survival in RCC. By the end of follow-up 86% of CRCC patients with tumors expressing GRs were alive as compared to 54% of patients whose tumors were negative.

Since GRs are cytoplasmic receptors, which are translocated to the nuclei upon activation, the predominantly nuclear immunoreactivity of GRs suggests that these receptors are activated in RCCs. Association of GR expression with less aggressive behavior also suggests the tumor-suppressive role of GRs. Signaling through GRs in renal cancer cells involves suppression of other transcription factors, including nuclear factor *k*B, AP-1, CREB, CCAAT enhancer binding protein (C/EBP), signal transduction activator of transcription (STAT), p53, Smad, *etc* (De Bosscher et al. 2003). Treatment of RCC cell lines with glucocorticoids (dexamethasone) inhibits the activation of nuclear factor kB and its downstream products including IL-2, IL-6, IL-8, and vascular endothelial growth factor which have been demonstrated to promote growth of RCC cell lines (Arai et al. 2008; Iwai et al. 2004; Miki et al. 1989; Takenawa et al. 1995). Glucocorticoids have long been used as anti-inflammatory drugs, and have been beneficial in the treatment of hematopoietic neoplasms (multiple myeloma) and solid malignancies such as hormone-refractory prostate cancer (Greenstein et al. 2002; Storlie et al. 1995). Although glucocorticoids have not been implicated in the treatment of patients with renal cancer, there are few case reports describing the beneficial effects of incidental glucocorticoid treatment in metastatic RCC (Christophersen et al. 2006; Omland & Fossa 1989; Tanaka et al. 2003). Palliation treatment with oral dexamethasone was associated with complete regression of pulmonary and brain metastases (Omland & Fossa 1989). In another case, multiple lung and bone metastases of RCC completely

Development of antibodies against human GR enabled immunohistochemical and Western blot assessment of GR protein expression. In addition, molecular studies utilizing reverse transcriptase polymerase chain reaction (RT-PCR) revealed that most commonly used RCC cell lines express high levels of GR. In a study by Arai et al., two RCC cell lines OUR-10 and NC65 expressed high levels of GR, whereas Caki-1 cell exhibited low levels of GR expression by Western blot (Arai et al. 2008). Iwai et al. demonstrated GR mRNA expression in the A498, RCC270, Caki1, and ACHN renal carcinoma cells. A498 and RCC270 expressed especially high levels of the GR gene (Iwai et al. 2004). Recently, using tissue microarray technology and real-time RT-PCR we described the immunohistochemical and mRNA expression of GRs in different histologic subtypes of RCNs including clear cell RCC, papillary RCC, chromophobe RCC, and oncocytoma (Yakirevich et al. 2011). We found that GRs are strongly expressed in the majority of clear cell RCCs (66%), in 26% of papillary RCCs, and in only 6% of chromophobe RCC and 14% of oncocytomas. Within the clear cell carcinoma group, most positive cases (87%) demonstrated strong expression, whereas only 1 papillary RCC, 1 chromophobe RCC and none of the oncocytomas demonstrated strong immunoreactivity. In this study we used commercially available rabbit-antihuman GR polyclonal antibody PA1-511A from Affinity Bioreagents (Golden, CO) which recognizes both the - and - isoforms of GR. In order to recognize specific isoform expressed in RCC, we measured both isoforms by quantitative real-time PCR and demonstrated that RCCs express GR- isoform. We found that GR expression is associated with tumors of low nuclear grade (Fuhrman grade 1 and 2) and low stage (stage 1 and 2). Although GR expression was demonstrated predominantly in clear cell RCC group, the loss of GR expression in high-grade tumors and overlap with other histologic subtypes of RCCs limit the diagnostic utility of this marker. GR appears to be a marker of less aggressive behavior in RCC as there is significant correlation between GR expression and overall survival in RCC. By the end of follow-up 86% of CRCC patients with tumors expressing GRs were alive

Since GRs are cytoplasmic receptors, which are translocated to the nuclei upon activation, the predominantly nuclear immunoreactivity of GRs suggests that these receptors are activated in RCCs. Association of GR expression with less aggressive behavior also suggests the tumor-suppressive role of GRs. Signaling through GRs in renal cancer cells involves suppression of other transcription factors, including nuclear factor *k*B, AP-1, CREB, CCAAT enhancer binding protein (C/EBP), signal transduction activator of transcription (STAT), p53, Smad, *etc* (De Bosscher et al. 2003). Treatment of RCC cell lines with glucocorticoids (dexamethasone) inhibits the activation of nuclear factor kB and its downstream products including IL-2, IL-6, IL-8, and vascular endothelial growth factor which have been demonstrated to promote growth of RCC cell lines (Arai et al. 2008; Iwai et al. 2004; Miki et al. 1989; Takenawa et al. 1995). Glucocorticoids have long been used as anti-inflammatory drugs, and have been beneficial in the treatment of hematopoietic neoplasms (multiple myeloma) and solid malignancies such as hormone-refractory prostate cancer (Greenstein et al. 2002; Storlie et al. 1995). Although glucocorticoids have not been implicated in the treatment of patients with renal cancer, there are few case reports describing the beneficial effects of incidental glucocorticoid treatment in metastatic RCC (Christophersen et al. 2006; Omland & Fossa 1989; Tanaka et al. 2003). Palliation treatment with oral dexamethasone was associated with complete regression of pulmonary and brain metastases (Omland & Fossa 1989). In another case, multiple lung and bone metastases of RCC completely

as compared to 54% of patients whose tumors were negative.

regressed after palliative treatment with betamethasone (Tanaka et al. 2003). A 10 year complete remission of metastatic RCC to the liver and retroperitoneal lymph nodes was described in a patient who received palliative cortisone therapy (Christophersen et al. 2006). The mechanism of metastases regression in these cases is unknown and is not likely to be immune related, because glucocorticoids are known to suppress the immune system. These observations suggest that GR and its agonists may have a potential role in novel anti-cancer hormonal therapies in clear cell RCC.

#### **3. Mineralocorticoid receptor**

The mineralocorticoid receptor (MR) has long been considered as a secondary glucocorticoid receptor, even though specific roles of its natural ligand, aldosterone, have been well established since the purification of electrocortin more than 50 years ago. Aldosterone was initially restricted to the control of sodium reabsorption in the kidney, thereby being recognized as a major regulator of volume status and blood pressure. The cloning of a specific receptor for aldosterone (Arriza et al. 1987) definitively moved MR out of the shadow of GR and opened a new era of exciting biological, biochemical, and genetic studies that have provided important insights into the complexity of MR action. The MR is closely related to GR and is 94% homologous in the DNA binding domain and 57% homologous in the ligand binding domain, but only 15% homologous in the N-terminal region (Evans 1988). The MR has a similar affinity for the mineralocorticoid aldosterone and the glucocorticoids corticosterone and cortisol (Krozowski & Funder 1983). Although rats and mice synthesize only corticosterone, cortisol is the predominant glucocorticoid in humans and many other mammals, including rodents. Since the circulating levels of glucocorticoids are several orders of magnitude higher than those of aldosterone, the primary mineralocorticoid, glucocorticoid activation of MR may be functionally significant. Specificity is conferred by the enzyme 11-hydroxysteroid dehydrogenase type II (11- HSD2) which converts the cortisol to the less active compound cortisone, thus allowing aldosterone binding to MR. In the absence of ligand, MRs are located in both the cytosol and nucleus bound by a variety of chaperone proteins, including hsp90. Upon exposure to either aldosterone or corticosterone, most MRs are found in the nucleus, where they bind to hormone-response elements and mediate gene expression of signaling proteins regulating water and electrolyte transport including K-ras, serine-threonine kinase Sgk1, and corticosteroid hormone-induced factor (Connell & Davies 2005). The most recent role of aldosterone in renal and cardiac fibrosis has indicated a pro-fibrotic role for MR and the product of 11-HSD2, cortisone or 11-dehydro-corticosterone in the regulation of this process (Brem et al.).

#### **3.1 Expression of MR in the normal kidney**

In contrast to GRs which are expressed in a broad variety of cells, expression of MRs is restricted to fewer cell types. The MR is expressed in so-called "classical" aldosterone target tissues, which are sodium-transporting epithelia (kidney, colon, pancreas, salivary, and sweat glands) and in a variety of non-epithelial target tissues such as the central nervous system, mononuclear lymphocytes, large blood vessels, and the heart (Arriza et al. 1987; Sasano et al. 1992). A general agreement exists that the distal nephron is an aldosteronespecific target site. Specific nuclear binding sites for aldosterone exist from the thick

Steroid Receptors in Renal Cell Carcinoma 107

Expression of ERs was extensively studied in hamster kidneys; however, the distribution of ER in normal hamster kidney is controversial. In a study by Bhat et al. who treated hamsters with estradiol to induce tumors, ER immunolocalization in normal kidneys of estrogentreated hamsters or in untreated controls was identified only in the renal glomerular pododcytes, mesangial and parietal cells and in several interstitial cell types but not in the tubular epithelia of the cortex (Bhat et al. 1993). In addition, arterial cells, including pericytes and endothelial cells of the arteriolae rectae and endothelial cells of the arterial vasa recta, strongly expressed ER. The receptor distribution in kidneys of untreated female hamsters matched that of males, but the intensity of staining was higher than in male kidneys. Another study confirmed immunohistochemical expression of ER in interstitial cells and localized these cells to the corticomedullary junction (Li et al. 2001). The authors found that estrogen treatment causes a significant increase in ER- positive interstitial cells compared to untreated controls and hypothesized that renal tumors arise from a subset of multipotential interstitial cells driven to proliferate by estrogens. However, in contrast to the study by Bhat et al., in this study ER expression was consistently demonstrated in nuclei of

Initial biochemical studies of ER status in renal tumors were performed in early 1980s by the dextran-coated charcoal method and the sucrose gradient centrifugation assay. These biochemical assays were based on cytosol preparations containing high, but unknown levels of plasma contamination. Furthermore, there was significant inconsistency in the number of tumor cells present within the specimens (Karr et al. 1983). Therefore, the level and frequency of ER expression in human kidney tumors were highly variable. Hemstreet et al. reported detectable ERs in 30% of the tumors compared to 40% of normals, whereas in other studies utilizing similar biochemical techniques ERs were not detected or detected in a rather low percentage of 4-9% of tumors (Hemstreet et al. 1980; Karr et al. 1983; Pearson et al. 1981). In a more recent immunohistochemical analysis of steroid hormone expression in tissue microarrays containing 182 RCCs of different histologic subtypes, Langer et al. demonstrated ER immunoreactivity in less than 10% of tumor cells in only 2 of 182 of patients (1.1%), including one clear cell RCC and one chromophobe RCC (Langner et al. 2004). Thus, the biochemical and immunohistochemical results provide evidence that ER is

Recently, several benign renal tumors, characterized by the presence of stroma that resembles ovarian, endometrial, and mullerian-like, have been described, including cystic nephroma, mixed epithelial and stromal tumor (MEST) and angiomyolipomas with epithelial cysts (AMLEC) (Fine et al. 2006; Turbiner et al. 2007). Adsay et al. detected ERs in nuclei of the spindle cells in seven of 12 MESTs (Adsay et al. 2000). The staining was strong and diffuse and was present predominantly in the areas with long, slender, fibrocyte-like cells. In three of these cases, the epithelial cells also exhibited a cytoplasmic reaction with antibody to ER. Distinctive clinical and pathologic features characterize these lesions. Most of the patients in study of Adsay et al. were middle-aged (perimenopausal) females (mean age, 56 years) who had a long-term history of estrogen use. The only male patient also had a history of diethylstilbestrol exposure for 7 years followed by 4 years of lupron therapy for

**4.1 Expression of ER in the normal kidney** 

proximal tubules and disappeared after estrogen treatment.

not expressed or very rare expressed in low levels in RCCs.

**4.2 Expression of ER in kidney tumors** 

ascending limb of Henle's loop (cortical part) to the distal collecting duct in rabbit and rat kidneys (Farman & Bonvalet 1983; Farman et al. 1982). MR is expressed in the distal tubules, the connecting tubules, and along the collecting ducts at the mRNA level in rat and rabbit kidneys (Escoubet et al. 1996; Todd-Turla et al. 1993) and at the protein level in rabbit kidneys (Lombes et al. 1990). Immunohistochemical studies showed that in normal human kidney MR is expressed in the distal convoluted tubules, collecting ducts, and loops of Henle with predominant nuclear localization (Hirasawa et al. 1997; Sasano et al. 1992; Yakirevich et al. 2008).

#### **3.2 Expression of MR in kidney tumors**

More than 30 years ago Rafestin-Oblin et al. demonstrated the presence of high-affinity sites for aldosterone in normal human kidneys using a ligand-binding assay. In RCCs the cytosol and nuclear aldosterone binding was significantly lower than in normal tissues (Rafestin-Oblin et al. 1979). However, this study focused exclusively on clear cell RCCs. Recently using immunohistochemistry we analyzed tissue microarray specimens from patients with different histologic subtypes of renal cell neoplasms, and in addition, we quantitated MR mRNA by real time RT-PCR (Yakirevich et al. 2008). Most of the chromophobe RCC (90%) and oncocytomas (93%) strongly expressed MR. No MR immunoreactivity was detected in clear cell RCC, including clear cell carcinoma with predominantly granular cytoplasm, or in papillary RCC. The MR+ immunophenotype of chromophobe carcinoma and oncocytoma reflects their histogenetic origin from phenotypically similar distal convoluted tubules and collecting ducts, whereas absence of immunoreactivity in clear cell RCC is consistent with its origin from proximal convoluted tubules. As we described in the previous section, proximal tubules and histogenetically related clear cell RCCs express high levels of GR. MR appears to be a sensitive and specific marker of the distal nephron and its related neoplasms (chromophobe RCC and oncocytoma) and may be considered in the immunohistochemical panel to more accurately subtype renal cell tumors.

#### **4. Estrogen receptor**

The effects of estrogens are mediated by estrogen receptors (ERs). ERs were discovered in the 1960's by Jensen and Jacobson (Jensen et al. 2010). The basic structure of ER protein is similar to other steroid receptors and contains a DNA binding domain, transcription modulating domain, and steroid hormone binding domain. There are two ER types encoded on different chromosomes: ER- cloned in 1986 and ER-, which was discovered in 1996 (Greene et al. 1986; Kuiper et al. 1996). ER- is expressed in a variety of human organs, mainly reproductive, including the mammary gland, ovary, uterus, and vagina (Muramatsu & Inoue 2000). ER- is expressed in genitourinary human tissues such as prostate, ovary, testis, bladder, uterus, and renal pelvis, in the central nervous system, and is especially increased compared to ER- in various fetal tissues such as adrenals (Gustafsson 1999). The affinity of ER- to bind estradiol-17 is similar to the ER- form. However, ER- binds both androgens and phytoestrogens with greater affinity. The main physiologic role of ERs is implicated in the control of proliferation, differentiation, and development of many tissues. In contrast to the beneficial physiologic effects, ERs may also promote the development and growth of variety of cancers, including breast, endometrial and ovarian carcinomas in humans (Speirs et al. 1999) and renal tumors in Syrian hamsters (Li et al. 2001).

ascending limb of Henle's loop (cortical part) to the distal collecting duct in rabbit and rat kidneys (Farman & Bonvalet 1983; Farman et al. 1982). MR is expressed in the distal tubules, the connecting tubules, and along the collecting ducts at the mRNA level in rat and rabbit kidneys (Escoubet et al. 1996; Todd-Turla et al. 1993) and at the protein level in rabbit kidneys (Lombes et al. 1990). Immunohistochemical studies showed that in normal human kidney MR is expressed in the distal convoluted tubules, collecting ducts, and loops of Henle with predominant nuclear localization (Hirasawa et al. 1997; Sasano et al. 1992;

More than 30 years ago Rafestin-Oblin et al. demonstrated the presence of high-affinity sites for aldosterone in normal human kidneys using a ligand-binding assay. In RCCs the cytosol and nuclear aldosterone binding was significantly lower than in normal tissues (Rafestin-Oblin et al. 1979). However, this study focused exclusively on clear cell RCCs. Recently using immunohistochemistry we analyzed tissue microarray specimens from patients with different histologic subtypes of renal cell neoplasms, and in addition, we quantitated MR mRNA by real time RT-PCR (Yakirevich et al. 2008). Most of the chromophobe RCC (90%) and oncocytomas (93%) strongly expressed MR. No MR immunoreactivity was detected in clear cell RCC, including clear cell carcinoma with predominantly granular cytoplasm, or in papillary RCC. The MR+ immunophenotype of chromophobe carcinoma and oncocytoma reflects their histogenetic origin from phenotypically similar distal convoluted tubules and collecting ducts, whereas absence of immunoreactivity in clear cell RCC is consistent with its origin from proximal convoluted tubules. As we described in the previous section, proximal tubules and histogenetically related clear cell RCCs express high levels of GR. MR appears to be a sensitive and specific marker of the distal nephron and its related neoplasms (chromophobe RCC and oncocytoma) and may be considered in the immunohistochemical

The effects of estrogens are mediated by estrogen receptors (ERs). ERs were discovered in the 1960's by Jensen and Jacobson (Jensen et al. 2010). The basic structure of ER protein is similar to other steroid receptors and contains a DNA binding domain, transcription modulating domain, and steroid hormone binding domain. There are two ER types encoded on different chromosomes: ER- cloned in 1986 and ER-, which was discovered in 1996 (Greene et al. 1986; Kuiper et al. 1996). ER- is expressed in a variety of human organs, mainly reproductive, including the mammary gland, ovary, uterus, and vagina (Muramatsu & Inoue 2000). ER- is expressed in genitourinary human tissues such as prostate, ovary, testis, bladder, uterus, and renal pelvis, in the central nervous system, and is especially increased compared to ER- in various fetal tissues such as adrenals (Gustafsson 1999). The affinity of ER- to bind estradiol-17 is similar to the ER- form. However, ER- binds both androgens and phytoestrogens with greater affinity. The main physiologic role of ERs is implicated in the control of proliferation, differentiation, and development of many tissues. In contrast to the beneficial physiologic effects, ERs may also promote the development and growth of variety of cancers, including breast, endometrial and ovarian carcinomas in

humans (Speirs et al. 1999) and renal tumors in Syrian hamsters (Li et al. 2001).

Yakirevich et al. 2008).

**4. Estrogen receptor** 

**3.2 Expression of MR in kidney tumors** 

panel to more accurately subtype renal cell tumors.

#### **4.1 Expression of ER in the normal kidney**

Expression of ERs was extensively studied in hamster kidneys; however, the distribution of ER in normal hamster kidney is controversial. In a study by Bhat et al. who treated hamsters with estradiol to induce tumors, ER immunolocalization in normal kidneys of estrogentreated hamsters or in untreated controls was identified only in the renal glomerular pododcytes, mesangial and parietal cells and in several interstitial cell types but not in the tubular epithelia of the cortex (Bhat et al. 1993). In addition, arterial cells, including pericytes and endothelial cells of the arteriolae rectae and endothelial cells of the arterial vasa recta, strongly expressed ER. The receptor distribution in kidneys of untreated female hamsters matched that of males, but the intensity of staining was higher than in male kidneys. Another study confirmed immunohistochemical expression of ER in interstitial cells and localized these cells to the corticomedullary junction (Li et al. 2001). The authors found that estrogen treatment causes a significant increase in ER- positive interstitial cells compared to untreated controls and hypothesized that renal tumors arise from a subset of multipotential interstitial cells driven to proliferate by estrogens. However, in contrast to the study by Bhat et al., in this study ER expression was consistently demonstrated in nuclei of proximal tubules and disappeared after estrogen treatment.

#### **4.2 Expression of ER in kidney tumors**

Initial biochemical studies of ER status in renal tumors were performed in early 1980s by the dextran-coated charcoal method and the sucrose gradient centrifugation assay. These biochemical assays were based on cytosol preparations containing high, but unknown levels of plasma contamination. Furthermore, there was significant inconsistency in the number of tumor cells present within the specimens (Karr et al. 1983). Therefore, the level and frequency of ER expression in human kidney tumors were highly variable. Hemstreet et al. reported detectable ERs in 30% of the tumors compared to 40% of normals, whereas in other studies utilizing similar biochemical techniques ERs were not detected or detected in a rather low percentage of 4-9% of tumors (Hemstreet et al. 1980; Karr et al. 1983; Pearson et al. 1981). In a more recent immunohistochemical analysis of steroid hormone expression in tissue microarrays containing 182 RCCs of different histologic subtypes, Langer et al. demonstrated ER immunoreactivity in less than 10% of tumor cells in only 2 of 182 of patients (1.1%), including one clear cell RCC and one chromophobe RCC (Langner et al. 2004). Thus, the biochemical and immunohistochemical results provide evidence that ER is not expressed or very rare expressed in low levels in RCCs.

Recently, several benign renal tumors, characterized by the presence of stroma that resembles ovarian, endometrial, and mullerian-like, have been described, including cystic nephroma, mixed epithelial and stromal tumor (MEST) and angiomyolipomas with epithelial cysts (AMLEC) (Fine et al. 2006; Turbiner et al. 2007). Adsay et al. detected ERs in nuclei of the spindle cells in seven of 12 MESTs (Adsay et al. 2000). The staining was strong and diffuse and was present predominantly in the areas with long, slender, fibrocyte-like cells. In three of these cases, the epithelial cells also exhibited a cytoplasmic reaction with antibody to ER. Distinctive clinical and pathologic features characterize these lesions. Most of the patients in study of Adsay et al. were middle-aged (perimenopausal) females (mean age, 56 years) who had a long-term history of estrogen use. The only male patient also had a history of diethylstilbestrol exposure for 7 years followed by 4 years of lupron therapy for

Steroid Receptors in Renal Cell Carcinoma 109

distinguishing oncocytoma from chromophobe RCC. The presence of PR in oncocytoma and chromophobe RCC provides additional support to the histopathogenetic relationship

Androgens are essential for differentiation and growth of male reproductive organs and for various biological effects in the kidney, brain, liver, muscle, bone and skin. Androgens include testosterone and dihydrotestosterone and mediate their biologic effect through the androgen receptor (AR). The *AR* gene is located on chromosome Xq11-12 (Brown et al. 1989; Lubahn et al. 1988). Males have a single copy of the gene allowing phenotypic manifestation of any genetic alteration. Transcription of the *AR* gene is cell-specific and modified by age, androgen and other steroid hormones (Gelmann 2002). Androgen is best known to influence development and growth of prostate cancer. However, its metabolic role in cancer is not limited to the prostate and a number of studies utilizing animal models combined with clinical and epidemiologic data suggest a role for androgen in RCC (Concolino, Marocchi,

AR is ubiquitously expressed in the whole body with studies showing detectable levels of protein and mRNA in adrenal glands, uterus, aorta, adipose tissue, kidney, spleen, heart, lung, large intestine, stomach, small intestine and liver (Kimura et al. 1993; Ruizeveld de Winter et al. 1991; Takeda et al. 1990). In normal kidneys AR expression was consistently demonstrated to be present in the nuclei of distal tubule cells (Kimura et al. 1993; Li et al. 2010). Additionally, a study by Takeda et al. showed AR immunoreactivity not only in the distal tubule but also in the proximal tubule and focal parietal expression in the Bowman's

The hormone dependence of RCC has been established in animal models and in humans for many years (Bloom 1973; Concolino, Marocchi, Conti et al. 1978; Concolino, Marocchi, Tenaglia et al. 1978; Li et al. 1977). In humans, extensive research on AR in RCC has shown variable results (Concolino et al. 1981; Jakse & Muller-Holzner 1988; Karr et al. 1983; Klotzl et al. 1987; Nakano et al. 1984; Noronha & Rao 1985) In a case series study by Brown et al., that included 12 primary clear cell RCCs and 5 clear cell RCCs metastatic to the central nervous system, AR immunoreactivity was present in five primary and one metastatic RCC (Brown et al. 1998). A more recent study by Langner et al. demonstrated that AR immunoreactivity was not detectable in non-tumoral kidney tissue (Langner et al. 2004). However, AR was found in 15% of patients with RCC and inversely correlated with histopathologic stage, with 27% of pT1 tumors being positive versus 4% of pT3 tumors. Furthermore, expression of AR was higher in pT1a tumors compared to pT1b (32% vs. 17%). Additionally, AR expression inversely correlated with nuclear grade with 21% positivity in nuclear grades 1 and 2 and 7% in nuclear grades 3 and 4. Univariate analysis showed a longer disease free survival in patients with AR positive tumors compared to patients with

between renal oncocytoma and chromophobe RCC.

**6. Androgen receptor** 

Conti et al. 1978; Karr et al. 1983).

capsule (Takeda et al. 1990).

**6.2 Expression of AR in kidney tumors** 

**6.1 Expression of AR in the normal kidney** 

prostatic adenocarcinoma. These clinical findings, combined with frequent ER expression detected by immunohistochemistry raise the possibility of hormonal mechanism of pathogenesis of these tumors. It is plausible that the spindle cells of these tumors arise from a "periductal fetal mesenchyma" present in epithelial structures of organs such as kidney, pancreas, and liver. The primitive mesenchyme may have the capacity to interact with epithelia. Alterations of hormonal milieu (perimenopausal changes or therapeutic hormones with unopposed estrogens) may induce proliferation of this mesenchyme, which in turn activates the growth of epithelial component.

#### **5. Progesterone receptor**

The progesterone receptor (PR) has two predominant isoforms: PR-, and PR-, which are produced from a single gene by alternative promoter usage (Jeltsch et al. 1986). These isoforms have similar steroid hormone and DNA binding activities, but PR- has a much higher transcriptional activating potential. Clinically, PR expression is routinely assessed by immunohistochemistry using an antibody that recognizes both PR- and PR-.

#### **5.1 Expression of PR in the normal kidney**

No detectable PR staining was seen in renal sections from untreated castrated male hamsters in a study by Bhat et al. (Bhat et al. 1993). However, after estrogen treatment, PR expression was detected in single interstitial cells. The pattern of PR immunoreactivity was largely confined to interstitial cells located at the renal corticomedullary region, similar to ER expressing cells described above. PRs were identified in normal human kidneys by biochemical and more recently immunohistochemical techniques (Hemstreet et al. 1980; McDonald et al. 1983). Interesting, in normal human kidneys PRs were detected by immunohistochemistry in interstitial stromal cells, some tubules, and mesangial cells of glomeruli in two of seven cases (Tickoo et al. 2008).

#### **5.2 Expression of PR in kidney tumors**

Expression of PR in kidney tumors was studied in parallel with ER analysis. The level and frequency of PR in human kidney tumors is highly variable when analyzed biochemically varying from 0 to 23% (Hemstreet et al. 1980; Karr et al. 1983; Pearson et al. 1981). Immunohistochemical analysis of steroid hormone expression in tissue microarrays containing 182 RCCs of different histologic subtypes demonstrated PR immunoreactivity in less than 10% of tumor cells in only two of 182 patients, including one clear cell RCC and one papillary RCC (Langner et al. 2004). PRs were found in stromal cells of renal neoplasms with ovarian-like stroma, although less frequently as compared to ER (Adsay et al. 2000). More recently Mai et al. identified PR immunoreactivity of tumor cells and stromal cells within the neoplasm and/or surrounding capsule in renal oncocytoma and chromophobe RCC (Mai et al. 2008). This immunoreactivity was not seen in other tumors with oncocytic/eosinophilic cytoplasm, such as papillary RCC with eosinophilc cytoplasm or clear cell RCC with eosinophilic cytoplasm. PR appears to be a sensitive and highly specific marker for renal oncocytoma and a highly specific marker for chromophobe RCC. It was demonstrated that PR immunoreactivity is more extensive in oncocytoma than in chromophobe RCC, therefore, the extent of PR immunoreactivity could be useful in distinguishing oncocytoma from chromophobe RCC. The presence of PR in oncocytoma and chromophobe RCC provides additional support to the histopathogenetic relationship between renal oncocytoma and chromophobe RCC.

#### **6. Androgen receptor**

108 Emerging Research and Treatments in Renal Cell Carcinoma

prostatic adenocarcinoma. These clinical findings, combined with frequent ER expression detected by immunohistochemistry raise the possibility of hormonal mechanism of pathogenesis of these tumors. It is plausible that the spindle cells of these tumors arise from a "periductal fetal mesenchyma" present in epithelial structures of organs such as kidney, pancreas, and liver. The primitive mesenchyme may have the capacity to interact with epithelia. Alterations of hormonal milieu (perimenopausal changes or therapeutic hormones with unopposed estrogens) may induce proliferation of this mesenchyme, which in turn

The progesterone receptor (PR) has two predominant isoforms: PR-, and PR-, which are produced from a single gene by alternative promoter usage (Jeltsch et al. 1986). These isoforms have similar steroid hormone and DNA binding activities, but PR- has a much higher transcriptional activating potential. Clinically, PR expression is routinely assessed by

No detectable PR staining was seen in renal sections from untreated castrated male hamsters in a study by Bhat et al. (Bhat et al. 1993). However, after estrogen treatment, PR expression was detected in single interstitial cells. The pattern of PR immunoreactivity was largely confined to interstitial cells located at the renal corticomedullary region, similar to ER expressing cells described above. PRs were identified in normal human kidneys by biochemical and more recently immunohistochemical techniques (Hemstreet et al. 1980; McDonald et al. 1983). Interesting, in normal human kidneys PRs were detected by immunohistochemistry in interstitial stromal cells, some tubules, and mesangial cells of

Expression of PR in kidney tumors was studied in parallel with ER analysis. The level and frequency of PR in human kidney tumors is highly variable when analyzed biochemically varying from 0 to 23% (Hemstreet et al. 1980; Karr et al. 1983; Pearson et al. 1981). Immunohistochemical analysis of steroid hormone expression in tissue microarrays containing 182 RCCs of different histologic subtypes demonstrated PR immunoreactivity in less than 10% of tumor cells in only two of 182 patients, including one clear cell RCC and one papillary RCC (Langner et al. 2004). PRs were found in stromal cells of renal neoplasms with ovarian-like stroma, although less frequently as compared to ER (Adsay et al. 2000). More recently Mai et al. identified PR immunoreactivity of tumor cells and stromal cells within the neoplasm and/or surrounding capsule in renal oncocytoma and chromophobe RCC (Mai et al. 2008). This immunoreactivity was not seen in other tumors with oncocytic/eosinophilic cytoplasm, such as papillary RCC with eosinophilc cytoplasm or clear cell RCC with eosinophilic cytoplasm. PR appears to be a sensitive and highly specific marker for renal oncocytoma and a highly specific marker for chromophobe RCC. It was demonstrated that PR immunoreactivity is more extensive in oncocytoma than in chromophobe RCC, therefore, the extent of PR immunoreactivity could be useful in

immunohistochemistry using an antibody that recognizes both PR- and PR-.

activates the growth of epithelial component.

**5.1 Expression of PR in the normal kidney** 

glomeruli in two of seven cases (Tickoo et al. 2008).

**5.2 Expression of PR in kidney tumors** 

**5. Progesterone receptor** 

Androgens are essential for differentiation and growth of male reproductive organs and for various biological effects in the kidney, brain, liver, muscle, bone and skin. Androgens include testosterone and dihydrotestosterone and mediate their biologic effect through the androgen receptor (AR). The *AR* gene is located on chromosome Xq11-12 (Brown et al. 1989; Lubahn et al. 1988). Males have a single copy of the gene allowing phenotypic manifestation of any genetic alteration. Transcription of the *AR* gene is cell-specific and modified by age, androgen and other steroid hormones (Gelmann 2002). Androgen is best known to influence development and growth of prostate cancer. However, its metabolic role in cancer is not limited to the prostate and a number of studies utilizing animal models combined with clinical and epidemiologic data suggest a role for androgen in RCC (Concolino, Marocchi, Conti et al. 1978; Karr et al. 1983).

#### **6.1 Expression of AR in the normal kidney**

AR is ubiquitously expressed in the whole body with studies showing detectable levels of protein and mRNA in adrenal glands, uterus, aorta, adipose tissue, kidney, spleen, heart, lung, large intestine, stomach, small intestine and liver (Kimura et al. 1993; Ruizeveld de Winter et al. 1991; Takeda et al. 1990). In normal kidneys AR expression was consistently demonstrated to be present in the nuclei of distal tubule cells (Kimura et al. 1993; Li et al. 2010). Additionally, a study by Takeda et al. showed AR immunoreactivity not only in the distal tubule but also in the proximal tubule and focal parietal expression in the Bowman's capsule (Takeda et al. 1990).

#### **6.2 Expression of AR in kidney tumors**

The hormone dependence of RCC has been established in animal models and in humans for many years (Bloom 1973; Concolino, Marocchi, Conti et al. 1978; Concolino, Marocchi, Tenaglia et al. 1978; Li et al. 1977). In humans, extensive research on AR in RCC has shown variable results (Concolino et al. 1981; Jakse & Muller-Holzner 1988; Karr et al. 1983; Klotzl et al. 1987; Nakano et al. 1984; Noronha & Rao 1985) In a case series study by Brown et al., that included 12 primary clear cell RCCs and 5 clear cell RCCs metastatic to the central nervous system, AR immunoreactivity was present in five primary and one metastatic RCC (Brown et al. 1998). A more recent study by Langner et al. demonstrated that AR immunoreactivity was not detectable in non-tumoral kidney tissue (Langner et al. 2004). However, AR was found in 15% of patients with RCC and inversely correlated with histopathologic stage, with 27% of pT1 tumors being positive versus 4% of pT3 tumors. Furthermore, expression of AR was higher in pT1a tumors compared to pT1b (32% vs. 17%). Additionally, AR expression inversely correlated with nuclear grade with 21% positivity in nuclear grades 1 and 2 and 7% in nuclear grades 3 and 4. Univariate analysis showed a longer disease free survival in patients with AR positive tumors compared to patients with

Steroid Receptors in Renal Cell Carcinoma 111

level seems not to be affected by the Fuhrman nuclear grade, increased VDR immunoreactivity was observed in sarcomatous and poorly differentiated areas of RCC and

A different study by Blomberg Jensen et al. showed that VDR mRNA was detected in all normal kidney samples while almost undetectable in clear cell RCC with similar results confirmed by Western blot (Blomberg Jensen et al. 2010). Additionally, in this study, the authors investigated the expression of Vitamin D activating enzymes including CYP2R1, CYP27A1, and CYP27B1. The 1 -hydroxylase (CYP27B1) was present in all normal samples with varying degrees of expression levels, the lowest expression in atrophic kidneys. By immunohistochemistry and in-situ hybridization, expression of CYP2R1 and CYP27A1 was localized to the distal tubule, collecting ducts and minimal expression in the proximal tubule. Expression of CYP27B1 was more prominent in the proximal tubule. Expression of these enzymes was diminished in clear cell RCC along with decreased expression of VDR (Blomberg Jensen et al. 2010). Papillary RCC is positive for VDR in the great majority of cases. This recapitulates more closely the phenotype of distal tubules. Similarly, chromophobe carcinoma and oncocytomas are also positive for VDR. Staining of chromophobe carcinoma accentuates the cell membrane while in oncocytomas it is stronger in the perinuclear area (Liu et al. 2006). Collecting duct carcinoma is thought to derive from the principal cells of the collecting duct of Bellini. Consistent with other tumors of origin from the distal nephron, three out of three collecting duct carcinomas tested were positive

Currently, immunohistochemistry for vitamin D is not routinely used for diagnostic purposes. However, several findings described above could eventually prove to have diagnostic utility in anatomic pathology. Because almost all clear cell RCC proved to be negative by immunohistochemistry (with the exception of some high grade tumors, or tumor present within vascular lumens), a positive VDR immunohistochemistry result should alert the pathologist about a potential problem in the classification of a tumor

A frequent problem in the diagnosis of renal tumors is the distinction between oncocytomas and eosinophilic chromophobe carcinoma (Takahashi et al. 2003; Young et al. 2001). This distinction is critical as these tumors have completely different prognostic and therapeutic clinical implications. Results reported in the literature indicate that both tumors are immunoreactive for VDR with a difference in the localization of the stain. While oncocytomas stained preferably in the perinuclear area, chromophobe carcinoma showed

Positive stain for VDR in papillary RCC could help differentiate this tumor from clear cell RCC with papillary features, which will be negative in the great majority of cases. VDR expression in CPRCC has not been tested; however, since these tumors are CK7 positive, it is likely that they are VDR positive as well, consistent with distal nephron phenotype. Only three cases of collecting duct carcinoma have been tested for VDR immunoreactivity and all of them turned positive. Differential diagnosis of these tumors could be challenging due to their infrequent presentations. Main differential diagnoses include adenocarcinoma or urothelial carcinoma with glandular differentiation. Although there is lack of information in

in metastatic tumors or in intravascular tumor islands (Liu et al. 2006).

for VDR by immunohistochemistry (Liu et al. 2006).

thought to be clear cell RCC (Liu et al. 2006).

accentuated stain of the cell membrane (Liu et al. 2006).

AR negative tumors (Langner et al. 2004). These results reflect similar trends observed with GRs in RCC (Yakirevich et al. 2011), however, the diagnostic, prognostic or therapeutic utility of AR analysis in RCC is uncertain and might require further investigations.

#### **7. Vitamin D receptor**

Vitamin D is a lipid-soluble compound whose major function is the maintenance of adequate plasma levels of calcium and phosphorus, important for bone mineralization, neuromuscular transmission and general cellular metabolism. Vitamin D receptor (VDR) is present in various tissues that do not participate in calcium metabolism and regulates the expression of hundreds of genes that control cell proliferation, differentiation and angiogenesis. Low levels of vitamin D have been associated with increased incidence of colon, prostate and breast cancer (Thacher & Clarke 2011). Recent studies suggest that vitamin D may be inversely associated with the risk of RCC. (Bosetti et al. 2007; Ikuyama et al. 2002; Karami et al. 2008; Obara, Suzuki et al. 2007). Vitamin D receptor is expressed in malignant tumors, including RCC, and mediates the biological actions of 1,25(OH)2D3 (Lamprecht & Lipkin 2003). In this section, we will review the current literature on the relevance of vitamin D and its receptor in RCC.

#### **7.1 Expression of VDR in the normal kidney**

The kidney is a major organ for vitamin D metabolism and calcium homeostasis. Activation of vitamin D involves conversion of 7-dehydrocholesterol to cholecalciferol by UVB radiation in the skin. Cholecalciferol is metabolized by the 25-hydroxylases (CYP2R1 and CYP27A1) in the liver to 25-hydroxycholecalciferol (25(OH)D3). 25(OH)D3 then undergoes glomerular filtration and is subsequently converted to the active form calcitriol (1,25(OH)2D3) by the 1-hydroxylase (CYP27B1) located primarily in the proximal tubule. Calcitriol binds to an intracellular receptor (VDR), a ligand dependent transcription factor belonging to the class II nuclear receptor subfamily. The effect of calcitriol is negatively controlled by CYP24A1 (Fleet 2008; Nykjaer et al. 1999). Immunohistochemistry studies of non-tumoral kidney show expression of VDR predominantly in the distal tubules and collecting ducts with only faint or lack of stain in the proximal tubule cells (Blomberg Jensen et al. 2010; Liu et al. 2006; Obara, Konda et al. 2007). This expression pattern is consistent with studies that demonstrate that vitamin-D induced calcium re-absorption occurs in the distal tubules (Li & Christakos 1991).

#### **7.2 Expression of VDR in kidney tumors**

In keeping with absence of VDR expression in the proximal tubule, a study by Liu et al. showed that clear cell RCC is generally negative for VDR by immunohistochemistry and showed decreased mRNA level compared to non-tumoral kidney control tissue by RT-PCR (Liu et al. 2006). When whole sections of tumors were stained, expression of VDR was present only focally in the peripheral region of the tumor. Previously, a study by Madej et al. showed that expression of VDR in clear cell RCC was similar to control tissue by Western and Northern blot analysis (Madej et al. 2003). This discrepancy could be due to a difference in the degree of differentiation of the tumors analyzed in each study. While the expression

AR negative tumors (Langner et al. 2004). These results reflect similar trends observed with GRs in RCC (Yakirevich et al. 2011), however, the diagnostic, prognostic or therapeutic

Vitamin D is a lipid-soluble compound whose major function is the maintenance of adequate plasma levels of calcium and phosphorus, important for bone mineralization, neuromuscular transmission and general cellular metabolism. Vitamin D receptor (VDR) is present in various tissues that do not participate in calcium metabolism and regulates the expression of hundreds of genes that control cell proliferation, differentiation and angiogenesis. Low levels of vitamin D have been associated with increased incidence of colon, prostate and breast cancer (Thacher & Clarke 2011). Recent studies suggest that vitamin D may be inversely associated with the risk of RCC. (Bosetti et al. 2007; Ikuyama et al. 2002; Karami et al. 2008; Obara, Suzuki et al. 2007). Vitamin D receptor is expressed in malignant tumors, including RCC, and mediates the biological actions of 1,25(OH)2D3 (Lamprecht & Lipkin 2003). In this section, we will review the current literature on the

The kidney is a major organ for vitamin D metabolism and calcium homeostasis. Activation of vitamin D involves conversion of 7-dehydrocholesterol to cholecalciferol by UVB radiation in the skin. Cholecalciferol is metabolized by the 25-hydroxylases (CYP2R1 and CYP27A1) in the liver to 25-hydroxycholecalciferol (25(OH)D3). 25(OH)D3 then undergoes glomerular filtration and is subsequently converted to the active form calcitriol (1,25(OH)2D3) by the 1-hydroxylase (CYP27B1) located primarily in the proximal tubule. Calcitriol binds to an intracellular receptor (VDR), a ligand dependent transcription factor belonging to the class II nuclear receptor subfamily. The effect of calcitriol is negatively controlled by CYP24A1 (Fleet 2008; Nykjaer et al. 1999). Immunohistochemistry studies of non-tumoral kidney show expression of VDR predominantly in the distal tubules and collecting ducts with only faint or lack of stain in the proximal tubule cells (Blomberg Jensen et al. 2010; Liu et al. 2006; Obara, Konda et al. 2007). This expression pattern is consistent with studies that demonstrate that vitamin-D induced calcium re-absorption occurs in the

In keeping with absence of VDR expression in the proximal tubule, a study by Liu et al. showed that clear cell RCC is generally negative for VDR by immunohistochemistry and showed decreased mRNA level compared to non-tumoral kidney control tissue by RT-PCR (Liu et al. 2006). When whole sections of tumors were stained, expression of VDR was present only focally in the peripheral region of the tumor. Previously, a study by Madej et al. showed that expression of VDR in clear cell RCC was similar to control tissue by Western and Northern blot analysis (Madej et al. 2003). This discrepancy could be due to a difference in the degree of differentiation of the tumors analyzed in each study. While the expression

utility of AR analysis in RCC is uncertain and might require further investigations.

**7. Vitamin D receptor** 

relevance of vitamin D and its receptor in RCC.

**7.1 Expression of VDR in the normal kidney** 

distal tubules (Li & Christakos 1991).

**7.2 Expression of VDR in kidney tumors** 

level seems not to be affected by the Fuhrman nuclear grade, increased VDR immunoreactivity was observed in sarcomatous and poorly differentiated areas of RCC and in metastatic tumors or in intravascular tumor islands (Liu et al. 2006).

A different study by Blomberg Jensen et al. showed that VDR mRNA was detected in all normal kidney samples while almost undetectable in clear cell RCC with similar results confirmed by Western blot (Blomberg Jensen et al. 2010). Additionally, in this study, the authors investigated the expression of Vitamin D activating enzymes including CYP2R1, CYP27A1, and CYP27B1. The 1 -hydroxylase (CYP27B1) was present in all normal samples with varying degrees of expression levels, the lowest expression in atrophic kidneys. By immunohistochemistry and in-situ hybridization, expression of CYP2R1 and CYP27A1 was localized to the distal tubule, collecting ducts and minimal expression in the proximal tubule. Expression of CYP27B1 was more prominent in the proximal tubule. Expression of these enzymes was diminished in clear cell RCC along with decreased expression of VDR (Blomberg Jensen et al. 2010). Papillary RCC is positive for VDR in the great majority of cases. This recapitulates more closely the phenotype of distal tubules. Similarly, chromophobe carcinoma and oncocytomas are also positive for VDR. Staining of chromophobe carcinoma accentuates the cell membrane while in oncocytomas it is stronger in the perinuclear area (Liu et al. 2006). Collecting duct carcinoma is thought to derive from the principal cells of the collecting duct of Bellini. Consistent with other tumors of origin from the distal nephron, three out of three collecting duct carcinomas tested were positive for VDR by immunohistochemistry (Liu et al. 2006).

Currently, immunohistochemistry for vitamin D is not routinely used for diagnostic purposes. However, several findings described above could eventually prove to have diagnostic utility in anatomic pathology. Because almost all clear cell RCC proved to be negative by immunohistochemistry (with the exception of some high grade tumors, or tumor present within vascular lumens), a positive VDR immunohistochemistry result should alert the pathologist about a potential problem in the classification of a tumor thought to be clear cell RCC (Liu et al. 2006).

A frequent problem in the diagnosis of renal tumors is the distinction between oncocytomas and eosinophilic chromophobe carcinoma (Takahashi et al. 2003; Young et al. 2001). This distinction is critical as these tumors have completely different prognostic and therapeutic clinical implications. Results reported in the literature indicate that both tumors are immunoreactive for VDR with a difference in the localization of the stain. While oncocytomas stained preferably in the perinuclear area, chromophobe carcinoma showed accentuated stain of the cell membrane (Liu et al. 2006).

Positive stain for VDR in papillary RCC could help differentiate this tumor from clear cell RCC with papillary features, which will be negative in the great majority of cases. VDR expression in CPRCC has not been tested; however, since these tumors are CK7 positive, it is likely that they are VDR positive as well, consistent with distal nephron phenotype. Only three cases of collecting duct carcinoma have been tested for VDR immunoreactivity and all of them turned positive. Differential diagnosis of these tumors could be challenging due to their infrequent presentations. Main differential diagnoses include adenocarcinoma or urothelial carcinoma with glandular differentiation. Although there is lack of information in

Steroid Receptors in Renal Cell Carcinoma 113

and reduction of cyclin A staining in the tumors. These results show a promising potential of vitamin D derived compounds as targeted therapy for RCC patients (Lambert et al. 2010).

Retinoids are a family of molecules related to vitamin A that include retinoic acid (RA) and all-trans retinoid. Retinoids participate in diverse functions in many organ systems during development and in adulthood including vision, neural function and immune response. Extensive research also supports a role of retinoids in cell proliferation and differentiation through cell cycle signaling promoting block in G1 phase of cell cycle, by directly or indirectly modulating cyclins, CDKs, and cell-cycle inhibitors (Mongan & Gudas 2007; Tang & Gudas 2011). There are two distinct retinoid nuclear receptor systems, the RARs types -, - and -, and RXRs types -, - and - (Pemrick et al. 1994). RARs form heterodimers with RXRs and act by binding to retinoic acid response elements (RARE) located in the promoter regions of RA-target genes and modulate transcription rates (Altucci & Gronemeyer 2001). In addition to its role in senescence and cell differentiation, retinoic acid can follow an alternative pathway by binding to a so-called orphan nuclear receptor, PPAR-/- to

Studies evaluating the expression of RARs and RXRs indicate that expression of a given receptor subtype is cell type specific and that retinoic acid effect in different cell types are linked to specific receptor type (Geradts et al. 1993; Kakizuka et al. 1991; Moasser et al. 1994; Moasser et al. 1995; Sheikh et al. 1994; Swisshelm et al. 1994). Information on expression of RARs in normal kidney derives primarily from normal controls used in studies for various purposes. RAR- mRNA has consistently been found to be expressed in normal kidney tissue samples (Goelden et al. 2005; Vanderleede et al. 1995). Additionally, expression of RARs and RXRs were studied in podocytes, which expressed most isoforms of retinoic acid receptors (RAR) and RXRs with the exception of RXR- (He et al. 2007). Obara et al. detected expression of RXR- and- in nuclei of proximal tubule cells, while RXR- expression was

Dysregulation of each RA receptor has been found in association with different types of cancer. RAR- is dysregulated in acute promyelocytic leukemia (APL). Majority of APL cases present a chromosomal translocation that fuses the promyelocytic leukemia gene, *PML*

a combination of retinoid and chemotherapy. In contrast to RAR-, RAR- is involved in solid tumorigenesis including RCC (Argiles et al. 1994; Berg et al. 1999; Goelden et al. 2005; Hoffman et al. 1996). The RAR- gene maps on the short arm of chromosome 3, a region frequently deleted in cancer (Houle et al. 1993). Several studies demonstrated decreased or undetectable levels of RAR- mRNA in tissue or cell lines derived from different tumors including lung (Suh et al. 2002; Zhang et al. 1994), prostate (Nakayama et al. 2001), breast (Swisshelm et al. 1994), ovary (Sabichi et al. 1998), colon (Cote et al. 1998), head and neck

genes [t(15;17)(22;q11.2-12)] which can be effectively treated and cured with

present in proximal tubule cells and interstitial cells (Obara, Konda et al. 2007).

**8.2 Expression of retinoic acid receptors in kidney tumors** 

and the *RAR-*

promote cell survival under certain conditions (Schug et al. 2007).

**8.1 Expression of retinoic acid receptors in the normal kidney** 

**8. Retinoic acid receptors** 

the literature regarding expression of VDR receptor in urothelial carcinoma with glandular differentiation, studies on normal urothelium and urothelial neoplasms have shown consistent positivity for VDR for which it seems unlikely that it would have utility in the differential diagnosis on this context (Hermann & Andersen 1997; Konety et al. 2001).

The anti-cancer effect of vitamin D includes inhibition of cell proliferation and induction of apoptosis (Blutt et al. 2000; Rashid et al. 2001; Zhuang & Burnstein 1998). Expression of VDR as detected by immunohistochemistry was not associated with survival in a cohort of 68 RCC patients (Obara, Konda et al. 2007). This could be due to a small number of patients studied or secondary to other possible alterations within the signaling pathway that could interfere with the normal function of the receptor. Different studies have shown consistently that VDR-DNA complexes are decreased in RCC, even in the presence of exogenous vitamin D (Madej et al. 2003; Trydal et al. 1988). This functional impairment could be secondary to suboptimal VDR heterodimerization with its partners in tumor cells. Before binding to DNA, VDR heterodimerizes with retinoid X receptor (RXR), its obligate partner (Barsony & Prufer 2002; Prufer & Barsony 2002). Retinoid X receptors are part of the retinoic acid receptor systems and share with retinoic acid part of the signaling pathways. Notably, positive RXR- staining in RCC correlates with prolonged overall 5-years survival (Obara, Konda et al. 2007).

Calcitriol has anti-proliferative properties in a variety of malignant cell types (Getzenberg et al. 1997; Reichel et al. 1989). The anti-neoplastic activity of VDR ligands was first described in 1981 in a study showing differentiating properties of calcitriol in mouse myeloid leukemia cells (Abe et al. 1981). Since then, a number of studies have demonstrated the invitro and in-vivo anti-cancer potential of vitamin D in models of bladder, breast, colon, endometrium, lung, pancreas, prostate and squamous cell carcinoma, sarcomas of the soft tissues and bone, neuroblastoma, glioma, melanoma, and other malignancies (Beer & Myrthue 2004; Trump et al. 2004; Trump et al. 2006).

Calcitriol treatment of cells inhibited cell growth and clonogenicity of the RCC cell line derived from a pulmonary metastasis of RCC, (Nagakura et al. 1986). In a different study, BALB/c mice were inoculated with murine renal cancer Renca and graded doses of calcitriol were given intraperitoneally. Vitamin D inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner. Furthermore, vitamin D treated mice showed reduced pulmonary and hepatic metastates (Fujioka et al. 1998). Despite these and other promising results in cell culture and in murine models, the utility of vitamin D therapy in humans has been challenged by its hypercalcemic toxic effect (Fakih et al. 2007; Muindi et al. 2009). To try to bypass this toxicity, researchers have explored alternative vitamin D like molecules. A recent published study investigated the in-vitro and in-vivo effect of 1,25-dihydroxyvitamin D3-3-bromoacetate [1,25(OH)2D3-3-BE], an alkylating derivative of 1,25(OH)2D3 (Lambert et al. 2010). This study reports that 1,25(OH)2D3-3-BE is significantly more potent than an equivalent concentration of 1,25(OH)2D3 in inhibiting growth of A498 and Caki 1 human kidney cancer cells. The mechanisms behind cell growth inhibition of cell-cycle progression include downregulating cyclin A and induction of apoptosis through caspase activity. When compared to calcitriol, 1,25(OH)2D3-3-BE was more potent at reducing tumor size, which was accompanied by an increase in apoptosis and reduction of cyclin A staining in the tumors. These results show a promising potential of vitamin D derived compounds as targeted therapy for RCC patients (Lambert et al. 2010).

#### **8. Retinoic acid receptors**

112 Emerging Research and Treatments in Renal Cell Carcinoma

the literature regarding expression of VDR receptor in urothelial carcinoma with glandular differentiation, studies on normal urothelium and urothelial neoplasms have shown consistent positivity for VDR for which it seems unlikely that it would have utility in the

The anti-cancer effect of vitamin D includes inhibition of cell proliferation and induction of apoptosis (Blutt et al. 2000; Rashid et al. 2001; Zhuang & Burnstein 1998). Expression of VDR as detected by immunohistochemistry was not associated with survival in a cohort of 68 RCC patients (Obara, Konda et al. 2007). This could be due to a small number of patients studied or secondary to other possible alterations within the signaling pathway that could interfere with the normal function of the receptor. Different studies have shown consistently that VDR-DNA complexes are decreased in RCC, even in the presence of exogenous vitamin D (Madej et al. 2003; Trydal et al. 1988). This functional impairment could be secondary to suboptimal VDR heterodimerization with its partners in tumor cells. Before binding to DNA, VDR heterodimerizes with retinoid X receptor (RXR), its obligate partner (Barsony & Prufer 2002; Prufer & Barsony 2002). Retinoid X receptors are part of the retinoic acid receptor systems and share with retinoic acid part of the signaling pathways. Notably, positive RXR- staining in RCC correlates with prolonged overall 5-years survival (Obara,

Calcitriol has anti-proliferative properties in a variety of malignant cell types (Getzenberg et al. 1997; Reichel et al. 1989). The anti-neoplastic activity of VDR ligands was first described in 1981 in a study showing differentiating properties of calcitriol in mouse myeloid leukemia cells (Abe et al. 1981). Since then, a number of studies have demonstrated the invitro and in-vivo anti-cancer potential of vitamin D in models of bladder, breast, colon, endometrium, lung, pancreas, prostate and squamous cell carcinoma, sarcomas of the soft tissues and bone, neuroblastoma, glioma, melanoma, and other malignancies (Beer &

Calcitriol treatment of cells inhibited cell growth and clonogenicity of the RCC cell line derived from a pulmonary metastasis of RCC, (Nagakura et al. 1986). In a different study, BALB/c mice were inoculated with murine renal cancer Renca and graded doses of calcitriol were given intraperitoneally. Vitamin D inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner. Furthermore, vitamin D treated mice showed reduced pulmonary and hepatic metastates (Fujioka et al. 1998). Despite these and other promising results in cell culture and in murine models, the utility of vitamin D therapy in humans has been challenged by its hypercalcemic toxic effect (Fakih et al. 2007; Muindi et al. 2009). To try to bypass this toxicity, researchers have explored alternative vitamin D like molecules. A recent published study investigated the in-vitro and in-vivo effect of 1,25-dihydroxyvitamin D3-3-bromoacetate [1,25(OH)2D3-3-BE], an alkylating derivative of 1,25(OH)2D3 (Lambert et al. 2010). This study reports that 1,25(OH)2D3-3-BE is significantly more potent than an equivalent concentration of 1,25(OH)2D3 in inhibiting growth of A498 and Caki 1 human kidney cancer cells. The mechanisms behind cell growth inhibition of cell-cycle progression include downregulating cyclin A and induction of apoptosis through caspase activity. When compared to calcitriol, 1,25(OH)2D3-3-BE was more potent at reducing tumor size, which was accompanied by an increase in apoptosis

differential diagnosis on this context (Hermann & Andersen 1997; Konety et al. 2001).

Konda et al. 2007).

Myrthue 2004; Trump et al. 2004; Trump et al. 2006).

Retinoids are a family of molecules related to vitamin A that include retinoic acid (RA) and all-trans retinoid. Retinoids participate in diverse functions in many organ systems during development and in adulthood including vision, neural function and immune response. Extensive research also supports a role of retinoids in cell proliferation and differentiation through cell cycle signaling promoting block in G1 phase of cell cycle, by directly or indirectly modulating cyclins, CDKs, and cell-cycle inhibitors (Mongan & Gudas 2007; Tang & Gudas 2011). There are two distinct retinoid nuclear receptor systems, the RARs types -, - and -, and RXRs types -, - and - (Pemrick et al. 1994). RARs form heterodimers with RXRs and act by binding to retinoic acid response elements (RARE) located in the promoter regions of RA-target genes and modulate transcription rates (Altucci & Gronemeyer 2001). In addition to its role in senescence and cell differentiation, retinoic acid can follow an alternative pathway by binding to a so-called orphan nuclear receptor, PPAR-/- to promote cell survival under certain conditions (Schug et al. 2007).

#### **8.1 Expression of retinoic acid receptors in the normal kidney**

Studies evaluating the expression of RARs and RXRs indicate that expression of a given receptor subtype is cell type specific and that retinoic acid effect in different cell types are linked to specific receptor type (Geradts et al. 1993; Kakizuka et al. 1991; Moasser et al. 1994; Moasser et al. 1995; Sheikh et al. 1994; Swisshelm et al. 1994). Information on expression of RARs in normal kidney derives primarily from normal controls used in studies for various purposes. RAR- mRNA has consistently been found to be expressed in normal kidney tissue samples (Goelden et al. 2005; Vanderleede et al. 1995). Additionally, expression of RARs and RXRs were studied in podocytes, which expressed most isoforms of retinoic acid receptors (RAR) and RXRs with the exception of RXR- (He et al. 2007). Obara et al. detected expression of RXR- and- in nuclei of proximal tubule cells, while RXR- expression was present in proximal tubule cells and interstitial cells (Obara, Konda et al. 2007).

#### **8.2 Expression of retinoic acid receptors in kidney tumors**

Dysregulation of each RA receptor has been found in association with different types of cancer. RAR- is dysregulated in acute promyelocytic leukemia (APL). Majority of APL cases present a chromosomal translocation that fuses the promyelocytic leukemia gene, *PML* and the *RAR-* genes [t(15;17)(22;q11.2-12)] which can be effectively treated and cured with a combination of retinoid and chemotherapy. In contrast to RAR-, RAR- is involved in solid tumorigenesis including RCC (Argiles et al. 1994; Berg et al. 1999; Goelden et al. 2005; Hoffman et al. 1996). The RAR- gene maps on the short arm of chromosome 3, a region frequently deleted in cancer (Houle et al. 1993). Several studies demonstrated decreased or undetectable levels of RAR- mRNA in tissue or cell lines derived from different tumors including lung (Suh et al. 2002; Zhang et al. 1994), prostate (Nakayama et al. 2001), breast (Swisshelm et al. 1994), ovary (Sabichi et al. 1998), colon (Cote et al. 1998), head and neck

Steroid Receptors in Renal Cell Carcinoma 115

Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S., and

Altucci, L., and Gronemeyer, H. 2001. The promise of retinoids to fight against cancer. *Nat* 

Amin, M. B., Tamboli, P., Javidan, J., Stricker, H., de-Peralta Venturina, M., Deshpande, A.,

Argiles, A., Ootaka, T., Hill, P. A., Nikolic-Paterson, D. J., Hutchinson, P., Kraft, N. E., and

Arriza, J. L., Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B. L., Housman, D. E., and

Atkins, M. B., Hidalgo, M., Stadler, W. M., Logan, T. F., Dutcher, J. P., Hudes, G. R., Park, Y.,

Atzpodien, J., Kirchner, H., Jonas, U., Bergmann, L., Schott, H., Heynemann, H., Fornara, P.,

Barsony, J., and Prufer, K. 2002. Vitamin D receptor and retinoid X receptor interactions in

Baylis, C., Handa, R. K., and Sorkin, M. 1990. Glucocorticoids and control of glomerular

Beato, M., Herrlich, P., and Schutz, G. 1995. Steroid hormone receptors: many actors in

alpha,25-dihydroxyvitamin D3. *Proc Natl Acad Sci U S A* 78 (8):4990-4. Adsay, N. V., Eble, J. N., Srigley, J. R., Jones, E. C., and Grignon, D. J. 2000. Mixed epithelial

and stromal tumor of the kidney. *Am J Surg Pathol* 24 (7):958-70.

30951). *J Clin Oncol* 23 (18):4172-8.

*Rev Cancer* 1 (3):181-93.

*Cancer Invest* 26 (1):35-40.

237 (4812):268-75.

*Oncol* 22 (7):1188-94.

motion. *Vitam Horm* 65:345-76.

search of a plot. *Cell* 83 (6):851-7.

filtration rate. *Semin Nephrol* 10 (4):320-9.

carcinoma. *J Clin Oncol* 22 (5):909-18.

31.

of interferon Alfa-2a with and without 13-cis-retinoic acid in patients with progressive metastatic renal cell Carcinoma: the European Organisation for Research and Treatment of Cancer Genito-Urinary Tract Cancer Group (EORTC

Suda, T. 1981. Differentiation of mouse myeloid leukemia cells induced by 1

and Menon, M. 2002. Prognostic impact of histologic subtyping of adult renal epithelial neoplasms: an experience of 405 cases. *Am J Surg Pathol* 26 (3):281-91. Arai, Y., Nonomura, N., Nakai, Y., Nishimura, K., Oka, D., Shiba, M., Nakayama, M.,

Takayama, H., Mizutani, Y., Miki, T., and Okuyama, A. 2008. The growthinhibitory effects of dexamethasone on renal cell carcinoma in vivo and in vitro.

Atkins, R. C. 1994. Regulation of human renal adenocarcinoma cell growth by retinoic acid and its interactions with epidermal growth factor. *Kidney Int* 45 (1):23-

Evans, R. M. 1987. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. *Science*

Liou, S. H., Marshall, B., Boni, J. P., Dukart, G., and Sherman, M. L. 2004. Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell

Loening, S. A., Roigas, J., Muller, S. C., Bodenstein, H., Pomer, S., Metzner, B., Rebmann, U., Oberneder, R., Siebels, M., Wandert, T., Puchberger, T., and Reitz, M. 2004. Interleukin-2- and interferon alfa-2a-based immunochemotherapy in advanced renal cell carcinoma: a Prospectively Randomized Trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN). *J Clin* 

(Xu et al. 1994) and cervix (Geisen et al. 1997). RAR- mRNA was decreased or not detectable in 11 of 12 RCC cell lines (Hoffman et al. 1996). These cell lines were either resistant or minimally inhibited when treated with 13-*cis*-RA (13-CRA). Conversely, chromophobe RCC shows much higher levels of expression of RAR- with a ratio of tumor/normal of over 36 (Goelden et al. 2005). In clear cell RCC, immunoreactivity for RXR was observed in up to 70% of the cases, RXR- was present in 47% of cases, and RXR stain was seen in 85% of cases, in a study that included 49 CRCCs (Obara, Konda et al. 2007). Only expression of RXR- was found to correlate inversely with pathological and clinical stage. While all subtypes of RXRs showed variable nuclear or cytoplasmic stain, subcellular location did not correlate with any prognostic variables. Additionally, this study suggests a prolonged overall 5-year survival of patients with tumors that are RXR- positive (Obara, Konda et al. 2007).

In clinical trials, the effect of RA in RCC patients has been tested in patients with metastatic disease. A randomized clinical trial of 284 patients evaluated response to treatment with IFN2a plus 13-cis-retinoic acid (13-CRA) or treatment with IFN2a alone (Motzer et al. 2000). This study showed no difference in the overall survival but median duration of response (complete and partial combined) in the group treated with the combination was 33 months versus 22 months for the second group. Nineteen percent of patients treated with IFN2a plus 13-CRA were progression-free at 24 months, compared with 10% of patients treated with IFN2a alone (Motzer et al. 2000). However, a separate clinical trial that involved 320 patients concluded that progression-free and overall survival for patients with progressive metastatic RCC treated with IFN2a plus 13-CRA were significantly longer compared with patients on IFN alone (Aass et al. 2005). Another clinical trial that included three different treatment regimens: *a*) triple combination of IL-2, IFN2a, and fluorouracil; *b*) triple combination of group *a* and additional 13-CRA; *c*) control group treated with IFN and vinblastine. Progression-free and overall survival were significantly longer in groups *a* and *b* but there was no significant survival advantage for patients receiving 13-CRA (Atzpodien et al. 2004). These studies suggest that there is some beneficial effect of retinoids treatment, in at least a subset of patients with RCC.

#### **9. Conclusion**

Steroid receptors are differentially expressed in the normal kidney and in renal cell neoplasms. Several steroid receptors, such as MR, PR, and vitamin D receptor may be included in diagnostic immunohistochemical panels in order to more accurately subtype renal cell tumors. Although ER is not detected in significant amounts in RCCs, it is expressed by stromal cells in several benign renal neoplasms and may be involved in their pathogenesis. GR and AR appear to be markers of less aggressive behavior in clear cell RCC. Finally, steroid receptors and their downstream signaling mechanisms may have a potential role in novel anticancer hormonal therapies in RCCs.

#### **10. References**

Aass, N., De Mulder, P. H., Mickisch, G. H., Mulders, P., van Oosterom, A. T., van Poppel, H., Fossa, S. D., de Prijck, L., and Sylvester, R. J. 2005. Randomized phase II/III trial

(Xu et al. 1994) and cervix (Geisen et al. 1997). RAR- mRNA was decreased or not detectable in 11 of 12 RCC cell lines (Hoffman et al. 1996). These cell lines were either resistant or minimally inhibited when treated with 13-*cis*-RA (13-CRA). Conversely, chromophobe RCC shows much higher levels of expression of RAR- with a ratio of tumor/normal of over 36 (Goelden et al. 2005). In clear cell RCC, immunoreactivity for RXR was observed in up to 70% of the cases, RXR- was present in 47% of cases, and RXR stain was seen in 85% of cases, in a study that included 49 CRCCs (Obara, Konda et al. 2007). Only expression of RXR- was found to correlate inversely with pathological and clinical stage. While all subtypes of RXRs showed variable nuclear or cytoplasmic stain, subcellular location did not correlate with any prognostic variables. Additionally, this study suggests a prolonged overall 5-year survival of patients with tumors that are RXR- positive (Obara,

In clinical trials, the effect of RA in RCC patients has been tested in patients with metastatic disease. A randomized clinical trial of 284 patients evaluated response to treatment with IFN2a plus 13-cis-retinoic acid (13-CRA) or treatment with IFN2a alone (Motzer et al. 2000). This study showed no difference in the overall survival but median duration of response (complete and partial combined) in the group treated with the combination was 33 months versus 22 months for the second group. Nineteen percent of patients treated with IFN2a plus 13-CRA were progression-free at 24 months, compared with 10% of patients treated with IFN2a alone (Motzer et al. 2000). However, a separate clinical trial that involved 320 patients concluded that progression-free and overall survival for patients with progressive metastatic RCC treated with IFN2a plus 13-CRA were significantly longer compared with patients on IFN alone (Aass et al. 2005). Another clinical trial that included three different treatment regimens: *a*) triple combination of IL-2, IFN2a, and fluorouracil; *b*) triple combination of group *a* and additional 13-CRA; *c*) control group treated with IFN and vinblastine. Progression-free and overall survival were significantly longer in groups *a* and *b* but there was no significant survival advantage for patients receiving 13-CRA (Atzpodien et al. 2004). These studies suggest that there is some beneficial effect of retinoids

Steroid receptors are differentially expressed in the normal kidney and in renal cell neoplasms. Several steroid receptors, such as MR, PR, and vitamin D receptor may be included in diagnostic immunohistochemical panels in order to more accurately subtype renal cell tumors. Although ER is not detected in significant amounts in RCCs, it is expressed by stromal cells in several benign renal neoplasms and may be involved in their pathogenesis. GR and AR appear to be markers of less aggressive behavior in clear cell RCC. Finally, steroid receptors and their downstream signaling mechanisms may have a potential

Aass, N., De Mulder, P. H., Mickisch, G. H., Mulders, P., van Oosterom, A. T., van Poppel,

H., Fossa, S. D., de Prijck, L., and Sylvester, R. J. 2005. Randomized phase II/III trial

Konda et al. 2007).

**9. Conclusion** 

**10. References** 

treatment, in at least a subset of patients with RCC.

role in novel anticancer hormonal therapies in RCCs.

of interferon Alfa-2a with and without 13-cis-retinoic acid in patients with progressive metastatic renal cell Carcinoma: the European Organisation for Research and Treatment of Cancer Genito-Urinary Tract Cancer Group (EORTC 30951). *J Clin Oncol* 23 (18):4172-8.


Steroid Receptors in Renal Cell Carcinoma 117

Christophersen, A. O., Lie, A. K., and Fossa, S. D. 2006. Unexpected 10 years complete

Concolino, G., Marocchi, A., Conti, C., Tenaglia, R., Di Silverio, F., and Bracci, U. 1978.

Concolino, G., Marocchi, A., Tenaglia, R., Di Silverio, F., and Sparano, F. 1978. Specific progesterone receptor in human renal cancer. *J Steroid Biochem* 9 (5):399-402. Concolino, G., Marocchi, A., Toscano, V., and Di Silverio, F. 1981. Nuclear androgen

Connell, J. M., and Davies, E. 2005. The new biology of aldosterone. *J Endocrinol* 186

Cote, S., Sinnett, D., and Momparler, R. L. 1998. Demethylation by 5-aza-2'-deoxycytidine of

Eble, J. N., Sauter, G. , Epstein, J. I., and Sesterhenn, I., eds. 2004. *World Health Organization* 

Escoubet, B., Coureau, C., Blot-Chabaud, M., Bonvalet, J. P., and Farman, N. 1996.

Escudier, B., Pluzanska, A., Koralewski, P., Ravaud, A., Bracarda, S., Szczylik, C., Chevreau,

Evans, R. M. 1988. The steroid and thyroid hormone receptor superfamily. *Science* 240

Fakih, M. G., Trump, D. L., Muindi, J. R., Black, J. D., Bernardi, R. J., Creaven, P. J., Schwartz,

Farman, N., and Bonvalet, J. P. 1983. Aldosterone binding in isolated tubules. III. Autoradiography along the rat nephron. *Am J Physiol* 245 (5 Pt 1):F606-14. Farman, N., Vandewalle, A., and Bonvalet, J. P. 1982. Aldosterone binding in isolated

beta gene in human colon carcinoma cells. *Anticancer Drugs* 9 (9):743-50. De Bosscher, K., Vanden Berghe, W., and Haegeman, G. 2003. The interplay between the

mechanisms for gene repression. *Endocr Rev* 24 (4):488-522.

kidney, heart, and colon. *Am J Physiol* 270 (5 Pt 1):C1343-53.

renal cell carcinoma. *J Steroid Biochem* 15:397-402.

*genital organs*. Lyon, France: IARC Press.

trial. *Lancet* 370 (9605):2103-11.

nephron. *Am J Physiol* 242 (1):F63-8.

(4854):889-95.

*Res* 13 (4):1216-23.

*Oncol* 45 (2):226-8.

2):4340-4.

(1):1-20.

remission after cortisone mono-therapy in metastatic renal cell carcinoma. *Acta* 

Human renal cell carcinoma as a hormone-dependent tumor. *Cancer Res* 38 (11 Pt

receptor as marker of responsiveness to medroxyprogesterone acetate in human

specific 5-methylcytosine sites in the promoter region of the retinoic acid receptor

glucocorticoid receptor and nuclear factor-kappaB or activator protein-1: molecular

*classification of tumours: pathology and genetics of tumours of the urinary system and male* 

Corticosteroid receptor mRNA expression is unaffected by corticosteroids in rat

C., Filipek, M., Melichar, B., Bajetta, E., Gorbunova, V., Bay, J. O., Bodrogi, I., Jagiello-Gruszfeld, A., and Moore, N. 2007. Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma: a randomised, double-blind phase III

J., Brattain, M. G., Hutson, A., French, R., and Johnson, C. S. 2007. A phase I pharmacokinetic and pharmacodynamic study of intravenous calcitriol in combination with oral gefitinib in patients with advanced solid tumors. *Clin Cancer* 

tubules I. Biochemical determination in proximal and distal parts of the rabbit


Beer, T. M., and Myrthue, A. 2004. Calcitriol in cancer treatment: from the lab to the clinic.

Berg, W. J., Nanus, D. M., Leung, A., Brown, K. T., Hutchinson, B., Mazumdar, M., Xu, X. C.,

Bhat, H. K., Hacker, H. J., Bannasch, P., Thompson, E. A., and Liehr, J. G. 1993. Localization

Blomberg Jensen, M., Andersen, C. B., Nielsen, J. E., Bagi, P., Jorgensen, A., Juul, A., and

Bloom, H. J. 1973. Proceedings: Hormone-induced and spontaneous regression of metastatic

Blutt, S. E., McDonnell, T. J., Polek, T. C., and Weigel, N. L. 2000. Calcitriol-induced

Bojar, H., Maar, K., and Staib, W. 1979. The endocrine background of human renal cell

Boross, M., Kinsella, J., Cheng, L., and Sacktor, B. 1986. Glucocorticoids and metabolic

Bosetti, C., Scotti, L., Maso, L. D., Talamini, R., Montella, M., Negri, E., Ramazzotti, V.,

Brem, A. S., Morris, D. J., Ge, Y., Dworkin, L. D., Tolbert, E., and Gong, R. 2010. Direct

Brown, C. J., Goss, S. J., Lubahn, D. B., Joseph, D. R., Wilson, E. M., French, F. S., and

Brown, D. F., Dababo, M. A., Hladik, C. L., Eagan, K. P., White, C. L., 3rd, and Rushing, E. J.

Campen, T. J., Vaughn, D. A., and Fanestil, D. D. 1983. Mineralo- and glucocorticoid effects

Chen, L., Weiss, F. R., Chaichik, S., and Keydar, I. 1980. Steroid receptors in human renal

cancer: a case-control study from Italy. *Int J Cancer* 120 (4):892-6.

11{beta}-HSD Activity. *Am J Physiol Renal Physiol*. 298: F1178–87.

on renal excretion of electrolytes. *Pflugers Arch* 399 (2):93-101.

retinoic acid and interferon-alpha-2a. *Clin Cancer Res* 5 (7):1671-5.

Lotan, R., Reuter, V. E., and Motzer, R. J. 1999. Up-regulation of retinoic acid receptor beta expression in renal cancers in vivo correlates with response to 13-cis-

of estrogen receptors in interstitial cells of hamster kidney and in estradiol-induced renal tumors as evidence of the mesenchymal origin of this neoplasm. *Cancer Res* 53

Leffers, H. 2010. Expression of the vitamin D receptor, 25-hydroxylases, 1alphahydroxylase and 24-hydroxylase in the human kidney and renal clear cell cancer. *J* 

apoptosis in LNCaP cells is blocked by overexpression of Bcl-2. *Endocrinology* 141

carcinoma. IV. Glucocorticoid receptors as possible mediators of progestogen

acidosis-induced renal transports of inorganic phosphate, calcium, and NH4. *Am J* 

Franceschi, S., and La Vecchia, C. 2007. Micronutrients and the risk of renal cell

Fibrogenic Effects of Aldosterone on Normotensive Kidney: An Effect Modified by

Willard, H. F. 1989. Androgen receptor locus on the human X chromosome: regional localization to Xq11-12 and description of a DNA polymorphism. *Am J* 

1998. Hormone receptor immunoreactivity in hemangioblastomas and clear cell

*Mol Cancer Ther* 3 (3):373-81.

*Steroid Biochem Mol Biol* 121 (1-2):376-82.

renal cancer. *Cancer* 32 (5):1066-71.

action. *Urol Int* 34 (5):330-8.

*Physiol* 250 (5 Pt 2):F827-33.

*Hum Genet* 44 (2):264-9.

renal cell carcinomas. *Mod Pathol* 11 (1):55-9.

carcinoma. *Isr J Med Sci* 16 (11):756-60.

(22):5447-51.

(1):10-7.


Steroid Receptors in Renal Cell Carcinoma 119

He, J. C., Lu, T. C., Fleet, M., Sunamoto, M., Husain, M., Fang, W., Neves, S., Chen, Y.,

Hemstreet, G. P., 3rd, Wittliff, J. L., Sarrif, A. M., Hall, M. L., 3rd, McRae, L. J., and Durant, J.

Hermann, G. G., and Andersen, C. B. 1997. Transitional cell carcinoma express vitamin D

Hirasawa, G., Sasano, H., Takahashi, K., Fukushima, K., Suzuki, T., Hiwatashi, N., Toyota,

Hoffman, A. D., Engelstein, D., Bogenrieder, T., Papandreou, C. N., Steckelman, E., Dave,

Houle, B., Rochette-Egly, C., and Bradley, W. E. 1993. Tumor-suppressive effect of the

Hudes, G., Carducci, M., Tomczak, P., Dutcher, J., Figlin, R., Kapoor, A., Staroslawska, E.,

Ikuyama, T., Hamasaki, T., Inatomi, H., Katoh, T., Muratani, T., and Matsumoto, T. 2002.

Interferon-alpha and survival in metastatic renal carcinoma: early results of a randomised

Iwai, A., Fujii, Y., Kawakami, S., Takazawa, R., Kageyama, Y., Yoshida, M. A., and Kihara,

Jeltsch, J. M., Krozowski, Z., Quirin-Stricker, C., Gronemeyer, H., Simpson, R. J., Garnier, J.

Jensen, E. V., Jacobson, H. I., Walf, A. A., and Frye, C. A. 2010. Estrogen action: a historic

carcinoma cells by glucocorticoids. *Mol Cell Endocrinol* 226 (1-2):11-7. Jakse, G., and Muller-Holzner, E. 1988. Hormone receptors in renal cancer: an overview.

autologous normal kidney. *Int J Cancer* 26 (6):769-75.

receptors. *Scand J Urol Nephrol* 31 (2):161-6.

*Endocrinol Metab* 82 (11):3859-63.

(1):93-102.

(6):1077-82.

*Sci U S A* 90 (3):985-9.

*Engl J Med* 356 (22):2271-81.

Japanese. *Endocr J* 49 (4):433-8.

*Semin Surg Oncol* 4 (3):161-4.

receptor. *Proc Natl Acad Sci U S A* 83 (15):5424-8.

353 (9146):14-7.

99 (2):151-62.

Shankland, S., Iyengar, R., and Klotman, P. E. 2007. Retinoic acid inhibits HIV-1 induced podocyte proliferation through the cAMP pathway. *J Am Soc Nephrol* 18

R. 1980. Comparison of steroid receptor levels in renal-cell carcinoma and

T., Krozowski, Z. S., and Nagura, H. 1997. Colocalization of 11 beta-hydroxysteroid dehydrogenase type II and mineralocorticoid receptor in human epithelia. *J Clin* 

A., Motzer, R. J., Dmitrovsky, E., Albino, A. P., and Nanus, D. M. 1996. Expression of retinoic acid receptor beta in human renal cell carcinomas correlates with sensitivity to the antiproliferative effects of 13-cis-retinoic acid. *Clin Cancer Res* 2

retinoic acid receptor beta in human epidermoid lung cancer cells. *Proc Natl Acad* 

Sosman, J., McDermott, D., Bodrogi, I., Kovacevic, Z., Lesovoy, V., Schmidt-Wolf, I. G., Barbarash, O., Gokmen, E., O'Toole, T., Lustgarten, S., Moore, L., and Motzer, R. J. 2007. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. *N* 

Association of vitamin D receptor gene polymorphism with renal cell carcinoma in

controlled trial. Medical Research Council Renal Cancer Collaborators. 1999. *Lancet*

K. 2004. Down-regulation of vascular endothelial growth factor in renal cell

M., Krust, A., Jacob, F., and Chambon, P. 1986. Cloning of the chicken progesterone

perspective on the implications of considering alternative approaches. *Physiol Behav*


Fine, S. W., Reuter, V. E., Epstein, J. I., and Argani, P. 2006. Angiomyolipoma with epithelial

Fleet, J. C. 2008. Molecular actions of vitamin D contributing to cancer prevention. *Mol* 

Freiberg, J. M., Kinsella, J., and Sacktor, B. 1982. Glucocorticoids increase the Na+-H+

Fuller, P. J. 1991. The steroid receptor superfamily: mechanisms of diversity. *FASEB J* 5

Fuzesi, L., Gunawan, B., Bergmann, F., Tack, S., Braun, S., and Jakse, G. 1999. Papillary renal

Geisen, C., Denk, C., Gremm, B., Baust, C., Karger, A., Bollag, W., and Schwarz, E. 1997.

Geradts, J., Chen, J. Y., Russell, E. K., Yankaskas, J. R., Nieves, L., and Minna, J. D. 1993.

Getzenberg, R. H., Light, B. W., Lapco, P. E., Konety, B. R., Nangia, A. K., Acierno, J. S., Dhir,

Gobbo, S., Eble, J. N., Grignon, D. J., Martignoni, G., MacLennan, G. T., Shah, R. B., Zhang,

Goelden, U., Ukena, S. N., Pfoertner, S., Hofmann, R., Buer, J., and Schrader, A. J. 2005.

Greene, G. L., Gilna, P., Waterfield, M., Baker, A., Hort, Y., and Shine, J. 1986. Sequence and

Greenstein, S., Ghias, K., Krett, N. L., and Rosen, S. T. 2002. Mechanisms of glucocorticoidmediated apoptosis in hematological malignancies. *Clin Cancer Res* 8 (6):1681-94. Gustafsson, J. A. 1999. Estrogen receptor beta--a new dimension in estrogen mechanism of

down-regulated in cervical carcinoma cells. *Cancer Res* 57 (8):1460-7. Gelmann, E. P. 2002. Molecular biology of the androgen receptor. *J Clin Oncol* 20 (13):3001-

(5):593-9.

(15):3092-9.

15.

45.

(4742):1150-4.

action. *J Endocrinol* 163 (3):379-83.

*Aspects Med* 29 (6):388-96.

cell carcinoma. *J Urol* 160 (1):247-51.

*Histopathology* 35 (2):157-61.

*Growth Differ* 4 (10):799-809.

prostate model system. *Urology* 50 (6):999-1006.

for therapeutic intervention? *Exp Oncol* 27 (3):220-4.

cysts (AMLEC): a distinct cystic variant of angiomyolipoma. *Am J Surg Pathol* 30

exchange and decrease the Na+ gradient-dependent phosphate-uptake systems in renal brush border membrane vesicles. *Proc Natl Acad Sci U S A* 79 (16):4932-6. Fujioka, T., Hasegawa, M., Ishikura, K., Matsushita, Y., Sato, M., and Tanji, S. 1998.

Inhibition of tumor growth and angiogenesis by vitamin D3 agents in murine renal

cell carcinoma with clear cell cytomorphology and chromosomal loss of 3p.

High-level expression of the retinoic acid receptor beta gene in normal cells of the uterine cervix is regulated by the retinoic acid receptor alpha and is abnormally

Human lung cancer cell lines exhibit resistance to retinoic acid treatment. *Cell* 

R., Shurin, Z., Day, R. S., Trump, D. L., and Johnson, C. S. 1997. Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat

S., Brunelli, M., and Cheng, L. 2008. Clear cell papillary renal cell carcinoma: a distinct histopathologic and molecular genetic entity. *Am J Surg Pathol* 32 (8):1239-

RAR-beta(1) overexpression in chromophobe renal cell carcinoma: a novel target

expression of human estrogen receptor complementary DNA. *Science* 231


Steroid Receptors in Renal Cell Carcinoma 121

Li, J. J., Weroha, S. J., Davis, M. F., Tawfik, O., Hou, X., and Li, S. A. 2001. ER and PR in

Li, J. Y., Zhou, T., Gao, X., Xu, C., Sun, Y., Peng, Y., Chang, Z., Zhang, Y., Jiang, J., Wang, L.,

Li, S. A., Li, J. J., and Villee, C. A. 1977. Significance of the progesterone receptor in the

Liu, S. H., Otal-Brun, M., and Webb, T. E. 1980. Glucocorticoid receptors in human tumors.

Liu, W., Tretiakova, M., Kong, J., Turkyilmaz, M., Li, Y. C., and Krausz, T. 2006. Expression of vitamin D3 receptor in kidney tumors. *Hum Pathol* 37 (10):1268-78. Lombes, M., Farman, N., Oblin, M. E., Baulieu, E. E., Bonvalet, J. P., Erlanger, B. F., and

Lubahn, D. B., Joseph, D. R., Sullivan, P. M., Willard, H. F., French, F. S., and Wilson, E. M.

Madej, A., Puzianowska-Kuznicka, M., Tanski, Z., Nauman, J., and Nauman, A. 2003.

Mai, K. T., Teo, I., Belanger, E. C., Robertson, S. J., Marginean, E. C., and Islam, S. 2008.

McDonald, M. W., Diokno, A. C., Seski, J. C., and Menon, K. M. 1983. Measurement of

Miki, S., Iwano, M., Miki, Y., Yamamoto, M., Tang, B., Yokokawa, K., Sonoda, T., Hirano, T.,

Mishina, T., Scholer, D. W., and Edelman, I. S. 1981. Glucocorticoid receptors in rat kidney

Moasser, M. M., DeBlasio, A., and Dmitrovsky, E. 1994. Response and resistance to retinoic

Moasser, M. M., Reuter, V. E., and Dmitrovsky, E. 1995. Overexpression of the retinoic acid

Mongan, N. P., and Gudas, L. J. 2007. Diverse actions of retinoid receptors in cancer

growth factor in renal cell carcinomas. *FEBS Lett* 250 (2):607-10.

in human renal clear cell cancer. *Nephron Exp Nephrol* 93 (4):e150-7.

aldosterone. *Proc Natl Acad Sci U S A* 87 (3):1086-8.

to the X chromosome. *Science* 240 (4850):327-30.

carcinoma. *Histopathology* 52 (3):277-82.

teratocarcinomas. *Oncogene* 9 (3):833-40.

carcinoma cells. *Oncogene* 10 (8):1537-43.

prevention and treatment. *Differentiation* 75 (9):853-70.

*Surg Oncol* 22 (3):164-6.

45.

(9):4006-14.

*Urol* 184 (6):2360-3.

*Y Acad Sci* 286:369-83.

*Cancer Lett* 10 (3):269-75.

renomedullary interstitial cells during Syrian hamster estrogen-induced tumorigenesis: evidence for receptor-mediated oncogenesis. *Endocrinology* 142

and Hou, J. 2010. Testosterone and androgen receptor in human nephrolithiasis. *J* 

estrogen-induced and -dependent renal tumor of the Syrian golden hamster. *Ann N* 

Gasc, J. M. 1990. Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of

1988. Cloning of human androgen receptor complementary DNA and localization

Vitamin D receptor binding to DNA is altered without the change in its expression

Progesterone receptor reactivity in renal oncocytoma and chromophobe renal cell

progesterone receptor in human renal cell carcinoma and normal renal tissue. *J* 

and Kishimoto, T. 1989. Interleukin-6 (IL-6) functions as an in vitro autocrine

cortical tubules enriched in proximal and distal segments. *Am J Physiol* 240 (1):F38-

acid are mediated through the retinoic acid nuclear receptor gamma in human

receptor gamma directly induces terminal differentiation of human embryonal


Kakizuka, A., Miller, W. H., Jr., Umesono, K., Warrell, R. P., Jr., Frankel, S. R., Murty, V. V.,

Karami, S., Brennan, P., Hung, R. J., Boffetta, P., Toro, J., Wilson, R. T., Zaridze, D.,

Karr, J. P., Pontes, J. E., Schneider, S., Sandberg, A. A., and Murphy, G. P. 1983. Clinical

Kimura, N., Mizokami, A., Oonuma, T., Sasano, H., and Nagura, H. 1993.

Konety, B. R., Lavelle, J. P., Pirtskalaishvili, G., Dhir, R., Meyers, S. A., Nguyen, T. S.,

Krozowski, Z. S., and Funder, J. W. 1983. Renal mineralocorticoid receptors and

Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. A. 1996. Cloning

Lambert, J. R., Eddy, V. J., Young, C. D., Persons, K. S., Sarkar, S., Kelly, J. A., Genova, E.,

Leveridge, M. J., and Jewett, M. A. 2011. Recent developments in kidney cancer. *Can Urol* 

Li, H., and Christakos, S. 1991. Differential regulation by 1,25-dihydroxyvitamin D3 of

carcinoma of the bladder in vitro and in vivo. *J Urol* 165 (1):253-8.

specificity. *Proc Natl Acad Sci U S A* 80 (19):6056-60.

182 tumors. *J Urol* 171 (2 Pt 1):611-4.

*Assoc J* 5 (3):195-203.

128 (6):2844-52.

in paraffin-embedded human tissues. *J Histochem Cytochem* 41 (5):671-8. Klotzl, G., Otto, U., Becker, H., and Klosterhalfen, H. 1987. Determination of androgen,

transcription factor, PML. *Cell* 66 (4):663-74.

*Environ Health A* 71 (6):367-72.

(2):117-24.

*Urol Int* 42 (2):100-4.

(12):5925-30.

Dmitrovsky, E., and Evans, R. M. 1991. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative

Navratilova, M., Chatterjee, N., Mates, D., Janout, V., Kollarova, H., Bencko, V., Szeszenia-Dabrowska, N., Holcatova, I., Moukeria, A., Welch, R., Chanock, S., Rothman, N., Chow, W. H., and Moore, L. E. 2008. Vitamin D receptor polymorphisms and renal cancer risk in Central and Eastern Europe. *J Toxicol* 

aspects of steroid hormone receptors in human renal cell carcinoma. *J Surg Oncol* 23

Immunocytochemical localization of androgen receptor with polyclonal antibody

progestin and estrogen receptors with two different assays in renal cell carcinoma.

Hershberger, P., Shurin, M. R., Johnson, C. S., Trump, D. L., Zeidel, M. L., and Getzenberg, R. H. 2001. Effects of vitamin D (calcitriol) on transitional cell

hippocampal corticosterone-binding species have identical intrinsic steroid

of a novel receptor expressed in rat prostate and ovary. *Proc Natl Acad Sci U S A* 93

Lucia, M. S., Faller, D. V., and Ray, R. 2010. A vitamin D receptor-alkylating derivative of 1alpha,25-dihydroxyvitamin D3 inhibits growth of human kidney cancer cells and suppresses tumor growth. *Cancer Prev Res (Phila)* 3 (12):1596-607. Lamprecht, S. A., and Lipkin, M. 2003. Chemoprevention of colon cancer by calcium, vitamin D and folate: molecular mechanisms. *Nat Rev Cancer* 3 (8):601-14. Langner, C., Ratschek, M., Rehak, P., Schips, L., and Zigeuner, R. 2004. Steroid hormone

receptor expression in renal cell carcinoma: an immunohistochemical analysis of

calbindin-D9k and calbindin-D28k gene expression in mouse kidney. *Endocrinology*


Steroid Receptors in Renal Cell Carcinoma 123

Obara, W., Suzuki, Y., Kato, K., Tanji, S., Konda, R., and Fujioka, T. 2007. Vitamin D receptor

Omland, H., and Fossa, S. D. 1989. Spontaneous regression of cerebral and pulmonary metastases in renal cell carcinoma. *Scand J Urol Nephrol* 23 (2):159-60. Pearson, J., Friedman, M. A., and Hoffman, P. G., Jr. 1981. Hormone receptors in renal cell

Pemrick, S. M., Lucas, D. A., and Grippo, J. F. 1994. The retinoid receptors. *Leukemia* 8 Suppl

Porta, C., Tortora, G., Linassier, C., Papazisis, K., Awada, A., Berthold, D., Maroto, J. P.,

Prufer, K., and Barsony, J. 2002. Retinoid X receptor dominates the nuclear import and export of the unliganded vitamin D receptor. *Mol Endocrinol* 16 (8):1738-51. Pujols, L., Mullol, J., Roca-Ferrer, J., Torrego, A., Xaubet, A., Cidlowski, J. A., and Picado, C.

Rafestin-Oblin, M. E., Roth-Meyer, C., Claire, M., Michaud, A., Baviera, E., Brisset, J. M., and

Rashid, S. F., Moore, J. S., Walker, E., Driver, P. M., Engel, J., Edwards, C. E., Brown, G.,

Reichel, H., Koeffler, H. P., and Norman, A. W. 1989. The role of the vitamin D endocrine

Revollo, J. R., and Cidlowski, J. A. 2009. Mechanisms generating diversity in glucocorticoid

Roland, B. L., Krozowski, Z. S., and Funder, J. W. 1995. Glucocorticoid receptor,

Sabichi, A. L., Hendricks, D. T., Bober, M. A., and Birrer, M. J. 1998. Retinoic acid receptor

retinoid N-(4-hydroxyphenyl) retinamide. *J Natl Cancer Inst* 90 (8):597-605. Sasano, H., Fukushima, K., Sasaki, I., Matsuno, S., Nagura, H., and Krozowski, Z. S. 1992.

mineralocorticoid receptors, 11 beta-hydroxysteroid dehydrogenase-1 and -2 expression in rat brain and kidney: in situ studies. *Mol Cell Endocrinol* 111 (1):R1-7. Ruizeveld de Winter, J. A., Trapman, J., Vermey, M., Mulder, E., Zegers, N. D., and van der

Kwast, T. H. 1991. Androgen receptor expression in human tissues: an

beta expression and growth inhibition of gynecologic cancer cells by the synthetic

Immunolocalization of mineralocorticoid receptor in human kidney, pancreas, salivary, mammary and sweat glands: a light and electron microscopic

and tissues. *Am J Physiol Cell Physiol* 283 (4):C1324-31.

system in health and disease. *N Engl J Med* 320 (15):980-91.

immunohistochemical study. *J Histochem Cytochem* 39 (7):927-36.

immunohistochemical study. *J Endocrinol* 132 (2):305-10.

receptor signaling. *Ann N Y Acad Sci* 1179:167-78.

adenocarcinoma? *Clin Sci (Lond)* 57 (5):421-5.

*Oncogene* 20 (15):1860-72.

cell carcinoma in a Japanese population. *Int J Urol* 14 (6):483-7.

*Chemother Pharmacol* 6 (2):151-4.

3:S1-10.

*Oncol*.

gene polymorphisms are associated with increased risk and progression of renal

carcinoma. Their utility as predictors of response to endocrine therapy. *Cancer* 

Powles, T., and De Santis, M. 2011. Maximising the duration of disease control in metastatic renal cell carcinoma with targeted agents: an expert agreement. *Med* 

2002. Expression of glucocorticoid receptor alpha- and beta-isoforms in human cells

Corvol, P. 1979. Are mineralocorticoid receptors present in human renal

Uskokovic, M. R., and Campbell, M. J. 2001. Synergistic growth inhibition of prostate cancer cells by 1 alpha,25 Dihydroxyvitamin D(3) and its 19-norhexafluoride analogs in combination with either sodium butyrate or trichostatin A.


Morris, D. J., and Souness, G. W. 1992. Protective and specificity-conferring mechanisms of

Motzer, R. J., Hudes, G. R., Curti, B. D., McDermott, D. F., Escudier, B. J., Negrier, S., Duclos,

Motzer, R. J., Michaelson, M. D., Redman, B. G., Hudes, G. R., Wilding, G., Figlin, R. A.,

Motzer, R. J., Murphy, B. A., Bacik, J., Schwartz, L. H., Nanus, D. M., Mariani, T., Loehrer, P.,

Muindi, J. R., Johnson, C. S., Trump, D. L., Christy, R., Engler, K. L., and Fakih, M. G. 2009.

Muramatsu, M., and Inoue, S. 2000. Estrogen receptors: how do they control reproductive and nonreproductive functions? *Biochem Biophys Res Commun* 270 (1):1-10. Nagakura, K., Abe, E., Suda, T., Hayakawa, M., Nakamura, H., and Tazaki, H. 1986.

Nakano, E., Tada, Y., Fujioka, H., Matsuda, M., Osafune, M., Kotake, T., Sato, B., Takaha, M.,

Nakayama, T., Watanabe, M., Yamanaka, M., Hirokawa, Y., Suzuki, H., Ito, H., Yatani, R.,

beta2 gene expression in human prostate cancers. *Lab Invest* 81 (7):1049-57. Naray-Fejes-Toth, A., and Fejes-Toth, G. 1990. Glucocorticoid receptors mediate

Noronha, R. F., and Rao, B. R. 1985. Increased dihydrotestosterone receptor levels in high-

Nykjaer, A., Dragun, D., Walther, D., Vorum, H., Jacobsen, C., Herz, J., Melsen, F.,

with clinical response to endocrine therapy. *J Urol* 132 (2):240-5.

B., Moore, L., O'Toole, T., Boni, J. P., and Dutcher, J. P. 2007. Phase I/II trial of temsirolimus combined with interferon alfa for advanced renal cell carcinoma. *J* 

Ginsberg, M. S., Kim, S. T., Baum, C. M., DePrimo, S. E., Li, J. Z., Bello, C. L., Theuer, C. P., George, D. J., and Rini, B. I. 2006. Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and plateletderived growth factor receptor, in patients with metastatic renal cell carcinoma. *J* 

Wilding, G., Fairclough, D. L., Cella, D., and Mazumdar, M. 2000. Phase III trial of interferon alfa-2a with or without 13-cis-retinoic acid for patients with advanced

A phase I and pharmacokinetics study of intravenous calcitriol in combination with oral dexamethasone and gefitinib in patients with advanced solid tumors. *Cancer* 

Inhibitory effect of 1 alpha,25-dihydroxyvitamin D3 on the growth of the renal

and Sonoda, T. 1984. Hormone receptor in renal cell carcinoma and correlation

and Shiraishi, T. 2001. The role of epigenetic modifications in retinoic acid receptor

mineralocorticoid-like effects in cultured collecting duct cells. *Am J Physiol* 259 (4 Pt

Christensen, E. I., and Willnow, T. E. 1999. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. *Cell* 96 (4):507-15. Obara, W., Konda, R., Akasaka, S., Nakamura, S., Sugawara, A., and Fujioka, T. 2007.

Prognostic significance of vitamin D receptor and retinoid X receptor expression in

mineralocorticoid action. *Am J Physiol* 263 (5 Pt 2):F759-68.

renal cell carcinoma. *J Clin Oncol* 18 (16):2972-80.

carcinoma cell line. *Kidney Int* 29 (4):834-40.

stage renal adenocarcinoma. *Cancer* 56 (1):134-7.

renal cell carcinoma. *J Urol* 178 (4 Pt 1):1497-503.

*Clin Oncol* 25 (25):3958-64.

*Clin Oncol* 24 (1):16-24.

2):F672-8.

*Chemother Pharmacol* 65 (1):33-40.


Steroid Receptors in Renal Cell Carcinoma 125

Tickoo, S. K., Gopalan, A., Tu, J. J., Harik, L. R., Al-Ahmadie, H. A., Fine, S. W., Olgac, S.,

Todd-Turla, K. M., Schnermann, J., Fejes-Toth, G., Naray-Fejes-Toth, A., Smart, A., Killen, P.

Trump, D. L., Muindi, J., Fakih, M., Yu, W. D., and Johnson, C. S. 2006. Vitamin D

Trydal, T., Bakke, A., Aksnes, L., and Aarskog, D. 1988. 1,25-Dihydroxyvitamin D3 receptor

Turbiner, J., Amin, M. B., Humphrey, P. A., Srigley, J. R., De Leval, L., Radhakrishnan, A.,

Vanderleede, B., Opdenoordt, T., Vandenbrink, C., Ebert, T., and Vandersaag, P. 1995.

Xu, X. C., Ro, J. Y., Lee, J. S., Shin, D. M., Hong, W. K., and Lotan, R. 1994. Differential

Yakirevich, E., Matoso, A., Sabo, E., Wang, L. J., Tavares, R., Meitner, P., Morris, D. J.,

transcriptase polymerase chain reaction study. *Hum Pathol*. 42 (11):1684-92. Yakirevich, E., Morris, D. J., Tavares, R., Meitner, P. A., Lechpammer, M., Noble, L., de

Yang, J. C., Sherry, R. M., Steinberg, S. M., Topalian, S. L., Schwartzentruber, D. J., Hwu, P.,

receptor mRNA along the nephron. *Am J Physiol* 264 (5 Pt 2):F781-91. Trump, D. L., Hershberger, P. A., Bernardi, R. J., Ahmed, S., Muindi, J., Fakih, M., Yu, W. D.,

studies. *J Steroid Biochem Mol Biol* 89-90 (1-5):519-26.

head and neck tissues. *Cancer Res* 54 (13):3580-7.

quantitative RT-PCR study. *Am J Surg Pathol* 32 (6):874-83.

renal cancer. *N Engl J Med* 349 (5):427-34.

obstruction. *Mod Pathol* 21 (1):60-5.

*Anticancer Res* 26 (4A):2551-6.

tissue. *Cancer Res* 48 (9):2458-61.

500.

(2):391-400.

and Reuter, V. E. 2008. Estrogen and progesterone-receptor-positive stroma as a non-tumorous proliferation in kidneys: a possible metaplastic response to

D., and Briggs, J. P. 1993. Distribution of mineralocorticoid and glucocorticoid

and Johnson, C. S. 2004. Anti-tumor activity of calcitriol: pre-clinical and clinical

compounds: clinical development as cancer therapy and prevention agents.

measurement in primary renal cell carcinomas and autologous normal kidney

and Oliva, E. 2007. Cystic nephroma and mixed epithelial and stromal tumor of kidney: a detailed clinicopathologic analysis of 34 cases and proposal for renal epithelial and stromal tumor (REST) as a unifying term. *Am J Surg Pathol* 31 (4):489-

Implication of retinoic Acid receptor-Beta in renal-cell carcinoma. *Int J Oncol* 6

expression of nuclear retinoid receptors in normal, premalignant, and malignant

Pareek, G., Delellis, R. A., and Resnick, M. B. 2011. Expression of the glucocorticoid receptor in renal cell neoplasms: an immunohistochemical and quantitative reverse

Rodriguez, A. F., Gomez-Sanchez, C. E., Wang, L. J., Sabo, E., Delellis, R. A., and Resnick, M. B. 2008. Mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type II expression in renal cell neoplasms: a tissue microarray and

Seipp, C. A., Rogers-Freezer, L., Morton, K. E., White, D. E., Liewehr, D. J., Merino, M. J., and Rosenberg, S. A. 2003. Randomized study of high-dose and low-dose interleukin-2 in patients with metastatic renal cancer. *J Clin Oncol* 21 (16):3127-32. Yang, J. C., Haworth, L., Sherry, R. M., Hwu, P., Schwartzentruber, D. J., Topalian, S. L.,

Steinberg, S. M., Chen, H. X., Rosenberg, S. A. 2003. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic


Schug, T. T., Berry, D. C., Shaw, N. S., Travis, S. N., and Noy, N. 2007. Opposing effects of

Sheikh, M. S., Shao, Z. M., Li, X. S., Dawson, M., Jetten, A. M., Wu, S., Conley, B. A., Garcia,

Speirs, V., Parkes, A. T., Kerin, M. J., Walton, D. S., Carleton, P. J., Fox, J. N., and Atkin, S. L.

Storlie, J. A., Buckner, J. C., Wiseman, G. A., Burch, P. A., Hartmann, L. C., and Richardson,

Suh, Y. A., Lee, H. Y., Virmani, A., Wong, J., Mann, K. K., Miller, W. H., Jr., Gazdar, A., and

Takahashi, M., Yang, X. J., Sugimura, J., Backdahl, J., Tretiakova, M., Qian, C. N., Gray, S. G.,

Takeda, H., Chodak, G., Mutchnik, S., Nakamoto, T., and Chang, C. 1990.

Tanaka, M., Fukuda, H., and Higashi, Y. 2003. [A case of complete regression of metastatic

Tang, X. H., and Gudas, L. J. 2011. Retinoids, retinoic acid receptors, and cancer. *Annu Rev* 

Thacher, T. D., and Clarke, B. L. 2011. Vitamin D insufficiency. *Mayo Clin Proc* 86 (1):50-60. Thomas, C. P., Liu, K. Z., and Vats, H. S. 2006. Medroxyprogesterone acetate binds the

collecting duct epithelia. *Am J Physiol Renal Physiol* 290 (2):F306-12.

polyclonal antibodies to androgen receptor. *J Endocrinol* 126 (1):17-25. Takenawa, J., Kaneko, Y., Okumura, K., Yoshida, O., Nakayama, H., and Fujita, J. 1995.

interleukin-6 receptor. *J Urol* 153 (3 Pt 1):858-62.

receptors. *Cell* 129 (4):723-33.

*Cancer J Clin* 61 (4):212-36.

(1):96-100.

(2):133-41.

*Pathol* 6:345-64.

8.

human breast cancer? *Cancer Res* 59 (3):525-8.

retinoic acid on cell growth result from alternate activation of two different nuclear

M., Rochefort, H., and Fontana, J. A. 1994. Retinoid-resistant estrogen receptornegative human breast carcinoma cells transfected with retinoic acid receptor-alpha acquire sensitivity to growth inhibition by retinoids. *J Biol Chem* 269 (34):21440-7. Siegel, R., Ward, E., Brawley, O., and Jemal, A. 2011. Cancer statistics, 2011: The impact of

eliminating socioeconomic and racial disparities on premature cancer deaths. *CA* 

1999. Coexpression of estrogen receptor alpha and beta: poor prognostic factors in

R. L. 1995. Prostate specific antigen levels and clinical response to low dose dexamethasone for hormone-refractory metastatic prostate carcinoma. *Cancer* 76

Kurie, J. M. 2002. Loss of retinoic acid receptor beta gene expression is linked to aberrant histone H3 acetylation in lung cancer cell lines. *Cancer Res* 62 (14):3945-9. Swisshelm, K., Ryan, K., Lee, X., Tsou, H. C., Peacocke, M., and Sager, R. 1994. Down-

regulation of retinoic acid receptor beta in mammary carcinoma cell lines and its up-regulation in senescing normal mammary epithelial cells. *Cell Growth Differ* 5

Knapp, R., Anema, J., Kahnoski, R., Nicol, D., Vogelzang, N. J., Furge, K. A., Kanayama, H., Kagawa, S., and Teh, B. T. 2003. Molecular subclassification of kidney tumors and the discovery of new diagnostic markers. *Oncogene* 22 (43):6810-

Immunohistochemical localization of androgen receptors with mono- and

Inhibitory effect of dexamethasone and progesterone in vitro on proliferation of human renal cell carcinomas and effects on expression of interleukin-6 or

renal cell carcinoma following corticosteroid treatment]. *Hinyokika Kiyo* 49 (4):225-8.

glucocorticoid receptor to stimulate alpha-ENaC and sgk1 expression in renal


**6** 

*Japan* 

**Anticancer Target Molecules Against** 

**the SCF Ubiquitin E3 Ligase in RCC:** 

Tomoaki Tanaka and Tatsuya Nakatani

**Potential Approaches to the NEDD8 Pathway** 

Several alternative treatments have recently been developed for metastatic renal cell carcinoma (RCC). Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic protein, which is responsible for increased vasculature and tumor-growth in RCC. Fundamentally, a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene induces overexpression of VEGF via accumulation of hypoxia-inducible factor (HIF)-1 in RCC. Several agents inhibiting the VEGF signaling cascade, such as sorafenib, sunitinib and bevacizumab, have been found to exert significant anti-tumor effects and provide meaningful clinical benefits. Furthermore, temsirolimus and everolimus, inhibitors of the mammalian target of rapamycin (mTOR), which block the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway involved in numerous cellular functions including cell proliferation, survival and angiogenesis, have been found to be effective agents against advanced RCC in the clinical setting. Although molecular targeting therapies against the VEGF or mTOR signaling pathway have revolutionized the treatment of advanced RCC, no

In this chapter, we focus on potent molecules and agents possibly suppressing the tumor growth of RCC via regulation of the ubiquitination-proteasome system. NEDD8, one of the ubiquitin-like proteins, reportedly forms conjugates with cullin family proteins and thereby activates the Skp1-Cullin-F-box (SCF) ubiquitin protein ligase complex that catalyzes the ubiquitination of many cell-cycle regulators, e.g. cyclin E, p21, p73 and p27. It is possible that negative regulation of NEDD8 and its conjugation system induces an antiproliferative action on RCC, secondary to inhibition of ubiqitin-proteasome activity. We previously showed that these negative regulator proteins, such as NEDD8 ultimate buster 1 (NUB1) and a dominant negative form (Ubc12 C111S) of NEDD8 E2 ligase, exhibited remarkable antitumor effects against some tumors, including RCC. Moreover, MLN4924, a potent and selective inhibitor of NEDD8-activating enzyme (NAE), was recently reported to disrupt SCF ubiquitin E3 ligase-induced protein turnover leading to apoptosis in tumor cells via deregulation of the cell cycle. This compound has already been applied in the clinical setting, e.g., malignant lymphoma. Thus, negative regulation of NEDD8 and its conjugation is an attractive anti-cancer strategy based on evidence obtained by basic research. We have

**1. Introduction** 

curative therapy has yet been established.

*Osaka City University Graduate School of Medicine, Department of Urology* 

