Diagnosis of Renal Cell Carcinoma

## **Chapter 1**

## Perspective Chapter: An Update on Renal Cell Carcinoma

*Jindong Chen*

## **Abstract**

Incidence and mortality of renal cell carcinoma (RCC) significantly vary worldwide. While RCC incidence has been increasing, its mortality rate has been decreasing. Smoking, obesity, hypertension, chronic kidney disease (CKD), ethnicity, location, and other environmental factors are reported to be associated with RCC. With the use of the improved diagnostic methods, including ultrasound, contrastenhanced ultrasound (CEUS), computed tomography (CT) scan, magnetic resonance imaging (MRI), and positron emission tomography (PET)/CT scan, the detection rate of RCC has significantly increased over the past decade. We have witnessed innovation in surgical techniques and robotic platforms with integration of imaging approaches, and urologists are now able to maximize functional and oncologic outcomes in nephron preservation and complication-free recovery. Thus, the paradigm in the surgical treatment of RCC has transformed and will continue to change in the future. In addition, targeted therapy, immunotherapy, and combination therapy are adopted to treat patients with advanced RCC. In recent years, the combination of immune checkpoint inhibition and antiangiogenic therapy is a very attractive combined therapeutic strategy for advanced/metastatic RCCs. Biomarkers, including epigenetic markers for RCC, have been increasing, which will be helpful to discover new therapeutic targets and related inhibitors for the treatment of advanced RCC.

**Keywords:** kidney cancer, renal cell carcinoma, RCC, genetics, targeted therapy, immunotherapy, combination therapy

## **1. Introduction**

Kidney cancer has been known to have distinct histological types with different genetic profiles. The most common type is renal cell carcinoma (RCC), representing more than 90% of all kidney cancers [1]. RCC is consisted of a few subtypes. The most common subtype is clear cell renal cell carcinoma (ccRCC), followed by papillary RCC (pRCC, type I and type II), chromophobe RCC (cRCC), collecting duct carcinoma (CDC), etc. Kidney cancer is the sixth most common malignant disease in men and tenth in women. It accounts for 5% of male and 3% female malignancies [2, 3]. The RCC incidence is constantly rising as well. In urology, kidney cancer is the third most common malignant disease. While kidney cancer occurs mostly in North American and European, the incidence of

renal cancer is relatively lower in Asian [4]. Based on the global cancer statistics in 2020 [5, 6], there were an estimated 431,300 people diagnosed with kidney cancer and approximately 179,400 deaths worldwide in 2020. It was recently estimated that there were 81,800 new kidney cancer cases and 14,890 deaths in the United States in 2023 [7]. Diagnosis of RCC at early stages is crucial in treating patients and improving survival rates. With the application of improved diagnostic techniques such as cross-section abdominal imaging [8], the detection rate of RCC has increased in recent years [9]. Choosing the best therapeutic strategy is essential for improving the outcome of patients with RCC.

## **2. Epidemiology of RCC**

RCC is the most lethal urogenital cancer, accounting for 2–3% of all cancers. The incidence of RCC is continuously rising worldwide, and it is higher in developed countries compared to developing countries, and higher in men than women (male to female ratio is 1.5:1). The mortality rate of RCC is 30–40% and is higher in men compared to women [3]. The majority of RCC patients are aged over 60 years. Since the advance in diagnostic methods and public consciousness of the importance of periodic health screening, the number of RCC patients diagnosed in the early stages keeps increasing. Thus, the incidence of RCC has been rising as well in the past three decades [9]. Even though, the mortality rate of RCC has been constantly decreasing, especially in developed countries, due to early therapy and progress of the therapeutic strategies [10]. However, despite the improvement in disease control and increased survival rate, metastases are often observed in many RCC patients [11]. As reported, 30–50% of patients with local RCC progress to metastasis. In addition, metastasis was observed in 20–30% of the RCC patients at the early stage of diagnosis, and nearly 40% of the patients with localized RCC tumors presented distant metastases even after surgery. The most common distant metastatic sites are the lungs, bones (vertebrae, proximal part of the femur, pelvic bones), lymphatic nodes brain, liver, the opposite kidney, and suprarenal glands [12]. The patients with RCC metastases suffer from pain, nerve compression, hypercalcemia, and pathological fractures. The occurrence of metastases often worsens the patients' prognosis [13]. The median survival for patients with RCC metastases is approximately 8 months and the mortality rate is 50% during the first year, and 10% for 5 years survival [10, 13, 14].

#### **3. Risk factors associated with RCC**

While age and gender are strongly associated with increased RCC incidence, hypertension, smoking, obesity, ethnicity, location, and history of using tobacco products increase the risk of RCC as well [3]. It was reported that smoking could increase the risk by about 54% in men and 22% in women. Smoking time and the daily number of cigarettes are directly associated with kidney cancer incidence for both men and women. In addition, some minor risk factors that may be related to RCC include acquired renal cystic disease, chronic kidney disease (CKD), endstage renal disease (ESRD), exposure to cadmium and trichloroethylene, consumption of red and processed meat, chronic use of palliatives, type-2 diabetes, vitamin D level, viral hepatitis, increased triglycerides, decreased physical activity, and

genetic syndromes [15]. Patients with renal insufficiency and in the terminal stage of the disease are four times more likely to develop kidney cancer. Measures of preventing RCC include smoking cessation, and reducing of the body mass index (BMI), etc.

## **4. Pathophysiology of RCC**

Since RCC is a heterogeneous cancer that may stem from different type of renal cells, histological subtyping of RCC may exert a crucial impact on the choice of therapeutic strategies and prognosis. Based on the histological features, RCC is divided into 16 subtypes, including clear cell RCC, pRCC (type I and II), cRCC, collecting duct RCC, multilocular cystic RCC, medullary carcinoma, mucinous tubular, spindle cell carcinoma, neuroblastoma-associated RCC, Xp11.2 translocations—TFE3 carcinoma, hereditary cancer syndromes, and unclassified lesion [16]. Of them, clear cell RCC is the most common subtype of RCC and accounts for 75–85%. The second most common one is pRCC that constitutes 10–15%. cRCC ranked third and represents 3–5% of kidney cancers. Both clear cell carcinoma and pRCC are thought to stem from the proximal tubule, while chromophore RCC originates from the distal connecting tubules (DCT) and collecting duct system, especially the intercalated cells [17]. While most clear cell RCCs are sporadic, approximately 5% of the clear cell RCC is hereditary, and usually related to hereditary syndromes such as Von Hippel–Lindau disease (VHL) and tuberous sclerosis (TS). Clear cell RCC tends to metastasize to the lymph nodes, the lungs, the liver, and bones. Clear cell RCC has poorer prognosis compared to pRCC and cRCC. On the basis of histological and genetic differences, pRCC is further divided into two subtypes: type I and type II. The type I pRCC cannot be distinguished from type II through routine imaging techniques. Papillary type I can be detected at an early stage, and thus has a better prognosis compared to type II. cRCC is commonly observed in patients aged more than 60, and less aggressive compared to clear cell RCC. Therefore, cRCC has the best prognosis among all RCCs [4].

## **5. Genetics of RCC**

The first categorization of kidney cancer based on molecular genetics was conducted by Heidelberg in 1997, and it was adopted in the WHO tumor classification of 2004, 2012 Vancouver ISUP [18], and the latest RCC classification of the WHO (2016) [16]. RCC has various genetic alterations, including gene mutations, epigenetic modifications, and chromosomal aberrations. While genetic alterations can cause both sporadic and hereditary types, patients with familial history (hereditary) represent only 3% of all kidney cancer cases. An increased number of RCC-related genes were identified in the past decades. Considering its high genetic heterogeneity, RCC can be caused by mutations in many genes and chromosomal abnormalities. Each subtype of RCC has its corresponding affected genes [19]. Clear cell RCC-related genes include *VHL*, *PBRM1, BAP1, STED2, JARID1c*, *FLCN*, and c-MYC. *VHL* mutations account for approximately 60% of clear cell RCC [20]. In addition, exome-sequencing has revealed that *PBRM1, SETD2, BAP1, and JARID1c* are associated with RCC as well (*PBRM1,* 40%, *SETD2,* 12%, *BAP1,*10%, and *KDM5C,*5%) [21]. Combinational mutations of the above genes were also detected in many clear cell RCCs. With the advent of new genetic tools, more RCC-related genes/biomarkers might be uncovered. pRCC is divided into two types: Type I and Type II. Although pRCC type I and type II are morphologically similar, they are cytologically different. Compare to pRCC II, pRCC type I has a low-grade tendency and better prognosis [22]. pRCC I is often caused by overexpression of the *MET* gene on chromosome 7q31, while pRCC II is less correlated to MET overexpression but associated with alterations in *FH*, *CDKN2A*, *SETD2*, *BAP1*, *PBRM1*, *FLCN*, *NRF2-ARE*, *TERT*, *TFE3*, and increased expression of *NF2* [23, 24]*.* cRCC is a relatively rare type of RCC that originates from the distal convoluted tubule. cRCC is associated with mutations in *PTEN*, *TP53*, *mTOR*, *c-kit*, *FAAH2*, *PDHB*, *PDXD1*, *ZNF765*, *PRKAG2*, *ARID1A*, and *ABHD3*. Of them, *PTEN* mutation is the most common event in cRCC [25]. In addition, mutations in *FLCN* appear to cause all types of RCC in *Flcn* knockout mouse models [26, 27].

Chromosomal aberration is also a common feature in RCC [28]. In clear cell RCC, the loss of the short arm of chromosome 3 is the most frequently occurred event, which is related to the loss of the *VHL* gene located on 3p [28, 29]. Other chromosomal aberrations include loss of chromosome Y, gaining 5q31, 8q, 4p, 14q, 9p, and trisomy of chromosome 7. Patients with gaining of 4p, 9p, and 14q have poor prognosis while gaining 5q31 is connected with prolonged survival in high-grade clear cell RCC. Deletion of the Y chromosome usually leads to clear cell RCC with distant metastasis. In addition, gaining chromosome 8q can cause metastasis of clear cell RCC, which may be associated with overexpression of C-MYC. For chromosomal abnormity, pRCC type I is also different from papillary type II. Trisomy of chromosomes (e.g., 3q, 7, 8, 12, 16, 17, 20) commonly appears in type I pRCC. In addition, loss of chromosome Y is an important feature of papillary type II in men. In contrast, gaining a chromosome 8q and losing 1p and 9p are frequently observed in RCC papillary type II. cRCC is also associated with chromosome aberrations, including loss of chromosomes 1, 2, 6, 10, 13, 17, and 21 [20]. Losing copies of chromosomes 1, 2, 6, 10, 13, and 17 are more commonly observed in the classical type of cRCC than the eosinophilic type [30]. In addition, gaining chromosome copies (e.g., 4, 7, 11, 12, 14q, and 18q) is related to cRCC as well [31].

## **6. Clinical diagnosis of RCC**

Since small RCC masses are usually asymptomatic, the majority of RCCs are accidentally detected by routine imaging for various medical screening purposes [32]. Patients diagnosed with RCC based on the symptoms account for only 30%, and approximately 20–30% of the patients present metastasis at the time of diagnosis. Flank pain, hematuria, and abdominal mass are usually considered as the classic symptoms for RCC diagnosis, but these symptoms are observed in only 4–17% of the cases. Other symptoms for diagnosis include fever, abdominal pain, anemia-induced fatigue, weight loss, bone pain, and cough caused by metastasis of cancer cells or lower limb edema, peripheral lymphadenopathy (LAP), and varicocele caused by inferior vena cava (IVC) or renal thrombosis [33]. Once signs and symptoms appear, further laboratory investigation should be performed. The laboratory investigation includes renal function tests, complete blood cell count (CBC), liver function tests, urinary analysis, thyroid function tests, and examination of the level of calcium, lactate dehydrogenase, and alkaline phosphatase.

Various preclinical imaging modalities are valuable tools for detecting renal masses. These imaging modalities include ultrasound, CEUS, computed tomography (CT) scan, magnetic resonance imaging (MRI), positron emission tomography

#### *Perspective Chapter: An Update on Renal Cell Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.112633*

(PET)/CT, etc. Many renal masses and benign cystic kidney lesions can be easily detected with ultrasound [34]. In addition, as an inexpensive and accurate imaging approach, contrast-enhanced ultrasound (CEUS) has been adopted to evaluate indeterminate renal lesions, though it may not effectively discriminate between malignant and benign renal masses. For more accurately identifying malignant masses, CT scan or MRI is required [35]. In some cases, both CT and MRI should be performed by contrast because contrast absorption is a key factor in determining malignant masses [36]. For small lesions (1–2 cm) and in cases of renal insufficiency, pregnant women, and patients allergic to contrast material, MRI should be conducted [37]. The primary purpose of imaging is to inspect the characteristics of the affected mass, identify possible abdominal metastases, mass expansion, and venous involvement for staging. PET scan, is not a standard scan strategy, is very helpful in the diagnosis and followup of RCC. Thus, routine use of PET/CT is also recommended [38]. Other imaging approaches, including advanced MRI techniques or the combination use of iodine PET and CT, may be performed to determine renal masses [39]. In addition, biopsy and histopathology are required to carry out in suspected masses before further treatment.

## **7. Treatment and management of RCC**

To date, surgery is still an essential treatment for RCC, and RE nephrectomy has been regarded as the standard of care for the management of renal tumors. For small renal lesions and surgery-induced chronic kidney disorders, active surveillance of nephron surgery and minimally invasive approaches have been adopted to limit the invasion and loss of kidney function in clinical [40]. RCC patients with severe comorbidities, elderly patients with small tumors ≤4 cm, or patients with a short life expectancy are recommended for active surveillance [41]. In some cases, active surveillance is also suggested to monitor the rate of the large tumor's growth. In addition, imaging can be performed for active surveillance in the first half year, and then, every 6 months at 2–3 years, and later, annually [42]. In addition, for treating small randomly detected tumors, minimally invasive procedures should be used. Cryotherapy (CRYO) and radiofrequency ablations (RFA) were suggested for patients with only one kidney or for those unable to undergo major surgery.

#### **7.1 Nephron preservation surgery**

While radical nephrectomy is the removal of the entire kidney and is suitable for patients with larger renal tumors (T2, >7 cm), nephron-sparing partial nephrectomy only removes the small localized renal tumor (T1, <7 cm) and preserves the parenchyma. Thus, in most cases including locally advanced and metastatic diseases, nephron-sparing partial nephrectomy is recommended except for those that technically impossible to be removed due to their unfavorable location.

The advent and improvement of robot-assisted surgical approaches and robotic platforms have helped to close the technical gap and allow for greater adoption of the laparoscopic approach and nephron-sparing surgery, leading to a revolution for surgical management of RCC in recent decades [43, 44]. As the use of robotic platforms combined with imaging techniques in surgery, larger, higher stage, and more complex renal lesions can now be treated in a minimally invasive fashion, which leads to decreased morbidity, shortened patients' hospitalization, and less side effects [45, 46]. Robot-assisted surgery has continued to evolve and has been playing an ever-expanding role in the treatment of RCC worldwide [45]. Laparoscopic partial nephrectomy and robot-assisted partial nephrectomy are becoming the standard treatment for patients eligible for nephron-sparing surgery. While the nephron-sparing surgery has been endorsed as the gold standard treatment of T1a tumors ≤4 cm in size and T1b lesions in the United States and Europe when technically possible with experienced surgeons [41, 47], robot-assisted partial nephrectomy has been adopted for treatment of cT2 RCC [48]. While new robotic platforms such as single-port (SP) robotic surgical system and multiport robotic surgical systems are currently under development, the popular da Vinci platform has significantly expanded the laparoscopic paradigm spectrum in the surgical treatment of RCC [49–51].

#### **7.2 Cytokine treatment of metastatic disease**

Before targeted therapy, patients with metastatic clear cell RCC were previously treated with immunosuppressive agents such as INFα and IL2, namely systemic therapy. In 1990s, systemic therapy with a high-dose IL2 turned into a commonly used modality for various cancer patients with metastasis. However, complete response was only observed in <10% of patients treated with a longer high dose of IL-2. It simultaneously caused severe toxic effects. Thus, high-dose IL2 treatment is not recommended for patients with metastatic RCC unless the patients are young and in very good conditions with low tumor volume [52].

Aldesleukin and INFα (along with Bevacizumab) are the only safe modulating drugs that are approved in selected metastatic RCC. INFα is adopted to treat RCC patients in various formulas with a response rate of 10–15% and a response time of 4 months. Patients who benefit from INF treatment were then subjected to nephrectomy. Although INFα treatment shows some efficacy in the treatment of patients with metastatic RCC, it is not suggested for treating RCC patients as a single drug.

#### **7.3 Targeted therapy and immunotherapy of metastatic RCC**

In the past decades, the most significant advance in the therapeutic strategy of metastatic RCC has been the development of treatments that specifically target the RCC-related biological pathways and related biomarkers [53, 54]. In the 1990s, cytokines, such as IFNα and IL-2, were used to treat metastatic clear cell RCC (**Figure 1**). In the 2000s, targeted therapies that target the VEGF/PDGFR/mTOR pathways replaced the cytokine therapies. In the targeted therapies, tyrosine kinase inhibitors (TKIs) are effective agents in the treatment of metastatic RCC, which was used as the first line and the second line treatment options. To date, five TKIs (e.g., cabozantinib, axitinib, pazopanib, sorafenib, and sunitinib) have been approved internationally for the targeted therapy of metastatic RCC (**Figure 1**). Cabozantinib targets the tyrosine-protein kinase receptor UFO and the MET receptor tyrosine kinase. Pazopanib targets the VEGFR, while axitinib inhibits VEGFR with improved specificity, sorafenib an multi-kinase inhibitor, inhibits RAF-1, B-RAF, VEGFR-2, VEGFR-3, PDGF-β, KIT, and FLT-3. Sunitinib targets VEGFR2(Flk-1) and PDGFR-β. Whereas, lenvatinib suppresses VEGFRs and fibroblast growth factor receptors [55, 56]. In addition, temsirolimus and everolimus, two mTOR inhibitors, have been approved for the treatment of advanced RCC as well. Bevacizumab, an anti-VEGF monoclonal antibody, is approved for the treatment of advanced RCC when it is used in combination with INF. In 2015, nivolumab, an anti-PD-1 immune checkpoint

#### **Figure 1.**

*Development of approved anti-RCC agents.*

inhibitor that prevents signaling through programmed cell death 1, has been approved for the first immunotherapy agent of metastatic RCC [57]. Later, ipilimumab, an anti-CTLA-4 monoclonal antibody, in combination with nivolumab was approved for the first-line treatment of advanced RCC [20, 58]. Recently, increasing attention has been paid to the combination therapy. The latest combination therapy strategies such as axitinib–avelumab (target VEGF and immune checkpoints) [59, 60], axitinib–pembrolizumab (target VEGFR) in combination with PD-L1 and PD-1, respectively, have been used to treat metastatic RCC. To date, immune checkpoint inhibition plus antiangiogenic therapy constitutes a very promising combined therapeutic strategy for advanced/metastatic RCCs.

In addition, some epigenetic markers have been proposed as promising epigenetic RCC markers based on DNA methylation, ncRNA expression, and histone modification [61–63]. Many epigenetic markers and epigenetic modifiers are likely candidates for clinical use, but further validation is needed [63, 64]. The development of epigenetic therapies, either alone or in combination with antiangiogenic agents and/or immune-checkpoint inhibitors, is a hopeful therapeutic strategy for RCC.

### **8. Conclusion**

RCC accounts for 4%of all malignant tumors. In urology, RCC is the third most common malignant tumor. Risk factors include positive family history, smoking, obesity, high blood pressure, etc. Although most cases of RCC are diagnosed accidentally, the improvement in diagnostic approaches has increased the detection rate over the past decades. Many RCC-related genes and predictive biomarkers are being identified and may further improve the diagnosis of RCC. In addition, choosing the best therapeutic strategy is critical to improve the outcome of RCC. The tumor size, the stage of the disorder, and the surgeon's experience are the determinant impact factors in the choice of the optimal treatment approach. While the open surgery is still reserved for locally advanced diseases, it is gradually displaced by robotic-assisted partial nephrectomy. Although the optimal first-line treatment, including targeted therapy,

immunotherapy, or combined therapy for metastatic RCC may differ by clinical risk group and efficacy endpoint, the first-line treatment landscape for metastatic RCC is rapidly evolving. Thus, knowledge of the latest advances in the diagnosis and management of RCC could assist the related researchers, physicians, and nephrologists, to better diagnose and treat RCC.

## **Author details**

Jindong Chen Exploring Health, LLC., Guangzhou, Guangdong, China

\*Address all correspondence to: jindong\_chen@hotmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[51] Valero R, Sawczyn G, Garisto J, Yau R, Kaouk J. Multiquadrant combined robotic radical prostatectomy and left partial nephrectomy: a combined procedure by a single approach. Actas Urológicas Españolas (English Edition). 2020;**44**(2):119-124

[52] McDermott DF, Regan MM, Clark JI, Flaherty LE, Weiss GR, Logan TF, et al. Randomized phase III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients with metastatic renal cell carcinoma. Journal of Clinical Oncology. 2005;**23**(1):133-141

[53] Dizman N, Philip EJ, Pal SK. Genomic profiling in renal cell carcinoma. Nature Reviews. Nephrology. 2020;**16**(8):435-451

[54] Osawa T, Takeuchi A, Kojima T, Shinohara N, Eto M, Nishiyama H.

Overview of current and future systemic therapy for metastatic renal cell carcinoma. Japanese Journal of Clinical Oncology. 2019;**49**(5):395-403

[55] Choueiri TK, Escudier B, Powles T, Mainwaring PN, Rini BI, Donskov F, et al. Cabozantinib versus everolimus in advanced renal-cell carcinoma. The New England Journal of Medicine. 2015;**373**(19):1814-1823

[56] Motzer RJ, Hutson TE, Glen H, Michaelson MD, Molina A, Eisen T, et al. Lenvatinib, everolimus, and the combination in patients with metastatic renal cell carcinoma: a randomised, phase 2, open-label, multicentre trial. The Lancet Oncology. 2015;**16**(15):1473-1482

[57] Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, et al. Nivolumab versus everolimus in advanced renal-cell carcinoma. The New England Journal of Medicine. 2015;**373**(19):1803-1813

[58] Jonasch E. NCCN guidelines updates: management of metastatic kidney cancer. Journal of the National Comprehensive Cancer Network. 2019;**17**(5.5):587-589

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[60] Rini BI, Plimack ER, Stus V, Gafanov R, Hawkins R, Nosov D, et al. Pembrolizumab plus axitinib versus sunitinib for advanced renal-cell carcinoma. The New England Journal of Medicine. 2019;**380**(12):1116-1127

[61] Angulo JC, Manini C, Lopez JI, Pueyo A, Colas B, Ropero S. The role of epigenetics in the progression of clear

*Perspective Chapter: An Update on Renal Cell Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.112633*

cell renal cell carcinoma and the basis for future epigenetic treatments. Cancers (Basel). 2021;**13**(9):2071

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[64] El Khoury LY, Fu S, Hlady RA, Wagner RT, Wang L, Eckel-Passow JE, et al. Identification of DNA methylation signatures associated with poor outcome in lower-risk Stage, Size, Grade and Necrosis (SSIGN) score clear cell renal cell cancer. Clinical Epigenetics. 2021;**13**(1):12

## **Chapter 2**

## Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas by Down-Regulating Differentiation

*Mary Taub*

## **Abstract**

L-2-Hydroxyglutarate (L2HG) overproducing Renal Cell Carcinomas (RCCs) arise in the kidney due to the genetic loss of L-2HG Dehydrogenase (L2HGDH), the enzyme responsible for the metabolism of L2HG. The overproduced 2-Hydroxyglutarate (2HG) promotes tumorigenesis by inhibiting α-ketoglutarate (αKG)-dependent dioxygenases, including Ten-eleven-Translocation 5-methylcytosine (5mC) dioxygenase (TET) enzymes as well as histone demethylases. The resulting epigenetic changes alter the phenotype of renal proximal tubule (RPT) cells, the cells of origin of RCCs. This report describes the consequences of increased L2HG on the differentiation of RPT cells, one of the initial steps in promoting tumorigenesis. Presumably, similar alterations promote the expansion of renal cancer stem-cells and tumorigenesis.

**Keywords:** oncometabolite, L-2-Hydroxyglutarate, renal cell carcinoma, renal proximal tubule, epigenetic, tubulogenesis, dedifferentiation, matrigel

## **1. Introduction**

Oncometabolites are components of intermediary metabolism whose abundance increases dramatically during the metabolic rewiring that occurs during tumorigenesis. These oncometabolites act to promote, and/or sustain tumor growth and metastasis. The hypothesis that metabolic rewiring plays a major role in tumorigenesis was initially supported by the work of Warburg in the 1920s. Warburg's experimental results indicated that aerobic glycolysis becomes the predominant means of generating the metabolic demands of developing tumors, rather than oxidative metabolism [1]. In recent years, the Warburg effect has been substantiated in studies of Clear Cell RCCs (ccRCCs) with von Hippel-Lindau (VHL) mutations, in addition to studies with other tumors [1]. In these tumors, aerobic glycolysis predominates over the tricarboxylic acid cycle (TCA) and oxidative phosphorylation. As a consequence,

these tumor cells use glucose more efficiently than normal cells, producing lactic acid. The lactic acid is used as an alternative source of acetyl-Coenzyme A (acetyl-CoA) for the production of fatty acids and cholesterol [2]. Glutamine, is another essential component for the production of fatty acids and the lipid droplets characteristic of ccRCCs [2]. Following its entry into the mitochondria, glutamine (Gln) is converted to glutamate (Glu) by glutaminase, followed by conversion to αKG, which as we will see, is not only an important intermediate in the TCA cycle, but is also a precursor for the oncometabolite 2HG.

Numerous investigations have shown that the Warburg effect arises due to major changes in the expression of a number of glycolytic and TCA cycle enzymes [3]. In the majority of ccRCCs, both alleles of the von Hippel-Lindau (VHL) gene are mutated, leading to dysregulation of Hypoxia-inducible factor 1 (HIF-1) transcriptional activity. HIF-1, a master regulator of oxygen homeostasis, is hydroxylated on its α subunit during normoxia by a ubiquitin protein ligase, whose activity depends upon VHL. In the absence of normal VHL, HIF-1 reprograms glucose and energy metabolism through its transcriptional activities, such that glycolysis and lactate production become predominant over respiration, even during normoxia [3].

More recent studies indicate that mutations which specifically effect the expression of 3 different types of metabolic enzymes (fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH)) similarly promote tumorigenesis because of the accumulation of "oncometabolites" [1]. Oncometabolites are defined as "small-molecule components (or enantiomers) of normal metabolism whose accumulation dysregulates signaling so as to establish a milieu that promotes carcinogenesis" [4]. The fumarate accumulation, which occurs as a consequence of FH mutations, results in the formation of hereditary papillary renal carcinomas, whereas the succinate accumulation, that occurs as a consequence of SDH mutations, results in the formation of hereditary paragangliomas (PGLs). In contrast, cancer-related mutations in the *IDH1* and *IDH2* genes, result in the formation of mutant IDH enzymes which produce D-2-hydroxyglutarate (D2HG) from αKG. This is particularly serious, because IDH1 and IDH2 mutations are associated with 70–80% glioblastomas, and 20% of acute myeloid leukemias (AMLs) [5]. All 3 oncometabolites, D2HG, fumarate and succinate, have been found to act by similar mechanisms, i.e. by inhibiting multiple αKG-dependent dioxygenases, which ultimately dysregulates signaling in a manner that promotes tumorigenesis [1, 5].

**Figure 1.**

*The D- and L- enantiomers of L-2Hydroxglutarate (L-2HG).2HG is a chiral molecule, with D-and Lenantiomers as illustrated above.*

#### *Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

Notably, oncometabolites such as 2HG have also been used as prognostic markers. In individuals with cancer-related IDH mutations, D2HG is produced to such an extent, that the elevated D2HG can be detected in serum [6]. Remarkedly, 87% of the AML patients with high serum 2HG had IDH1/IDH2 mutations, although only 29% of AML patients with moderately high 2HG had IDH1/IDH2 mutations. This latter finding suggests that other genetic events are also responsible for elevated 2HG. Of particular interest in these regards, 2HG is a chiral molecule, with both D- and L- enantiomers (as show in **Figure 1**). Both IDH1 and IDH2 mutations result in increased synthesis of the D- enantiomer (as shown in **Figure 2**). However, a loss of copy number of L2HGDH results in increased levels of the L- enantiomer of 2HG, rather than the D- enantiomer. For this reason, Struys [7] has stressed the importance of employing analytical methods that differentiate between L2HG and D2HG (e.g. chiral derivatization followed by Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC–MS/MS)), rather than measuring total 2HG.

During the initial time frame of the studies in which elevated 2HG was detected in serum, an inherited metabolic defect, L-2-Hydroxyglutarate aciduria (L2HGA) was being studied [8]. L2HGA is an autosomal recessive neurometabolic disorder, characterized by abnormalities of the subcortical cerebral white matter dentate nucleus, globus pallidus, putamen and caudate nucleus. The gene associated with this disorder was found to be a mutant *L2HGDH*, which was defective in metabolizing L2HG to αKG. Consistent with this observation, the biochemical hallmark of L2HGA is elevated levels of L2HG in the urine (such that L2HG levels are 10–300 fold more than normal controls). A consequence of L2HGA is an increased propensity for tumor formation. Kranendijk et al. [8] have reported that L2HG is produced from αKG by L-malate dehydrogenase (MDH), as a promiscuous side reaction (**Figure 3**). The primary

#### **Figure 2.**

*Effect of mutant Isocitrate dehydrogenase (IDH) on metabolism of D-2HG. Two different isoforms of IDH are present in mammalian cells, including cytosolic IDH1 and mitochondrial IDH2, which participated in the tricarboxylic acid (TCA) cycle. Normally, IDH1 or IDH2 have been found to synthesize alpha ketoglutarate from isocitrate. However, mutant IDH1 and IDH2 have been found to further synthesize D-2HG from alpha ketogutarate.*

#### **Figure 3.**

*Biosynthesis of L2HG from glutamine: Effect of a loss of L2HGDH copy number. The biosynthesis of L2HG depends upon glutamine (Gln), which is first metabolized to glutamine acid by Glutaminase, followed by the metabolism of alpha ketoglutarate. Alpha ketoglutarate generated either by the TCA cycle or in mitochondria or in the cytosol, is subsequently metabolized by either malate dehydrogenase (MDH) or by lactate dehydrogenase a (LDHA) into L2HG. When the copy number of L2HG Deydrogenase (L2HGDH) declines, the metabolism of L2HG slows, causing an increase in the level of L2HG.*

function of MDH is to convert L-malate to oxaloacetate. Lactate Dehydrogenase (LDH) has similarly been found to produce L2HG as a side reaction during oxygen deprivation under acidic conditions (**Figure 3**) [9]. In contrast, D2HG is produced from γ hydroxybutyrate by hydroxyacid-oxoacid transhydrogenase (HOT) [8].

As stated above, patients with L2HGA exhibit neurological disorders, associated with progressive damage to the brain. Biochemical alterations in brain tissues include reduced creatine kinase activity, oxidative stress, and increased Glutamate (Glu) uptake into synaptosomes and synaptic vesicles. Precisely why the brain is primarily affected is not well understood.

Subsequent to the findings that the D2HG enantiomer is elevated in glioblastomas and AMLs, other investigators found that other enantiomer, L2HG, is indeed elevated in another class of tumors, ccRCCs [10]. The elevated L2HG has been attributed to a loss of copies of the L2HGDH gene [11], encoding for the enzyme that metabolizes L2HG to αKG [10]. Elevated L2HG and reduced L2HGDH was observed in a number of human RCC cell lines, including A498, RXF-393 and Caki-1, and in addition was a common attribute of ccRCCs obtained from patients [10]. In order to determine whether the decreased L2HGDH contributes to tumorigenicity, A498 cells were transduced with a WT L2HGDH vector (generating A498-L2HGD). Not only were the L2HG levels reduced in A498-L2HGDH cells, but the volume of A498-L2HGDH tumors was reduced, as compared with controls [10]. L2HG levels are determined by its biosynthetic rate, as well as by its degradation. Shelar et al. [11] reported that L2HG in RCC cells is generated from αKG by MDH1 and MDH2, via promiscuous reactions.

#### **2. Epigenetic effects of L- and D-2HG**

Both L2HG and D2HG are competitive inhibitors of αKG-dependent dioxygenases. The specific αKG-dependent dioxygenase(s) which are the targets of elevated L2HG

#### *Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

in the brain of patients with L2HGA has not yet been identified. However, the most notable known targets of L2HG and D2HG in tumors are epigenetic targets, including αKG-dependent dioxygenases that regulate either DNA or histone demethylation. Included amongst these αKG-dependent dioxygenases that are inhibited by L2HG and D2HG are Jumonji domain-containing histone-lysine demethylases (Jmj-KDMs), that demethylate histones, as well as TETs, which demethylate 5-methyl-cytosine (5mC) residues in genomic DNA. Consequences of the inhibition of these dioxygenases by L2HG and D2HG include the increased methylation of histone marks, as well as an increase in 5mC residues in CpG islands [12]. While both enantiomers of 2HG inhibit Jmj-KDMs as well as TETs, overall L2HG is a more potent inhibitor than D2HG [13].

The Jmj-KDMs demethylate lysine residues on specific classes of methylated histones, their specificity being determined by specific reader domains present within each type of protein [14]. For example, JmjD2A and JmD3 remove a methyl group from the repressive histones H3K9me3, and H3K27me3, respectively, whereas JARID1A removes a methyl group from the activating histone H3K4me3 (**Table 1**) [14]. In this manner the Jmj-KDMs reverse the methylation events caused by corresponding Histone Lysine Methyltransferases (abbreviated as either KMTs or HMTs) (**Table 1**) [15].

The TETs demethylate 5mC residues in genomic DNA in a number of steps. Initially, TETs oxidize 5mC, generating 5-hydroxy-mC (5hmC), followed by further oxidation of 5hmC into 5-formylcytosine (5fC), and finally 5-carboxycytosine (5caC). Subsequently, the modified base is removed, and excision repair occurs [16]. Point mutations and deletion mutations are often observed in human cancers, particularly those affecting TET2 (as observed in AML). This latter observation is consistent with the hypothesis that the inactivation of TETs by D2HG plays a similar role in AML [16].

In their studies, Chowdhury et al. [13] found that 2HG is a weak antagonist of αKG (requiring a 100-fold molar excess of 2HG over αKG). This molar excess of 2HG can nevertheless be achieved in cells with IDH mutations. The concentration of D2HG increases to as high as 35 mM in cells with IDH mutations, exceeding the IC50 of 2HG for Jmj-KDMs [13]. In such IDH mutant cells, αKG is itself consumed, being the substrate of 2HG, further increasing the inhibition by 2HG. Although the evidence is convincing that D2HG and L2HG alter the epigenetic landscape in cells, inhibitory effects of 2HG on other classes of αKG-dependent dioxygenases, may also promote tumorigenesis. For example, L2HG inhibits Prolyl Hydroxylase 2 (PHD2), thereby preventing the hydroxylation of HIF1α, and the degradation of HIF1α by the


*Methylated Histones are listed in the top row, which have been methylated by the histone lysine methyltransferases (KMTs) in the first 2 rows listed directly below the particular KMT. In the third and fourth rows are listed the histone demethylases (in particular the Jmj-KDMs) which demethylate the histones listed in the same column as the particular Jm-KDMs.*

#### **Table 1.**

*Representative histone lysine Methyltransferases (KMTs), with associated Jmj-KDMs and histone substrates.*

proteosome [9]. Collagen hydroxylase is also inhibited by L2HG and D2HG, which perturbs basement membrane function [17]. Finally, D2HG and L2HG inhibit the repair of DNA alkylating damage (via ALK Homolog, i.e. ALKBH, enzymes), which promotes oncogenesis [17].

## **3. Effects of D-2HG on adipocyte and myocyte differentiation**

Of particular interest to this review are the L2HG mediated effects on renal differentiation, so as to increase the propensity of renal cells to become tumorigenic. However, previous studies concerning the effects of D2HG on the differentiation of other cell types will first be described, to facilitate our understanding of effects of L2HG on renal differentiation, including epigenetic changes. Both D2HG and L2HG inhibit αKG-dependent dioxygenases, although admittedly the 2 enantiomers have different binding affinities for a number of αKG-dependent dioxygenases. Nevertheless, previous studies of the effects of D2HG on differentiation in other tissues, will facilitate our understanding of how L2HG alters kidney development.

The effects of D2HG on differentiation were initially studied because gliomas with IDH mutations had a gene expression profile enriched for genes expressed in neural progenitor cells [18]. Moreover, increased levels of repressive H3K9me3 and H3K27me3 were observed in oligodendrogliomas with IDH1 mutations compared to tumors with wild type IDH1 [18]. Presumably, this was a consequence of the elevated D2HG in these tumors. Consistent with this hypothesis increased histone methylation was observed in 293 cells expressing a mutant IDH1 (or mutant IDH2) as compared to the wild type IDH allele.

In order to determine whether D2HG could block the differentiation of nontransformed cells, studies were conducted with 3T3-L1 cells which could be induced to differentiate into adipocytes using a differentiation cocktail [18]. 3T3-L1 cells transduced with an R172K mutant IDH2, overproduced D2HG, unlike cells transduced with WT IDH2 or empty vector. After induction of differentiation, the mutant IDH2 expressing 3T3-L1 cells had a markedly reduced ability to accumulate lipid droplets and were defective in the expression of transcription factors required for adipocyte differentiation, including CEBPA (CCAAT Enhancer Binding Protein α) and PPARγ (Peroxisome Proliferator-Activated Receptor γ, encoded by the *Pparg* gene). The impaired differentiation was associated with increased levels of H3K9me3 and H3K27me3. H3K9me3 in particular was located on the promoters of the *Cebpa* and *Pparg* genes. The increased H3K9me3 was attributed to the inhibition of the histone demethylase KDM4C by D2HG. Indeed, KDM4C was induced during the differentiation of 3T3L1 cells into adipocytes, and a knockdown of KDM4C with siRNA inhibited differentiation. Of particular interest in these regards KDM4C (which demethylates H3K9me3), is a member of the JHDM family of histone demethylases (i.e. JmjC domain-containing histone demethylases, or jmj-KDMs). Notably, H3K9me3 is the product of the G9a methyltransferase, which is known to produce repressive histones [14].

Subsequently, Schvartzman et al. [19] presented evidence indicating that the elevated D2HG (produced by oncogenic IDH1/2 mutations) similarly blocked the differentiation of 10T1/2 cell into myocytes by preventing H3K9 demethylation. Indeed, the HMT inhibitor UNC0638 (which blocks the H3K9 methylation by EHMT1/2) restored the ability of 10T1/2 cells expressing IDH2-R172K to form fused myotubes. Furthermore, similar results were obtained by a Clustered Regularly Interspaced

*Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

Short Palindromic Repeat (CRISPR) mediated deletion of EHMT1/2. The differentiation of 10T1/2 cells into myocytes depends upon the MyoD transcription factor. Very importantly, a Chromatin Immunoprecipitation Sequencing (ChIP-Seq) analysis indicated that the IDH2-R172K mutant did not have a global effect on chromatin accessibility in differentiating 10T1/2 cells. Instead, the IDH2-R172K mutant specifically prevented a MyoD-mediated increase in chromatin accessibility at myogenic regions. Thus, the authors conclude that the histone methylations that occur within genetic regions present within "facultative" heterochromatin are responsible for the 2HG-mediated block in differentiation, rather than random methylation events. Further studies of these 2HG-mediated blocks in differentiation, will reveal the precise nature of the 2HG-mediated genomic changes that contribute to tumorigenesis.

#### **4. Effects of L2HG on ccRCCs**

The transcriptional basis of nephron-specific gene expression patterns that emerge during nephrogenesis are incompletely understood. However, alterations in histone and DNA methylation caused by oncometabolites such as 2HG may very well upset the network of interactions established by transcription factors (TFs), so as to alter "normal" patterns of differentiated gene expression, and to cause "dedifferentiation." Nevertheless, Lindgren et al. [20] were able to identify remnants of cell type specific gene expression programs in a number of kidney cancers, and in this manner identify their lineage. Although many fundamental genetic alterations which contribute to the formation of RCCs have been identified, an understanding of the changes which occur in the specific cell subpopulation(s) that are the cells of origin of RCCs is nevertheless of importance. Lindgren et al. [20] were able to identify a gene cluster (cluster B) that was expressed in ccRCCs that corresponds with genes expressed in the renal proximal tubule (including trans-membrane transporters regulated by the Hepatocyte Nuclear Factor (HNF) TF family, most notably HNF4α which plays a role in RPT differentiation [21]). In addition, another gene cluster (cluster C) was over-expressed in ccRCCs (including genes regulated by HIF1α and expressed during hypoxia, angiogenesis as well as the epithelial-mesenchymal transition (EMT)). A number of the changes in gene cluster C are often associated the loss of VHL. In contrast, chromophobe RCCs expressed a Forkhead box protein L1 (FOXl1) gene signature. FOXI1 plays a critical role in the differentiation of intercalated cells in the connecting tubules and collecting ducts during renal development [22].

Although some remnants of normal transcription may remain, transcriptional changes that result in a loss of differentiation is a hallmark of many tumors. When considering ccRCCs in particular, evidence for changes in the activity of histone demethylases (including jmj-KDMs) has been obtained [23]. These changes alter the genetic landscape, resulting in the reduced expression of genes that encode for differentiated functions. Included amongst these genes, are genes encoding for renal transporters, proteins which maintain the polarized epithelial morphology, as well as proteins required for progression through the cell cycle, and apoptosis [24]. Gene silencing due to the hypermethylation of DNA and histones, is a strong candidate mechanism underlying the block in differentiation in ccRCCs. Of particular interest, is the role of elevated L2HG in such renal tumors, because increased L2HG is associated with increases in histone as well as DNA methylation. Indeed, Shelar et al. [11] observed increased H3K27 trimethylation in A498 RCC cells that have elevated levels of L2HG. In addition, there was a decrease in the expression of genes targeted by the

Polycomb Repressor Complex 2 (PRC2), including genes targeted by Suz12, that bear the H3K27me3 mark in human embryonic stem cells [11]. These results suggest there is an interrelationship between the changes in the gene expression profiles observed during embryonic development, and the gene expression changes caused by L2HG in renal carcinomas.

#### **5. Epigenetic and transcriptional changes during renal development**

Strikingly, during renal development, there is an interplay between the repressive effects of PRC2 proteins, and stimulatory effects of Trithorax proteins [25]. Repressive histones, including H3K9me2 and H3K27me3, are generated by PRC2 in the metanephric mesenchyme which surrounds the ureteric bud (which includes quiescent cells with low levels of the Six2 and Lhx1 transcription factors). As development proceeds, the mesenchyme condenses. The condensed mesenchyme becomes enriched with the activating histone H3K4me3, indicating it was poised for activation. Somewhat later, nascent nephrons emerge, which have high levels of H3K4me3 and low levels of repressive H3K9me3 and H3K27me3, activating such genes as Lhx1. After nascent nephron cells emerge, Notch2 appears, an important transcription factor in RPT development.

Of particular interest, are the developmental events that occur during segmentation of the nephron, because during this period Renal Proximal Tubules (RPTs) appear, RPTs being the cell of origin of ccRCCs and papillary RCCs [26, 27]. HNF TFs emerge during this developmental stage, including HNF4α, which facilitates the formation of H3K4me2 to maintain active chromatin during this developmental period [28]. HNF1α is similarly active during this developmental period, recruiting KDM6A (i.e. UTX), resulting in the demethylation of H3K27me3, thereby relieving polycomb repression [29]. In contrast, loss of HNF1α function (which has been observed in ccRCCs) [30] results in polycomb repression. A similar loss of HNF1α function has been reported in other cancers, including non-small cell lung cancers [31], oral squamous carcinomas [32], and pancreatic cancers [29]. In the case of pancreatic cancers, re-expression of HNF1α reactivated differentiated acinar cell programs, and in this manner suppressed the emergence of pancreatic cancers [29]. Presumably, there is a potential to similarly override 2HG mediated blocks in differentiation programs in other cancers, including RCCs, by re-establishing required regulation by HNFs. For this reason, an evaluation of the effects of elevated 2HG on the differentiation of renal cells is important, including effects of 2HG on such transcription factors as HNF1α.

### **6. Effects of L2HG on renal differentiation and development** *in vitro*

L2HG mediated effects on the differentiation of RPT cells have been examined, utilizing a well characterized primary culture system of normal renal cells. Primary cultures of kidney epithelial cells derived from purified rabbit RPTs were grown in serum free medium, supplemented with 5 μg/ml human insulin, 5 μg/ml human transferrin and 5 x 10<sup>−</sup><sup>8</sup> M hydrocortisone. Epithelial monolayers formed with a polarized morphology, and membrane transport systems distinctive of the RPT, including an apical Na<sup>+</sup> /glucose cotransport system (SGLT2), a Na+ /phosphate cotransport system (NPT2a), a Na+ /H+ antiport system (NHE3), and a basolateral p-Amino Hippurate (pAH) transport system (i.e. OAT1) (**Figure 4**) [33, 34]. The

*Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

#### **Figure 4.**

*Polarized morphology and transporters of primary proximal tubule (RPT) cells. A primary RPT cells form multicellular domes in culture. Groups of cells are elevated from the dish due to the accumulation of fluids between the monolayer and the dish, resulting from the transepithelial transport of fluids, and the resulting hydrostatic pressure. Two micrograoph fields are illustrated at t 100X. B. Transmission electron micrograph (TEM) of primary RPT cells. The TEM (illustrated at 2000X) shows the polarized morophology and interconnection of cells by tight junctions) from Taub et al. [34], reproduced with from Biotechniques, as agreed by future science, LTD) C. polarized transporters expressed in primary RPT cells as further defined in* **Table 2***.*

primary RPT cells also possess a parathyroid hormone (PTH) sensitive adenylate cyclase, Angiotensin II (Ang II) receptors [35], dopamine receptors, as well as α and β adrenergic receptors [36], which are critical for the regulation of ion transporters (**Figure 4**). Finally, the cultures retain normal metabolism, including gluconeogenesis, glutathione metabolism and other drug metabolic capabilities distinctive of the RPT (**Table 2**).

The effects of L2HG on primary RPT cells were examined both in 3-Dimensional (3D) matrigel cultures, as well as monolayer cultures. Such 3D culture systems are the method of choice for examining malignant cells *ex vivo* [37], including the effects of oncometabolites on differentiation (which relate to tumor progression). Matrigel, a reconstituted basement membrane from the Engelbreth-Holm-Swarm (EHS) tumor, is particularly important to use in 3D studies. Matrigel is a well-characterized 3D system used to study differentiation in both normal and malignant tissues [38]. Matrigel was initially employed to study baby mouse kidney epithelial cells *ex vivo*. Tubulogenesis was observed, provided that either Epidermal Growth Factor (EGF)


*Primary rabbit RPT cells are cultured in serum free medium supplemented with insulin, transferrin and hydrocortisone. The method for culturing the primary RPT cells and their extensive characterization has been described by Taub [33].*

#### **Table 2.**

*Characteristics of primary RPT cells in culture.*

or Transforming Growth Factor α (TGFα) were added to the culture medium [39]. Electron micrographs indicated that the baby mouse kidney tubules resembled collecting ducts [39].

Subsequently, a 3D system of renal proximal tubulogenesis was developed, using primary rabbit RPT cells [40]. The primary RPT cells form tubules in matrigel (**Figure 5**). The tubules possess transepithelial transport capacity, as indicated by their ability to secrete lucifer yellow (an organic anion) into the luminal space of the tubules [41]. This was indicated by the green fluorescence observed in the lumen of lucifer yellow treated cultures in matrigel. Tubulogenesis by RPTs was stimulated by EGF (similar to baby mouse kidney cells), as well as Hepatocyte Growth Factor (HGF). Because the RPT is the cell of origin of ccRCCs, this primary RPT cell culture system is an appropriate model system to examine effects of oncometabolite L2HG on RPT differentiation.

As described above, the elevated L2HG detected in RCCs, has been attributed to decreased expression of L2HGDH, the enzyme that breaks down L2HG to αKG. The results of a microarray study indicate that the expression of genes targeted by the Polycomb protein Suz-12 was reduced in L2HGDH deficient ccRCC cells, similar to the metanephric mesenchyme [42]. Thus, the hypothesis was examined, that reduced expression of L2HGDH blocks differentiation of renal cells.

In order to examine this hypothesis initially, primary RPT cell cultures were transduced with lentiviral particles containing a vector (pLKO-TRC) encoding either L2HGDH shRNA or control shRNA [43]. The effect on tubulogenesis in matrigel was examined. **Figure 6** shows the lack of tubules in cultures treated with L2HGDH shRNA, under conditions where L2HGDH mRNA was reduced by 80%. Tubulogenesis was similarly inhibited in primary RPT cells using L2HGDH siRNA, which similarly reduced L2HGDH mRNA by 80%. The effect of L2HGDH siRNA on intracellular L2HG and D2HG levels was examined by Gas Chromatography–Mass Spectrometry (GC–MS) analysis. The L2HG level increased more than 4-fold in

*Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

#### **Figure 5.**

*Tubule formation by primary PRT cells in Matrigel. A. Traverse section of a tubule. Lucifer yellow accumulation (green) in the lumen of a tubule in matrigel, with DIl (yellow), a membrane stain. B. Longitudinal section of another tubule, C. section of developing tubule. D Cross-section of a tubule examined by TEM showing a lumen (L), and a nucleus (N). Bar is 50 μM in a, B and C. Bar in D is 10 μm. From Han et al. [41] with permission from the Company of Biologists.*

primary RPTs treated with L2HGDH siRNA, L2HG becoming the predominant enantiomer.

Shelar et al. [11] observed that L2HG is primarily generated from Glutamine (Gln) in human ccRCCs, initially via the breakdown of Gln to Glu by glutaminase (as illustrated in **Figure 3**). Thus, the inhibition of Gln metabolism to Glu by glutaminase would be expected to reduce the L2HG level, and in this manner overcome the effect of an L2HGDH Knockdown (KD) on intracellular L2HG levels. In order to examine this hypothesis, the ability of primary RPT cells with an L2HGDH KD to form tubules in the presence of glutaminase inhibitor CB-839 was examined. The results (illustrated in **Figure 7**) indicated that CB-839 relieved the inhibition of tubulogenesis caused by an L2HGDH KD, presumably by reducing L2HG levels (a consequence of inhibiting the metabolism of Gln to Glu, and ultimately to L2HG). The involvement of the elevated L2HG in mediating the inhibition of tubulogenesis in cultures with an L2HGDH KD was further substantiated by the observed inhibitory effect of cell permeable L2HG octyl ester on tubulogenesis [43].

The block in tubulogenesis in primary RPT cells with an L2HGDH KD may very well be associated with a generalized loss of differentiated function. Consistent with this hypothesis, the expression of differentiated transporters was reduced in the primary RPT cells with an L2HGDH KD. This is exemplified by the reduction in the mRNA levels for the apical Na+ /Phosphate cotransporter (NPT2a), the Na+ /glucose cotransporter (SGLT2), and Aquaporin 1 (AQP1), as well as the basolateral pAH transporter (OAT1) in monolayers with an L2HGDH KD.

**Figure 6.**

*Effect of L2HGDH shRNA on Tubulogenesis, primary RPT cells cultures were transduced with lentivirus containing either L2HGDH shRNA or control TRC shRNA vectors. The cultures put into matrigel. Subsequently, tubules photographed (A, B, C, D) at 100X. Tubules were quantitated and L2HGDH mRNA level were determined. From Taub et al. [43] with permission of Frontiers.*

In matrigel cultures, the level of expression of NPT2a and SGLT2 increased relative to plastic in control cultures, consistent with the hypothesis that the overall state of differentiation was enhanced in matrigel. Nevertheless, the expression of these 2 transporters continued to be substantially reduced in cultures with an L2HGDH KD. In contrast, AQP1 expression increased in matrigel cultures with an L2HGDH KD. This observation can be explained by differences in transcriptional regulation between NPT2a and SGLT2 as compared with AQP1. Both NPT2a and SGLT2 regulation depends upon HNF1α which is down regulated in primary RPT cells with an

*Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

#### **Figure 7.**

*Effect of Glutaminase inhibitor CB-839 on tubulogenesis. A. Primary RPT cells were either a. transduced with lentivirus containing either L2HGDH shRNA or control TRC shRNA vectors, or B. transfected with L2HGDH shRNA or negative control siRNA. The cultures were passaged into matrigel, and treated with either with 1 μM CB-839 or untreated. Subsequently, the frequency of tubule formation was determined [41]. C. the effect of 1 μM CB-839 on the level of glutamine and glutamate was determined [41]. D. Model for the effect of CB-839 on L2HGDH levels. Values are averages (+/−) SEMs of triplicate determinations. With permission from Frontiers.*

L2HGDH KD, unlike AQP1, whose transcription is controlled by Tonicity Enhancer Binding Protein (TonEBP) and HIF1α. As described above, HNF1α is involved in the appearance of RPTs during kidney development. Thus, it will be of interest to determine whether during tubulogenesis in vitro, HNF1A recruits KDM6A, which relieves polycomb repression during this stage of kidney development by demethylating H3K27me3.

HNF1α has also been reported to play a role in maintaining the polarized morphology of epithelial cells. Indeed, inhibition of HNF1α gene expression with siRNA triggered the Epithelial to Mesenchymal Transition (EMT) in the liver cancer cell lines HEPG2 and HEP1B [44]. The expression of mRNAs encoding for such proteins as E-cadherin (CDH1) and plasminogen activator (PLAU) was similarly reduced in primary RPTs with an L2HGDH KD, as well as cell migration. Further studies are necessary to elucidate whether the downregulation of HNF1α under these conditions is responsible for these changes.

The downregulation of the expression of the differentiated transporters, and HNF1α in primary RPT cells with an L2HGDH KD is presumably a consequence of the inhibition of αKG dependent dioxygenases. Included amongst these αKG dependent dioxygenases are Jmj-KDM histone demethylases, as well as TET 5mC demethylases, which are inhibited not only in tumor cells with IDH1 and IDH2 mutations [45], but also in ccRCCs with reduced L2HGDH levels [11]. Consistent with this hypothesis, 5hmC blots of genomic DNA derived from primary RPT cells with an L2HGDH KD indicated that the level of 5hmC was reduced in primary RPT cells with an L2HGDH KD (vs. controls) [43]. In addition, Western blots indicated increases in the levels of a number of different classes of methylated histones in primary RPT cells with an L2HGD KD [43]. Not only was there an increase in the level of the activating histone, H3K4me3, but also an increase in the level of the repressive histone H3K27me3. Shelar et al. [11] similarly observed an increased level of H3K27me3 in A498 ccRCC cells as compared to A498 ccRCC cells which express exogenous L2HGDH. H3K27me3 is generated by EZH2, a component of the repressive PRC2 complex expressed in the primitive mesenchyme.

However, as described above, Schvartzman et al. [19] reported that the inhibitory effect of D2HG on the MyoD-mediated differentiation of myocytes was the specific consequence of increased H3K9 methylation generated by EHMT1/2. Schvartzman et al. [19] found that 5mC DNA hypermethylation could be excluded from in the differentiation block in 10 T1/2 cells, because the DNA methyltransferase inhibitor 5-azacytidine was unable to rescue the differentiation block caused by elevated D-2HG. The hypothesis that the inhibition of renal proximal tubulogenesis is similarly the specific consequence of increased H3K9 methylation cannot be excluded, in the absence of results with H3K9me3.

However, L2HG does not necessarily act on renal differentiation via the same mechanism as observed with D2HG in myocytes. Indeed, L2HG has a higher affinity than D2HG for a number of αKG-dependent dioxygenases, including TET 5hmC hydroxylases [45]. Furthermore, L2HG (unlike D2HG) is an effective inhibitor of such αKG dioxygenases as Prolyl Hydroxylase 2 (PDH2) (which promotes the degradation of HIF1α during normoxia) [17]. The effects of L2HG on other aspects of basic metabolism have not been extensively investigated. Thus, it is unclear whether L2HG, like D2HG causes a reduction in Nicotine Adenine Dinucleotide (NAD+ ) levels, associated with a reduction in the level of Naprt (Nicotinate Phosphoribosyltransferase), a rate limiting enzyme in the NAD+ salvage pathway that replenishes intracellular NAD+ levels [46]. In any case, the effects of L2HG on differentiation of RPT cells ultimately depend upon the unique sets of epigenetic mechanisms that regulate kidney development, unlike other tissues.

Nevertheless, the L2HG-mediated inhibition of kidney proximal tubulogenesis very likely results at least in part from the inhibition of Jmj-KDMs. Previous studies indicate that histone demethylases, as well as histone methyltransferases play an important role in kidney development. It is well-known, as stated above, that during kidney development there is an interplay between events mediated by repressive PRC2 and stimulatory Trithorax complexes [25]. Different classes of methylated histones are produced by these 2 sets of complexes. While the histone methyltransferase Ash21 (associated with Trithorax complexes) produces activating H3K4me histones, Ezh1, Ezh2 and Suz12 (associated with PRC2) produce repressive H3K9me2/3 and H3K27me3. During the segmentation of the nephron, H3K4me histone levels increase as proximal tubules appear, while H3K9me2/3 and H3K27me3 levels decline. Remarkably, an increase in the level of both H3K9me3 and H3K27me3 is associated with the block in the ability of 10T1/2 cells with a mutant IDH2-R172K to differentiate into myocytes.

#### *Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

A decline in H3K9me2/3 and H3K27me3 during kidney development depends not only upon the activity of the pertinent histone methyltransferases, but also upon the continued activity of pertinent JmjC domain containing histone demethylases. While KDM4A, B, C and D demethylate H3K9me3, the transcriptionally activating KDM7A demethylates H3K9me1/2. Similarly, the demethylation H3K27me3 involves KDM4D, as well as KDM7A. However, given that these KDMs are αKG dependent dioxygenases, they are subject to inhibition by elevated D- and L-2HG. In the case of 10T1/2 cells expressing a mutant IDH enzyme, inhibition of KDM4C (which normally demethylates H3K9me3) was proposed to be responsible for their inability to differentiate into myocytes. Consistent with this hypothesis, these investigators observed that the EHMT1/2 inhibitor UNC0638 restored the ability of 10T1/2 cells expressing IDH2- R172K to form fused myotubes. Similarly, primary RPT cells with an L2HGDH KD can overcome the block in tubulogenesis in the presence of UNC0638 (lowering levels of H3K9me3), although the cultures can also overcome the block in tubulogenesis in the presence of GSK343 (Taub, unpublished).

Shelar et al. [11] presented evidence that increased levels of methylated histones as well as DNA in ccRCCs with elevated L-2HG contribute to tumorigenesis. However, Shelar et al. [11] also provided evidence that the increased levels histone H3K27me3 in ccRCCS with elevated L-2HG are responsible for altering the genetic program of these tumor cells, and, as a consequence their state of differentiation. Indeed, the studies of Taub et al. [43] indicate that increased L2HG establishes a block in RPT differentiation, with associated epigenetic changes. Thus, it is of importance to consider the development of new avenues of epigenetic therapy for ccRCCs.

### **7. Conclusion**

Recent clinical trials of epigenetic-based therapies in RCCs have examined on the use of DNA Methyltransferase (DNMT) inhibitors such as decitabine and azacytidine [47]. DNA methylation of tumor suppressor genes in RCCs is thought to contribute tumorigenesis, and the hypermethylation of an EGF response element (recognized by Krueppel-like factor 5 (KLF5)) is associated with poor prognosis of RCC patients [47]. *In vitro* studies concerning the effects azacytidine with RCC cell lines have also shown promise [48], and combination therapies are in progress [47]. This adds to presently prescribed medications including VEGF inhibitors (e.g. sunitinib) mTOR inhibitors (e.g. everolimis), and HIF2α inhibitors (e.g. belzutifan) [49]. However metastatic ccRCC continues to carry a 5 year survival rate of 13% [49]. Thus, new treatment options are critical.

Epigenetic therapy is pertinent not only for ccRCCs with elevated L2HG, but also for ccRCCs with other genetic alterations (which may occur in addition to reduced L2HGH levels). Recent genome-wide sequencing studies have indicated that a number of epigenetic modifiers and chromatin remodelers are frequently altered in ccRCCs including PBRM1, SETD1, KDM5C, KDM6A, and BAP1. Notably, ccRCCs often have a 50 kb deletion on chromosome 3p where VHL, PBRM1, BAP1 and SETD2 are located, which has opened up the door to individualized epigenetic therapy. SETD1 in particular is an H3K36me3 histone methyltransferase that plays an important role in DNA repair and genomic stability [50]. While mutations in SETD1 result in reduced histone methylation, mutations in KDM5C and KDM6A often result in increased methylation of H3K4me3 and H3K27me3, respectively. The genes encoding for these 2 histone demethylases are located on the X chromosome, and thus, when altered, can a permit an escape from X-inactivation of tumor suppressor genes. Unlike patients with SETD mutations, RCC patients with KDM5C and KDM6A mutations would be expected to benefit from inhibitors of specific histone methyltransferases.

In addition to epigenetic therapies, therapies developed against cancer stem cells may also prove protective against ccRCCS with elevated L-2HG. Several theories have been proposed regarding the origin of cancer stem cells, including that, a) cancer stem cells arise from normal progenitor cells which become tumorigenic due to undefined mutation(s), and b) that cancer stem cells arise from normal somatic cells that acquire stem-like properties through similarly undefined genetic mutations [51]. Thus, the block in renal proximal differentiation caused by elevated L-2HG is a mechanism that potentially results in the development of renal cancer stem cells. Recently, Fendler et al. [52] isolated ccRCC cancer stem cells which depend upon signals sent through the Wingless-related integration site (WNT) and NOTCH networks, which direct the formation of RPTs during renal development. Further studies are needed to assess whether the WNT and NOTCH pathways are active in ccRCCs with elevated L2HG, and whether the targeting of such pathways can alleviate the differentiation block caused by increased L2HG in normal RPT cells.

## **Acknowledgements**

The Optical Imaging and Analysis Facility, The School of Dental Medicine, University at Buffalo, and The Confocal Microscopy and Flow Cytometry Center of the Jacobs School of Medicine and Biomedical Sciences of The University at Buffalo were instrumental in acquiring the results descried in the article. The research was supported in part by RO1CA200653.

## **Author details**

Mary Taub Biochemistry Department, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, USA

\*Address all correspondence to: biochtau@buffalo.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Oncometabolite L-2-Hydroxyglutarate Promotes Oncogenesis of Renal Cell Carcinomas… DOI: http://dx.doi.org/10.5772/intechopen.108992*

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## **Chapter 3**

## Clear Cell Renal Cancer, a Tumour with Neuroendocrine Features Originating from the Erythropoietin-Producing Cell

*Helge Waldum and Patricia Mjønes*

## **Abstract**

The dominating type of kidney cancer is the clear cell renal cell cancer (ccRCC), hitherto been thought to develop from proximal tubule cells. However, the ability of tubule cells to proliferate is at best controversial. ccRCCs show many peculiarities like erythrocytosis due to erythropoietin overproduction and a combination of early metastases and sometimes apparent dormancy and late recurrence, features in common with neuroendocrine tumours (NETs). We have shown that most ccRCCs express erythropoietin and the neuroendocrine marker neuron-specific enolase, and other neuroendocrine markers in a percentage of the cancers. Missense mutation in von Hippel–Lindau (VHL) factor is rather specific for ccRCC found in familial and sporadic forms. The function of VHL factor is together with other proteins to destroy hypoxia-inducible factors (HIFs), central in adaptation to hypoxia. Lack of functioning VHL factor results in continuous overstimulation of the erythropoietin-producing cell to release erythropoietin and parallelly to proliferate, and in long-term mutations and malignant transformation. Thus, ccRCC occurs about 30 years later in sporadic cases compared with familial von Hippel–Lindau syndrome, reflecting the time necessary for two versus one genetic change. Embryologically, there are many arguments favouring neural crest origin of the erythropoietin-producing cell.

**Keywords:** classification of kidney cancer, clear cell renal cell cancer, erythropoietin, erythropoietin-producing cell, neural crest, von Hippel–Lindau syndrome

## **1. Introduction**

The kidney is not among the most common locations for cancer development, but kidney cancers often affect middle-aged people, and the mortality is high. Clear cell renal cancer cell (ccRCC) (also called conventional RCC (cRCC)) makes up about 80% of renal malignancies [1] and is accordingly the most important renal cancer. ccRCC may be accompanied by erythrocytosis which has been presumed to be due to production of erythropoietin (EPO) by the erythropoietin-producing cell (EPC) localised in the kidney. Some years ago, we showed that virtually all ccRCC cancer

tumour cells expressed erythropoietin as well as the neuroendocrine marker neuronspecific enolase [2], which may suggest that the cell of origin of ccRCC is the EPC [3]. The present review is a follow-up further discussing the cell of origin of ccRCC.

#### **2. Kidney cancers**

Kidney cancers are classified according to presumed cell of origin with renal cell carcinomas making up about 90% [4]. Renal cell cancers consist of subgroups based on histological classification into ccRCC, papillary and chromophobe subgroups. ccRCC and papillary renal cell cancers (pRCCs) have been presumed to originate from proximal tubular cells. However, most cells in the adult kidney do not have the ability to divide and thus replace damaged or dead specialised cells [5]. Therefore, replacement of damaged nephrons does not occur after birth [6]. Generally, it may be noted that it seems strange that nephron cells with absent or at best low reproductive capacity should be the main origin of tumours. Based on gene expressions with similarities between proximal tubular cells and ccRCCs [7, 8], tubular cells have been thought to develop into ccRCC. However, discrepant expressions between normal proximal tubule cells and tumours presumed to originate from proximal tubule cells like ccRCC and pRCCs have been described [9]. In a review in 2012, it was written that the cells of origin RCCs "are far from established and only inferred by accumulated weight of marker similarities" [10]. Similarly, the degree of tubule regeneration and which cell type contributing to this process were discussed in a review in 2016 [11]. Recently, a novel stem cell subtype analysis for ccRCC based on stem cell markers was reported, but apparently not compared to any mature cells of the nephron [12]. It may, therefore, be concluded that there are uncertainties regarding the cell of origin of ccRCCs. Considering the classification of kidney tumours in general, the continuous changes and additions of new types [13, 14] indicate a weakness in the system and may suggest that the classifications are not rooted in biology. Our finding of erythropoietin and neuron-specific enolase expression in virtually all ccRCCs may indicate that the EPC is the cell of origin of ccRCC [2, 3].

#### **3. Erythropoietin (EPO) and the erythropoietin-producing cell (EPC)**

In the late part of nineteenth century, French scientists described the association between atmospheric pressure and the concentration of red blood cells [15]. Thus, the concentration of red blood cells increased in members of an expedition to the Andes mountains [16]. A factor in serum was suspected to mediate this effect, and this was shown to be true when serum from anaemic rabbits had erythropoietic activity in normal rabbits [17]. Finnish authors named the postulated substance erythropoietin [18], and the dominating role of the kidneys in the production of EPO was shown by reduced stimulation of erythropoiesis in nephrectomised animals [19]. However, some EPO production also occurs in the liver [15]. Subsequently, EPO was identified as a glycohormone [20]. It stimulates erythropoiesis by interaction with a receptor (REPO) localised on progenitor cells in the red cell line like erythroblasts. The EPC in the kidney was long disputed. However, it seems now that peritubular interstitial cells are established as the EPC [21]. Interestingly, these cells had a neuron-like morphology and expressed neuron genes [21]. In the liver, EPO production was also found in

*Clear Cell Renal Cancer, a Tumour with Neuroendocrine Features Originating… DOI: http://dx.doi.org/10.5772/intechopen.107051*

#### **Figure 1.**

*Clear cell renal cell carcinoma examined by haematoxylin and eosin (a), by immunohistochemistry for erythropoietin (b) and neuron-specific enolase (NSE)(c). From (2) APMIS 2017; 125: 213–222. The figure is reused under the terms of the creative commons public domains declaration and with permission from the publisher.*

cells surrounding the central vein in the liver [21], possibly stellate cells which have been shown to express EPO [22]. EPO has also been described in the brain [23].

The main stimulatory mechanism for EPO production and release is by the hypoxia-inducible factors (HIFs). There exist three O2 sensing HIF-α types (HIF-1α, HIF2α, and HIF-3α). HIF-1α and also HIF-2α affect many other processes than stimulation of EPO release including angiogenesis and other functions related to adaptation to hypoxia [15, 24]. HIF-2 seems to be the main regulator of EPO [25]. HIF-1α is ubiquitously expressed, while HIF-2α was initially reported in endothelial cells but has later been shown to be expressed in many other cell types [26].

EPO production in renal cancers, then named hypernephromas [27], was suspected based on erythrocytosis in some of the patients. A case report in 1977 described EPO production in a patient with ccRCC based on a biological mouse assay [28], and there is a report describing EPO production in a cell culture from a renal cell carcinoma [29]. EPO gene expression in ccRCC was reported to be much more common than the occurrence of erythrocytosis [30]. There are also studies evaluating the prognostic significance of EPO expression in renal cell cancers where one study did not find any effect [31], and the other reported reduced survival [32]. We cannot find that anybody had reflected on the EPC as the cell of origin in ccRCC before our paper in 2017 [2], where we found that most ccRCC expressed EPO in contrast to the other renal cell carcinomas which all were negative (**Figure 1**).

Before discussing cell of origin further, we will focus on clinical aspects of ccRCC, which also give strong indications of the central role of EPC in ccRCC carcinogenesis.

#### **4. Clinical aspects of ccRCC**

In the past, the classic symptom triad of RCC was haematuria, pain in the kidney area and a palpable tumour. However, nearly half of the patients do not have any symptoms suggesting renal illness [33], and in about half of the cases renal cancers are detected by ultrasonography or other imaging modalities done due to vague symptoms. Paraneoplastic syndromes are also an initial gateway to correct diagnosis. Among these syndromes, hypercalcemia, hypertension, polycythaemia, and Stauffer's syndrome (non-metastatic hepatic dysfunction [34]) are the most prevalent [35]. Hypercalcemia is due to parathyroid-related hormone (PTHrP), polycythaemia due to EPO, hypertension possibly due to renin, whereas the mechanism for Stauffer's

syndrome is unknown. PTHrP elevation has been attributed to vascular endothelial growth factor (VEGF) expression in ccRCC [36].

Elevated erythrocyte sedimentation rate has for long been recognised to be a feature of renal cancer, formerly called hypernephroma, now ccRCC. Rising erythrocyte sedimentation may be an early marker for renal cell cancer [37] and also an independent prognostic factor [38]. C-reactive protein is also a predictive factor for metastasis in patients after potentially curative surgery [39].

Macroscopically, ccRCCs often are yellowish and small tumours often with an apparent capsule. ccRCCs metastasize at an early stage, and metastases are often present at diagnosis [40]. RCCs may metastasize to uncommon places as a finger [41], the pituitary gland [42], and skeletal muscle [43]. Moreover, first metastasis may manifest itself many years after apparent curative surgery as shown in a case report where a brain metastasis developed 15 years after surgery [44]. Late metastasis has been explained by early dissemination and tumour cell dormancy [45, 46]. Another possibility may be that the cell of origin due to inherent qualities like low expression of factors contributing to cell adhesion may cause metastasis spread at an early phase of malignant development when the proliferation still is rather low. Such a phenomenon may explain the so-called dormancy [47]. As we see it, the possibility that some cancer cells go to sleep and awake after many years does not seem very plausible, although until now it has been the prevailing theory [45]. Production of substances affecting the vascular bed-like dilatation reducing blood flow rate as well as increasing the vascular permeability could also be involved. We have previously described that the enterochromaffin-like (ECL) cell in the oxyntic mucosa of the stomach lacks Ecadherin [48] and also releases histamine which has profound effect on the vasculature [49]. The ECL cell has a very slow proliferation, which made some conclude that this cell did not have the ability to divide [50] which is not correct [51]. There may be an apparent mismatch between the fact that tumours prone to early metastasis are among those with tendency to occurrence of late metastasis many years after other manifestations of malignancy. This is typically found in tumours developed from cells with the capacity to metastasize at an early phase of the malignant process when the proliferation is still slow. Neuroendocrine tumours (NETs) [52–55] and melanomas [56, 57] developed from melanocytes which also are of neural crest origin [58] like the ccRCCs (see later) are among the cancers where late metastasis occur [44]. The dormancy accordingly most probably reflects tumours originating from cells with low proliferation but the ability to metastasize after only minor genetic changes.

Another peculiarity with ccRCCs is the anecdotal spontaneous regression of metastases after surgical removal of the primary tumour, a phenomenon perhaps related to the abscopal phenomenon (regress of tumour metastases outside the area of irradiation of other metastases) [59, 60].

#### **5. Aetiology/pathogenesis**

ccRCC is the dominating kidney cancer and is also the most aggressive form. The incidence of ccRCC is nearly the double in men compared with women [4, 61]. An explanation of this sex difference in occurrence is not known. Otherwise, cigarette smoking, obesity, and hypertension have been all associated with a slight increased risk of ccRCC [62], but the exact mechanisms have not been clarified. In Japan, heavy smoking was found to increase the risk [63], but the mechanism for the slight carcinogenic effect on the kidneys have not been clarified. In inhalation studies on rats, we *Clear Cell Renal Cancer, a Tumour with Neuroendocrine Features Originating… DOI: http://dx.doi.org/10.5772/intechopen.107051*

examined the effect of nicotine added to the air in concentrations giving nicotine in blood exceeding that found in heavy smokers during greater part of 24 h for 24 months [64], or CO in a concentration giving about 15% carboxy-haemoglobin for most of 24 h for 18 months [65]. Although none of these studies were primarily done to explore possible mechanisms for tobacco smoking kidney carcinogenesis, the kidneys were examined macroscopically in both studies without finding any tumours. Thus, the mechanism for the effect of tobacco smoking on renal carcinogenesis is still unknown.

Obesity is an accepted and established role as a risk factor for ccRCC [62], but it is also associated with other types of cancers in other organs [66]. There exists a socalled obesity paradox between ccRCC and obesity, since obesity increases the occurrence and at the same time seems to improve the prognosis of the cancer [67]. Anyhow, a plausible mechanism for the carcinogenic effect of obesity is still not found. Likewise, on the background of the important role by the kidneys in regulation of blood pressure, it is not surprising that hypertension may be elevated in ccRCCs. However, again the mechanism for such a connection is not yet elucidated, although a role of the renin-angiotensin system has been examined.

On the other hand, a central role of the von Hippel–Lindau (VHL) tumour suppressor in pathogenesis of ccRCC is well established causing virtually all familial ccRCCs [68], but also the sporadic ones [69]. The average age at diagnosis of ccRCC as part of VHL syndrome is 37 years compared with 61 years of the sporadic form [40, 70]. Inactivation of VHL gene is only found in ccRCC of kidney cancers [40], and loss of functioning of both alleles of VHL gene may be common to all ccRCCs [40, 71]. VHL gene product (pVHL) binds to elongins making a complex which binds to the hypoxic-inducible factors, HIF-1 and HIF-2, which targets them to ubiquitinmediated proteolysis [72]. Pathological elongins can also be a factor contributing to lack of proteolysis of HIFs. HIFs are released during hypoxia, and HIF-2 is being the main stimulator of erythropoietin release [25]. Our experience is that it is a close correlation between regulation of function and growth [73], which in this case will indicate that HIF-2 will not only stimulate erythropoietin release but also proliferation of EPC. Lack of proteolysis of HIFs will accordingly lead to chronic overstimulation of proliferation explaining the carcinogenic effect.

#### **6. Neural crest origin of erythropoietin-producing cell**

Most ccRCCs express erythropoietin as well as neuron-specific enolase [2]. Based on our study, it seems that both clinically (overproduction of EPO in a proportion of the patients), the central and universal role of HIF in the carcinogenesis of familial as well as sporadic ccRCCs and the universal and specific expression of EPO in most ccRCCs, these tumours are of EPC origin. A case report from 1989 also describes EPC expressing cell as the cell of origin of a ccRCC [74]. Moreover, clinically ccRCCs have as outlined above, many similarities to neuroendocrine tumours in general. Furthermore, polycythaemia due to EPO production has been reported together with somatostatinoma, paraganglioma, and phaeochromocytoma [75, 76]. We found not only EPO but also NSE expression in virtually all ccRCCs, but both markers, were mostly negative in the other types of renal cell carcinomas [2]. NSE has had a poor reputation concerning specificity, but when we did a separate study on NSE specificity comparing NSE with many other neuroendocrine markers and applying histochemistry with the highest sensitivity available, we found that NSE was expressed in

all tumours which expressed another neuroendocrine marker [77]. Thus, NSE has unjustly been thought to be nonspecific due to its high sensitivity. Moreover, we detected synaptophysin expression in 6% and CD56, both neuroendocrine markers, in some ccRCCs [2]. It has to be underscored that during the process of malignant transformation, expression of markers from the cell of origin is gradually lost. Therefore, even expression in only a few percent of the tumour cells is of importance.

Interestingly, EPO production in neural and neural crest cells occurs in foetal life [78, 79]. In fact, EPO has been reported to play a role in the brain development [80]. Neural crest-derived cells have typically multipotential properties and play probably a very important role in carcinogenesis not only in the kidney but also for melanomas [81].

## **7. Conclusion**

Evidence suggests that ccRCC is derived from the EPC, which upon hyperstimulation by HIF not only increases its EPO production but also is stimulated to proliferate. Genetic changes in VHL, either familial or sporadic, leading to loss of proteolysis result in increased concentrations of HIF. The EPC expresses markers compatible with neuroendocrine origin. A change in the nomenclature of ccRCC should be considered.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Helge Waldum<sup>1</sup> \* and Patricia Mjønes1,2

1 Faculty of Medicine and Health Sciences, Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway

2 Department of Pathology, St. Olav's Hospital – Trondheim University Hospital, Trondheim, Norway

\*Address all correspondence to: helge.waldum@ntnu.no

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Clear Cell Renal Cancer, a Tumour with Neuroendocrine Features Originating… DOI: http://dx.doi.org/10.5772/intechopen.107051*

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Section 2
