**Cancer Stem Cells in Squamous Cell Carcinoma**

212 Squamous Cell Carcinoma

[83] Bektas-Kayhan K, Unur M, Boy-Metin Z, Cakmakoglu B. MCP-1 and CCR2 gene

[84] Amann B, Perabo FG, Wirger A, Hugenschmidt H, Schultze- Seemann W. Urinary

[85] Riethdorf L, Riethdorf S, Gutzlaff K, Prall F, Loning T. Differential expression of the

[86] Negus RP, Stamp GW, Relf MG et al. The detection and localization of monocyte

[87] Arenberg DA, Keane MP, DiGiovine B et al. Macrophage infiltration in human non-

[88] Ueno T, Toi M, Saji H et al. Significance of macrophage chemoattractant protein-1 in

[89] Soria G and Ben-Baruch A. The inflammatory chemokins CCL2 and CCL5 in breast

[90] Conti I, Rollins BJ. CCL2 (monocyte chemoattractant protein-1) and cancer. Semin

[91] Rovin BH, Lu L, Saxena R. A novel polymorphism in the *MCP-1* gene regulatory region

[92] Yeh CB, Tsai HT, Chen YC et al. Genetic polymorphism of CCR2-64I increased the susceptibility of hepatocellular carcinoma. J Surg Oncol 2010; 102: 264-70. [93] Ghilardi G, Biondi ML, Torre A, Battaglioli L, Scorza R. Breast cancer progression and

[94] Tse K, Tsang N, Chen K et al. MCP-1 promoter polymorphism at -2518 is associated

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[96] Vázquez-Lavista LG, Lima G, Gabilondo F and Llorente L Genetic association of

[98] Yang L, Shi GL, Song CX, XU SF. Relationship between genetic polymorphism of MCP-

[99] Nahon P, Sutton A, Rufat P et al. Chemokine system polymorphism, survival and

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levels of monocyte chemoattractant protein-1 correlate with tumour stage and

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monocyte chemoattractant protein 1 (MCP-1)-2518 polymorphism in Mexican patients with transitional cell carcinoma of the bladder. Urology 2009; 74: 414-8. [97] Attar R, Agachan B, Kuran SB et al. Association of *CCL2* and *CCR2* gene variants with

1 and non-small-cell lung cancer in Han nationality of North China. Genetics and

hepatocellular carcinoma occurrence in patients with hepatitis C virus-related

**13** 

*China* 

**Molecular Mechanisms Involving Therapeutic** 

**Cell Carcinoma (HNSCC) – Roles of Hypoxic** 

Locally advanced diseases accounting for most HNSCC have a poor prognosis. The main reason for this is that corresponding symptoms of HNSCC are not always obvious or ignored by patients at early stage, which is mostly reflected by the fact that more than 2/3 HNSCC patients present with stage III/IV disease (AL-Sarraf, 1987; Argiris, 2008). Patients characterized with advanced HNSCC are subjected to worse prognosis than those with confined disease, exhibiting 5-year survival of 10-40%, cure rate of 30% and median survival time of 6-10 months (Argiris, 2008; Vokes et al, 1993; Cohen et al, 2004). Our recent study in a large series (X. Li et al, 2009) demonstrated that overall survival rates of patients with distant metastases in clinic were 56.8% at 1 year, 9.1% at 3 years, and 6.8% at 5 years, respectively. In addition, traditional treatment related morbidities could negatively influence quality life of patients, which involves loss of speech, permamant tracheostomy or gastrostomy dependence, dysphagia and other systematic side effects. Therefore, it is necessary to seek novel strategies to cure advanced HNSCC aiming at organ preservation, prevention of metastases as well as second malignancies and

Surgical ablation plays a major role in the management of locoregional diseases of HNSCC. At early stage of HNSCC, current novel surgical alternatives, such as laser surgery, can achieve curable effects for 5-year survival rate of 80% even without prominent functional detriments. However, many tumors in the advanced stage are inoperable either due to the invasion of some major structures by tumor or due to the unfavorable general conditions of patients. Moreover, even with very skillful surgeons, some tumors remain after surgical resection, leading to postoperative recurrence if additional complimentary treatment is not carried out. In tumors with regional lymph node metastasis, extracapsular nodal spread always implicates poor prognosis even after a comprehensive neck dissection. For these conditions, adjuvant therapies such as radiotherapy, chemotherapy and chemoratiation are

**1. Introduction** 

improvement in quality of life.

**2. Necessities for adjuvant therapies in HNSCC** 

**Resistance in Head and Neck Squamous** 

**Microenvironment and Cancer Stem Cell** 

Xiaoming Li, Qingjia Sun and Yupeng Shen

*Bethune International Peace Hospital* 

## **Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell**

Xiaoming Li, Qingjia Sun and Yupeng Shen *Bethune International Peace Hospital China* 

## **1. Introduction**

Locally advanced diseases accounting for most HNSCC have a poor prognosis. The main reason for this is that corresponding symptoms of HNSCC are not always obvious or ignored by patients at early stage, which is mostly reflected by the fact that more than 2/3 HNSCC patients present with stage III/IV disease (AL-Sarraf, 1987; Argiris, 2008). Patients characterized with advanced HNSCC are subjected to worse prognosis than those with confined disease, exhibiting 5-year survival of 10-40%, cure rate of 30% and median survival time of 6-10 months (Argiris, 2008; Vokes et al, 1993; Cohen et al, 2004). Our recent study in a large series (X. Li et al, 2009) demonstrated that overall survival rates of patients with distant metastases in clinic were 56.8% at 1 year, 9.1% at 3 years, and 6.8% at 5 years, respectively. In addition, traditional treatment related morbidities could negatively influence quality life of patients, which involves loss of speech, permamant tracheostomy or gastrostomy dependence, dysphagia and other systematic side effects. Therefore, it is necessary to seek novel strategies to cure advanced HNSCC aiming at organ preservation, prevention of metastases as well as second malignancies and improvement in quality of life.

## **2. Necessities for adjuvant therapies in HNSCC**

Surgical ablation plays a major role in the management of locoregional diseases of HNSCC. At early stage of HNSCC, current novel surgical alternatives, such as laser surgery, can achieve curable effects for 5-year survival rate of 80% even without prominent functional detriments. However, many tumors in the advanced stage are inoperable either due to the invasion of some major structures by tumor or due to the unfavorable general conditions of patients. Moreover, even with very skillful surgeons, some tumors remain after surgical resection, leading to postoperative recurrence if additional complimentary treatment is not carried out. In tumors with regional lymph node metastasis, extracapsular nodal spread always implicates poor prognosis even after a comprehensive neck dissection. For these conditions, adjuvant therapies such as radiotherapy, chemotherapy and chemoratiation are

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

intrinsic responses underlying resistance under hypoxia.

**3.1 The general responses to hypoxia in tumor cells** 

heterogeneous population with hypoxia tolerance (see Fig. 1).

Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell 217

attributes to poor drugs delivery, leading to a common cause of chemoresistance. In addition, activated intrinsic pathways within tumor cells contribute to comprehensive radioand chemo-resistance under hypoxic condition. In this section, we will focus on these

General responses of tumor cells under hypoxia include translation inhibition, paradoxical translation and genetic instability. ATP defect caused by hypoxia invokes global translation inhibition for maintaining energy homeostasis. However, paradoxically, tumor cells activate some factors which are always unexpressed under normal conditions for adaptation to hypoxic stress. These proteins act as mediators of PH and metabolism, as well as function to propagate therapeutic resistance. In the long run, hypoxia-induced reactive oxygen species (ROS) and/or defective DNA repair induce mutagenesis of tumor cells to confer selection of

Fig. 1. General responses to hypoxia of tumor. Hypoxic tumor cells inhibit translation via mTOR pathways as well as UPR for energy homeostasis. Meanwhile paradoxically, they express some factors, such as HIF-1α and GRP78, to degrade nonfunctioning protein, regulate PH and counter apoptosis. These factors also act as resistance to therapy and metastasis. Hypoxia induced mutagenesis select clonal subset characterized with aggressive phenotypes, which confer more malignant biological behaviors including therapeutic

resistance.

due to undertake to increase the chance of cure or to prolong duration of survival of advanced cases.

#### **2.1 Traditional adjuvant therapies improve outcome of advanced HNSCC**

The participation of radiotherapy improved effects of surgery alone. In 2008, *Cancer* journal (Lavaf et al, 2008) reported a large-scale analysis with regard to effects of combined surgery and radiotherapy on survival of patients with lymph node-positive HNSCC patients. In 8795 patients meeting the inclusion criteria, 54.9% of 3-year overall survival and 43.2% of 5 year overall survival for adjuvant therapy could be gotten compared with 44.4% and 33.4% for surgery alone. More recently, a new analysis with large series (Shrime, 2010) reported that postoperative radiotherapy improved 5-year overall survival rate in patients with T1- 2N1 oral squamous carcinoma (41.4% for surgery alone vs. 54.2% for surgery plus radiotherapy). Although statistically significant, slight improvement in survival has to indicate the limitation of single radiotherapy addition, which appeals the need of chemotherapy in the management of advanced HNSCC. In 2009, the journal of *The Lancet Oncology* published a 10-year follow-up report of a trial for chemoradiotherapy for locally advanced head and neck cancer conducted by The UK Head and Neck (UKHAN) cancer group (Tobias et al, 2009). In this follow-up analysis, patients who did not undergo previous surgery benefited from scheduled simultaneous addition of chemicals to radiotherapy, exhibiting 4-7 years in the median overall survival. However, the median overall survival of patients undergoing surgery was still higher without substantial benefit from chemotherapy alone. Furthermore, sequent toxicity reactions, such as mucositis and xerostomia, are due to occur. All these findings suggest that the effects of traditional chemicals in treating the HNSCC are limited due to their unspecific hallmarks.

#### **2.2 Limitations of traditional adjuvant chemoradiation therapies**

As is known, HNSCC depend on many intrinsic or extrinsic factors to protect against traditional chemotherapeutic agents, such as cisplatin and 5- fluorouracil. As evidenced by clinical observations, HNSCC possesses a decreased sensitivity and increased resistance to chemo- and radiotherapy, giving rise to a poor tumor control efficacy of these treatment modalities. This situation is mostly reflected by the fact that many HNSCCs (including primary and recurrent carcinomas) have less or no response to the adopted treatment regimens in the course of chemotherapy and/or radiotherapy. For this reason, some tumors regenerate or relapse following a short- or long-term paracmasis during which time the tumor bulk contracts or even disappears visually in response to therapy. Therefore, chemoand radiotherapeutic resistance and post-treatment relapse has been always a puzzling problem that needs to be solved urgently.

#### **3. Role of hypoxia in therapeutic resistance in HNSCC**

The mechanisms underlying resistance to chemo- and/or radiotherapy by HNSCC are very complicated. Among various factors that are associated with therapeutic resistance in HNSCC, hypoxic microenvironment resulting from hypoxia in local cancer lesions is thought to be important one. It has been demonstrated that, most solid tumors have a lower pressure of oxygen (PO2) compared with normal tissues from which they originate. Hypoxia occurs due to rapid proliferation of cells and/or insufficient supplies of blood. The latter attributes to poor drugs delivery, leading to a common cause of chemoresistance. In addition, activated intrinsic pathways within tumor cells contribute to comprehensive radioand chemo-resistance under hypoxic condition. In this section, we will focus on these intrinsic responses underlying resistance under hypoxia.

## **3.1 The general responses to hypoxia in tumor cells**

216 Squamous Cell Carcinoma

due to undertake to increase the chance of cure or to prolong duration of survival of

The participation of radiotherapy improved effects of surgery alone. In 2008, *Cancer* journal (Lavaf et al, 2008) reported a large-scale analysis with regard to effects of combined surgery and radiotherapy on survival of patients with lymph node-positive HNSCC patients. In 8795 patients meeting the inclusion criteria, 54.9% of 3-year overall survival and 43.2% of 5 year overall survival for adjuvant therapy could be gotten compared with 44.4% and 33.4% for surgery alone. More recently, a new analysis with large series (Shrime, 2010) reported that postoperative radiotherapy improved 5-year overall survival rate in patients with T1- 2N1 oral squamous carcinoma (41.4% for surgery alone vs. 54.2% for surgery plus radiotherapy). Although statistically significant, slight improvement in survival has to indicate the limitation of single radiotherapy addition, which appeals the need of chemotherapy in the management of advanced HNSCC. In 2009, the journal of *The Lancet Oncology* published a 10-year follow-up report of a trial for chemoradiotherapy for locally advanced head and neck cancer conducted by The UK Head and Neck (UKHAN) cancer group (Tobias et al, 2009). In this follow-up analysis, patients who did not undergo previous surgery benefited from scheduled simultaneous addition of chemicals to radiotherapy, exhibiting 4-7 years in the median overall survival. However, the median overall survival of patients undergoing surgery was still higher without substantial benefit from chemotherapy alone. Furthermore, sequent toxicity reactions, such as mucositis and xerostomia, are due to occur. All these findings suggest that the effects of traditional chemicals in treating the

As is known, HNSCC depend on many intrinsic or extrinsic factors to protect against traditional chemotherapeutic agents, such as cisplatin and 5- fluorouracil. As evidenced by clinical observations, HNSCC possesses a decreased sensitivity and increased resistance to chemo- and radiotherapy, giving rise to a poor tumor control efficacy of these treatment modalities. This situation is mostly reflected by the fact that many HNSCCs (including primary and recurrent carcinomas) have less or no response to the adopted treatment regimens in the course of chemotherapy and/or radiotherapy. For this reason, some tumors regenerate or relapse following a short- or long-term paracmasis during which time the tumor bulk contracts or even disappears visually in response to therapy. Therefore, chemoand radiotherapeutic resistance and post-treatment relapse has been always a puzzling

The mechanisms underlying resistance to chemo- and/or radiotherapy by HNSCC are very complicated. Among various factors that are associated with therapeutic resistance in HNSCC, hypoxic microenvironment resulting from hypoxia in local cancer lesions is thought to be important one. It has been demonstrated that, most solid tumors have a lower pressure of oxygen (PO2) compared with normal tissues from which they originate. Hypoxia occurs due to rapid proliferation of cells and/or insufficient supplies of blood. The latter

**2.1 Traditional adjuvant therapies improve outcome of advanced HNSCC** 

HNSCC are limited due to their unspecific hallmarks.

problem that needs to be solved urgently.

**2.2 Limitations of traditional adjuvant chemoradiation therapies** 

**3. Role of hypoxia in therapeutic resistance in HNSCC** 

advanced cases.

General responses of tumor cells under hypoxia include translation inhibition, paradoxical translation and genetic instability. ATP defect caused by hypoxia invokes global translation inhibition for maintaining energy homeostasis. However, paradoxically, tumor cells activate some factors which are always unexpressed under normal conditions for adaptation to hypoxic stress. These proteins act as mediators of PH and metabolism, as well as function to propagate therapeutic resistance. In the long run, hypoxia-induced reactive oxygen species (ROS) and/or defective DNA repair induce mutagenesis of tumor cells to confer selection of heterogeneous population with hypoxia tolerance (see Fig. 1).

Fig. 1. General responses to hypoxia of tumor. Hypoxic tumor cells inhibit translation via mTOR pathways as well as UPR for energy homeostasis. Meanwhile paradoxically, they express some factors, such as HIF-1α and GRP78, to degrade nonfunctioning protein, regulate PH and counter apoptosis. These factors also act as resistance to therapy and metastasis. Hypoxia induced mutagenesis select clonal subset characterized with aggressive phenotypes, which confer more malignant biological behaviors including therapeutic resistance.

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

induces autophagic death and necrosis.

**3.3.1 HIF-1α**

**3.3 Factors expressed paradoxically under hypoxia** 

**3.3.2 Signal transducer and activator of transcription 3 (STAT3)** 

Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell 219

includes IRE-1 and ATF6 arms. Activated IRE-1 serves as an endoribonuclease to remove a 26 nucleotide intron from X-box binding protein 1 (XBP1) pre-mRNA. The resulting XBP1 protein by spliced XBP1 mRNA can activate lots of ER chaperones and enzymes to remove mis/unfolded protein and help ER-localized protein maturation as well as ER-associated degradation. Similar with XBP1, ATF6 needs cleavage for its activation. Briefly, upon UPR activation, ATF6 is transmitted from ER to Golgi apparatus where ATF6 completes its splicing process. Subsequently, cleaved ATF6 also activates target genes functioning in protein degradation and upregulation of molecular chaperones. As a matter of fact, their functions and target genes overlap one another. Overall, these sensors play a critical role in inhibition of mRNA and protein synthesis and upregulation of stress-adaptive factors. As a final step, unfolded protein is transported to cytoplasm and degraded via ubiquitinproteasome pathway (UPP) or autophagy. To our understanding, UPR is a double-edged sword. On the one hand, PERK--eIF2a-ATF4 pathway is a dominant UPR arm offering survival advantage under hypoxia. On the other hand, once severe stress persists, UPR will induce ER-stress-relared cell death (apoptosis, autophagy associated programmed death or necrosis). To date, GADD153/CHOP induced by ATF4, has been identified as a proapoptosis factor that can activate cascades of caspases, mediating type I programmed cell death (known as apoptosis). It remains to be determined by what mechanisms the UPR

HIFs are core factors regulating oxygen and energy supplies of the tumor bulk, by which tumor cells adapt to hypoxia through inducing expressions of related genes to overcome such an unfavorable low-oxygen condition. HIFs are members of bHLH-PAS protein family including HIF-α and HIF-β subunit, the former of which also includes HIF-1α, HIF-2α and HIF-3α. Functionally, HIF-α (HIF-1α, HIF-2α) can be stably sustained in the hypoxic niche. In the event of cell signaling, HIF-α (HIF-1α, HIF-2α) and HIF-β can form a heterodimer that binds to promoters or enhancers of target genes. Hypoxia not only induces the expression of HIF-1α, but also activates many specific biological effects of HIF-1α gene protein, which functions either to acquire the tolerance to hypoxia or to commit the capability of invasion, metastasis and therapeutic resistance: 1) inducing expression of carbonate dehydrates (CAH) to maintain a stable cytoplasmic PH to promote the survival ability of cancer cells in response to apoptosis-inducing factors; 2) upregulating the expression of MDR gene and its product, P-gp, resulting in resistance to multiple chemotherapeutic agents; 3) mediating the overexpression and activation of DNA kinase (DNA-PK), contributing essentially to the repair of DNA double-strand breaks (DSBs); 4) acting as an upstream regulator of genes encoding vascular endothelial growth factor (VEGF) as well as some key enzymes related to glycolysis, responsible for angiogenesis and glycometabolism within tumors; 5) promoting expressions of anti-apoptosis proteins such as Survivin and XIAP to inhibit the activation of pro-apoptosis proteins Bax and caspases, rendering the tumor cells the ability to escape from apoptosis.

STAT3 is an important factor overlapped by many intracellular signal pathways. It can be activated though Janus kinases (JAKs) or tyrosine kinase receptors such as EGFR. Upon

#### **3.2 Hypoxia-related translation inhibition**

#### **3.2.1 The mammalian target of rapamycin (mTOR) pathways**

Protein synthesis is processed as a result of energy-consumption. To date, emerging data have evidenced that inhibition of translation is an important category of cellular hypoxic tolerance, and the process occurs during the initial step of translation. The initiation of translation involves two components, the eukaryotic initiation factor 4F (eIF4F) as well as 43S preinitiation complex. The former consists of the cap binding protein eIF4E, a scaffolding protein eIF4G and an ATP-dependent helicase eIF4A (RNA helicase activity), among which eIF4E and eIF4G mostly participate in the regulation of initiation of translation. Under nutrient and/or oxygen repletion, mTOR phosphorylates eukaryotic initiation factor 4E binding protein 1 (4EBP1) that has a high affinity with eIF4E to low the affinity. Together with eIF4G, released eIF4E contributes to assembly of eIF4F complex that binds to the 5'm7GpppN cap structure of mRNA to facilitate the recruitment of 43S preinitiation complex that includes the 40S ribosomal subunit and the ternary complex (eIF2-GTP and the methionine-loaded initiator tRNA) for the start of translation initiation. The ribosomal constituents of preinitiation complex scan though the 5′ untranslated region (5'UTR) until the AUG initiation codon is recognized. Sequently, the 60S ribosomal subunit joins to form 80S ribosome for the elongation of translation. Lots of recent reports have implicated that hypoxia can disturb the process above via hypoxic activation of the tuberous sclerosis protein 1 (TSC1)–TSC2 complex-mediated downregulation of mTOR. Thereby, corresponding expressions of translation initiation-related proteins, such as phosphorylated 4EBP1 and eIF4F, other mTOR-mediated targets and S6 protein kinases (S6K 1 and 2), all of which also function in translation, would be inactivated accordingly. In addition, hypoxia also interferes with formation of preinitiation complex to inhibit translation via PERKmediated cascade of unfolded protein responses.

#### **3.2.2 Unfolded protein responses (UPR)**

UPR is an evolutionary conserved protective response to microenvironment stress. Because of abnormal vascular structure and aggressive cellular growth, hypoxia and glucose starvation always occur in tumor microenvironment, resulting in defective usage of energy in response to accumulation of many unfolded protein in endocytoplasmic reticulum (ER). Tumor cells must adapt to this stress though inhibiting mRNA and protein synthesis as well as degrading excessive useless protein, which is executed though activation of UPR. Glucose-regulated protein 78 (GRP78/BiP) is the core regulator in ER-stress and overexpressed in most tumor as a predictor of poor prognosis. Routinely, GRP78 binds to ER-stress sensors, IRE1α (inositol-requiring 1 alpha), PERK (double-strand RNA-activated protein kinase-like ER kinase), and ATF6 (activating transcription factor 6), to inactivate their downstream targets. Once ER-stress occurs, the role of GRP78 is shifted towards that of a chaperone. After dissociation, GRP78 handles unfolded protein to facilitate degradation and binds to Ca2+ to inhibit apoptosis. Importantly, the dissociation activates these integral ER membrane sensors PERK, IRE-1 and ATF6. Activated PERK phosphorylates eukaryotic initiation factor 2 subunit α (eIF2α), leading to either inhibition of global protein translational attenuation or paradoxical expression of the transcription factor 4 (ATF4) that immediately blocks eIF2α phosphorylation and sequently encodes genes upregulating stress-adaptive factors, such as GRP78 and hypoxia inducible factors **(**HIFs). UPR also includes IRE-1 and ATF6 arms. Activated IRE-1 serves as an endoribonuclease to remove a 26 nucleotide intron from X-box binding protein 1 (XBP1) pre-mRNA. The resulting XBP1 protein by spliced XBP1 mRNA can activate lots of ER chaperones and enzymes to remove mis/unfolded protein and help ER-localized protein maturation as well as ER-associated degradation. Similar with XBP1, ATF6 needs cleavage for its activation. Briefly, upon UPR activation, ATF6 is transmitted from ER to Golgi apparatus where ATF6 completes its splicing process. Subsequently, cleaved ATF6 also activates target genes functioning in protein degradation and upregulation of molecular chaperones. As a matter of fact, their functions and target genes overlap one another. Overall, these sensors play a critical role in inhibition of mRNA and protein synthesis and upregulation of stress-adaptive factors. As a final step, unfolded protein is transported to cytoplasm and degraded via ubiquitinproteasome pathway (UPP) or autophagy. To our understanding, UPR is a double-edged sword. On the one hand, PERK--eIF2a-ATF4 pathway is a dominant UPR arm offering survival advantage under hypoxia. On the other hand, once severe stress persists, UPR will induce ER-stress-relared cell death (apoptosis, autophagy associated programmed death or

necrosis). To date, GADD153/CHOP induced by ATF4, has been identified as a proapoptosis factor that can activate cascades of caspases, mediating type I programmed cell death (known as apoptosis). It remains to be determined by what mechanisms the UPR induces autophagic death and necrosis.

## **3.3 Factors expressed paradoxically under hypoxia**

#### **3.3.1 HIF-1α**

218 Squamous Cell Carcinoma

Protein synthesis is processed as a result of energy-consumption. To date, emerging data have evidenced that inhibition of translation is an important category of cellular hypoxic tolerance, and the process occurs during the initial step of translation. The initiation of translation involves two components, the eukaryotic initiation factor 4F (eIF4F) as well as 43S preinitiation complex. The former consists of the cap binding protein eIF4E, a scaffolding protein eIF4G and an ATP-dependent helicase eIF4A (RNA helicase activity), among which eIF4E and eIF4G mostly participate in the regulation of initiation of translation. Under nutrient and/or oxygen repletion, mTOR phosphorylates eukaryotic initiation factor 4E binding protein 1 (4EBP1) that has a high affinity with eIF4E to low the affinity. Together with eIF4G, released eIF4E contributes to assembly of eIF4F complex that binds to the 5'm7GpppN cap structure of mRNA to facilitate the recruitment of 43S preinitiation complex that includes the 40S ribosomal subunit and the ternary complex (eIF2-GTP and the methionine-loaded initiator tRNA) for the start of translation initiation. The ribosomal constituents of preinitiation complex scan though the 5′ untranslated region (5'UTR) until the AUG initiation codon is recognized. Sequently, the 60S ribosomal subunit joins to form 80S ribosome for the elongation of translation. Lots of recent reports have implicated that hypoxia can disturb the process above via hypoxic activation of the tuberous sclerosis protein 1 (TSC1)–TSC2 complex-mediated downregulation of mTOR. Thereby, corresponding expressions of translation initiation-related proteins, such as phosphorylated 4EBP1 and eIF4F, other mTOR-mediated targets and S6 protein kinases (S6K 1 and 2), all of which also function in translation, would be inactivated accordingly. In addition, hypoxia also interferes with formation of preinitiation complex to inhibit translation via PERK-

UPR is an evolutionary conserved protective response to microenvironment stress. Because of abnormal vascular structure and aggressive cellular growth, hypoxia and glucose starvation always occur in tumor microenvironment, resulting in defective usage of energy in response to accumulation of many unfolded protein in endocytoplasmic reticulum (ER). Tumor cells must adapt to this stress though inhibiting mRNA and protein synthesis as well as degrading excessive useless protein, which is executed though activation of UPR. Glucose-regulated protein 78 (GRP78/BiP) is the core regulator in ER-stress and overexpressed in most tumor as a predictor of poor prognosis. Routinely, GRP78 binds to ER-stress sensors, IRE1α (inositol-requiring 1 alpha), PERK (double-strand RNA-activated protein kinase-like ER kinase), and ATF6 (activating transcription factor 6), to inactivate their downstream targets. Once ER-stress occurs, the role of GRP78 is shifted towards that of a chaperone. After dissociation, GRP78 handles unfolded protein to facilitate degradation and binds to Ca2+ to inhibit apoptosis. Importantly, the dissociation activates these integral ER membrane sensors PERK, IRE-1 and ATF6. Activated PERK phosphorylates eukaryotic initiation factor 2 subunit α (eIF2α), leading to either inhibition of global protein translational attenuation or paradoxical expression of the transcription factor 4 (ATF4) that immediately blocks eIF2α phosphorylation and sequently encodes genes upregulating stress-adaptive factors, such as GRP78 and hypoxia inducible factors **(**HIFs). UPR also

**3.2 Hypoxia-related translation inhibition** 

mediated cascade of unfolded protein responses.

**3.2.2 Unfolded protein responses (UPR)** 

**3.2.1 The mammalian target of rapamycin (mTOR) pathways** 

HIFs are core factors regulating oxygen and energy supplies of the tumor bulk, by which tumor cells adapt to hypoxia through inducing expressions of related genes to overcome such an unfavorable low-oxygen condition. HIFs are members of bHLH-PAS protein family including HIF-α and HIF-β subunit, the former of which also includes HIF-1α, HIF-2α and HIF-3α. Functionally, HIF-α (HIF-1α, HIF-2α) can be stably sustained in the hypoxic niche. In the event of cell signaling, HIF-α (HIF-1α, HIF-2α) and HIF-β can form a heterodimer that binds to promoters or enhancers of target genes. Hypoxia not only induces the expression of HIF-1α, but also activates many specific biological effects of HIF-1α gene protein, which functions either to acquire the tolerance to hypoxia or to commit the capability of invasion, metastasis and therapeutic resistance: 1) inducing expression of carbonate dehydrates (CAH) to maintain a stable cytoplasmic PH to promote the survival ability of cancer cells in response to apoptosis-inducing factors; 2) upregulating the expression of MDR gene and its product, P-gp, resulting in resistance to multiple chemotherapeutic agents; 3) mediating the overexpression and activation of DNA kinase (DNA-PK), contributing essentially to the repair of DNA double-strand breaks (DSBs); 4) acting as an upstream regulator of genes encoding vascular endothelial growth factor (VEGF) as well as some key enzymes related to glycolysis, responsible for angiogenesis and glycometabolism within tumors; 5) promoting expressions of anti-apoptosis proteins such as Survivin and XIAP to inhibit the activation of pro-apoptosis proteins Bax and caspases, rendering the tumor cells the ability to escape from apoptosis.

#### **3.3.2 Signal transducer and activator of transcription 3 (STAT3)**

STAT3 is an important factor overlapped by many intracellular signal pathways. It can be activated though Janus kinases (JAKs) or tyrosine kinase receptors such as EGFR. Upon

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

resistant, invasive and metastatic phenotype (see Fig. 2).

pattern in a tumor.

Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell 221

recombination (HR) function, a main pathway to repair DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle. Secondly, cyclic hypoxia induces an enhanced metastasis. It was found that mice bearing melanoma xenografts exposed to cyclic hypoxia suffered from increased incidence of pulmonary metastases (Rofstad et al, 2010). Furthermore, tumor cells treated by cyclic hypoxia up-regulates the expression of VEGF-A, the ligand of VEGFR-1 on bone marrow derived cells conrmed to form ''premetastasis niche''. Therefore, induction of VEGF-A by hypoxia may be an important promoter of metastasis. Thirdly, cyclic hypoxia enhances metabolism of Tirapazimine (TPZ), an agent with specific hypoxic cytotoxicity, by intratumoral vessels adjacent to the populated tumor cells, which attenuates the effects of TPZ (Cárdenas-Navia et al, 2007). Finally, cyclic hypoxia is pervasive. As early as 1996, Kimura's group (Kimura et al, 1996) measured microvessel red cell ux (RCF) and perivascular PO2 in xenotransplant of R3230Ac mammary carcinomas using intratumoral dorsal ap window chambers. They found that the baseline RCF and PO2 underwent a highly dynamic process, demonstrating that uctuating hypoxia is a common phenomenon within a tumor. More recently, another group (Cárdenas-Navia et al, 2008) used phosphorescence lifetime imaging to detect uctuation of vascular PO2 in rat brosarcomas, 9L gliomas and R3230 mammary adenocarcinomas transplanted in dorsal skin-fold window chambers. By short interval periodic imaging, they found O2 delivery to tumors is constantly instable. In addition, hypoxia, including acute and chronic hypoxia, causes genomic instability. Cyclic hypoxia mostly induces DNA double-strand breaks (DSBs) by generating reactive oxygen species (ROS). Chronic hypoxia causes genomic instability due to the defective HR ability. Thereby, both types of hypoxia facilitate mutagenesis leading to clonal selection with therapy-

Fig. 2. Dynamic heterogeneity of hypoxia in solid tumor. Distinct hypoxia patterns exist in solid tumor. These patterns may overlap and co-exist in the same tumor bulk, which can be reflected though the 3D axis. Any point of this axis represents a combined type of hypoxia

phosphorylation at the Tyr705 residue, p-STAT3 translocates to nucleus to bind DNA for inducing the transcription of downstream targets. Emerging reports have demonstrated that STAT3 is associated with poor prognosis in many cancers including HNSCC. STAT3 induces resistance to therapy in tumors via activation of anti-apoptosis factors, such as Bcl-2, Bcl-xl as well as Survivin and downregulation of P53. Recently, a study demonstrated that STAT3 participates in inhibition of apoptosis caused by PIs in HNSCC (C. Li et al, 2009). Under hypoxia, STAT3 can be activated by ROS (Simon et al, 1998). Selvendiran et al (Selvendiran et al, 2009) found that STAT3 can be activated by production of ROS under 1% O2 in ovarian cancer. In their study, overexpressed STAT3 contributed to similar rate of proliferation as that under normoxia but increased drug resistance under hypoxia. Through blockage of STAT3 using RNAi technique, ovarian tumor cells with defective expression of STAT3 exhibited affected proliferation as well as increased sensitivity to traditional chemotherapeutics under hypoxia. In HNSCCs, STAT3 was also found to be constitutively activated and associated with cervical lymph node metastasis in laryngeal cancer. Silencing STAT3 gene with specific siRNA enhances the sensitivity of Hep-2 human laryngeal carcinoma cells to ionizing radiation both in vitro and in xenotransplanted mice model. (X. Li, et al, 2010a, 2010b)

#### **3.4 Hypoxic dynamic complication in solid tumor**

#### **3.4.1 Category of hypoxia**

In solid tumor, hypoxia can be categorized as chronic continuing hypoxia and cyclic hypoxia (also called intermittent hypoxia or fluctuating hypoxia) depending on distances of tumor cells from the adjacent vessels. The former is incurred from the condition that tumor cells locating far from vessels result to diffusion-limited and relatively stable delivery of oxygen. On the other hand, the latter characterized by acute hypoxia/reoxygenation is caused by status of nearby vessels that suffer from dynamic changes in blood perfusion not least as a result of the abnormal vasculature and the mechanical instability of microvessel walls caused by proliferating tumor cells and/or circulating blood cells. With regard to insufficiency in blood or oxygen supply, hypoxia is classified as mild hypoxia (PO2: 1-3%), moderate hypoxia (PO2: 0.1-1%) and severe hypoxia (PO2: 0-0.1%) ((Koumenis & Wouters, 2006). Additionally, in term of duration of persistent hypoxic condition, hypoxia is divided into acute hypoxia lasting several minutes to several hours as well as prolonged chronic hypoxia during which cells are exposed to hour-to-day intracellular low PO2. The complex nature of hypoxia and different responses to distinct hypoxia by tumor cells may explain why it is so difficult to antagonize hypoxia-induced therapeutic resistance in HNSCCs.

#### **3.4.2 Chronic versus cyclic**

In most lab experiments, there is an important difference ignored by us. That is the parameters of hypoxic condition selected by most investigators are usually simple and fairly stable. However, reoxygenation may occur during manipulation of assorted cells. To date, cyclic hypoxia has been less studied than chronic hypoxia. The setting of cyclic hypoxic condition was not consistent among various studies on cyclic hypoxia. Here we introduce the difference between the two as follows with special emphasis on the importance of cyclic hypoxia. Firstly, cyclic hypoxia confers more potential therapeutic resistance than chronic hypoxia. It has been demonstrated by many studies that increased expression of HIF-1α contributes to cyclic hypoxia-induced resistance. In addition, it has been conrmed that chronically hypoxic tumor cells are more susceptible to ionizing radiation (IR) or DNAdamaging drugs than acutely hypoxic tumor cells because of decreased homologous

phosphorylation at the Tyr705 residue, p-STAT3 translocates to nucleus to bind DNA for inducing the transcription of downstream targets. Emerging reports have demonstrated that STAT3 is associated with poor prognosis in many cancers including HNSCC. STAT3 induces resistance to therapy in tumors via activation of anti-apoptosis factors, such as Bcl-2, Bcl-xl as well as Survivin and downregulation of P53. Recently, a study demonstrated that STAT3 participates in inhibition of apoptosis caused by PIs in HNSCC (C. Li et al, 2009). Under hypoxia, STAT3 can be activated by ROS (Simon et al, 1998). Selvendiran et al (Selvendiran et al, 2009) found that STAT3 can be activated by production of ROS under 1% O2 in ovarian cancer. In their study, overexpressed STAT3 contributed to similar rate of proliferation as that under normoxia but increased drug resistance under hypoxia. Through blockage of STAT3 using RNAi technique, ovarian tumor cells with defective expression of STAT3 exhibited affected proliferation as well as increased sensitivity to traditional chemotherapeutics under hypoxia. In HNSCCs, STAT3 was also found to be constitutively activated and associated with cervical lymph node metastasis in laryngeal cancer. Silencing STAT3 gene with specific siRNA enhances the sensitivity of Hep-2 human laryngeal carcinoma cells to ionizing

radiation both in vitro and in xenotransplanted mice model. (X. Li, et al, 2010a, 2010b)

In solid tumor, hypoxia can be categorized as chronic continuing hypoxia and cyclic hypoxia (also called intermittent hypoxia or fluctuating hypoxia) depending on distances of tumor cells from the adjacent vessels. The former is incurred from the condition that tumor cells locating far from vessels result to diffusion-limited and relatively stable delivery of oxygen. On the other hand, the latter characterized by acute hypoxia/reoxygenation is caused by status of nearby vessels that suffer from dynamic changes in blood perfusion not least as a result of the abnormal vasculature and the mechanical instability of microvessel walls caused by proliferating tumor cells and/or circulating blood cells. With regard to insufficiency in blood or oxygen supply, hypoxia is classified as mild hypoxia (PO2: 1-3%), moderate hypoxia (PO2: 0.1-1%) and severe hypoxia (PO2: 0-0.1%) ((Koumenis & Wouters, 2006). Additionally, in term of duration of persistent hypoxic condition, hypoxia is divided into acute hypoxia lasting several minutes to several hours as well as prolonged chronic hypoxia during which cells are exposed to hour-to-day intracellular low PO2. The complex nature of hypoxia and different responses to distinct hypoxia by tumor cells may explain why it is so difficult to antagonize hypoxia-induced therapeutic resistance in HNSCCs.

In most lab experiments, there is an important difference ignored by us. That is the parameters of hypoxic condition selected by most investigators are usually simple and fairly stable. However, reoxygenation may occur during manipulation of assorted cells. To date, cyclic hypoxia has been less studied than chronic hypoxia. The setting of cyclic hypoxic condition was not consistent among various studies on cyclic hypoxia. Here we introduce the difference between the two as follows with special emphasis on the importance of cyclic hypoxia. Firstly, cyclic hypoxia confers more potential therapeutic resistance than chronic hypoxia. It has been demonstrated by many studies that increased expression of HIF-1α contributes to cyclic hypoxia-induced resistance. In addition, it has been conrmed that chronically hypoxic tumor cells are more susceptible to ionizing radiation (IR) or DNAdamaging drugs than acutely hypoxic tumor cells because of decreased homologous

**3.4 Hypoxic dynamic complication in solid tumor** 

**3.4.1 Category of hypoxia** 

**3.4.2 Chronic versus cyclic** 

recombination (HR) function, a main pathway to repair DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle. Secondly, cyclic hypoxia induces an enhanced metastasis. It was found that mice bearing melanoma xenografts exposed to cyclic hypoxia suffered from increased incidence of pulmonary metastases (Rofstad et al, 2010). Furthermore, tumor cells treated by cyclic hypoxia up-regulates the expression of VEGF-A, the ligand of VEGFR-1 on bone marrow derived cells conrmed to form ''premetastasis niche''. Therefore, induction of VEGF-A by hypoxia may be an important promoter of metastasis. Thirdly, cyclic hypoxia enhances metabolism of Tirapazimine (TPZ), an agent with specific hypoxic cytotoxicity, by intratumoral vessels adjacent to the populated tumor cells, which attenuates the effects of TPZ (Cárdenas-Navia et al, 2007). Finally, cyclic hypoxia is pervasive. As early as 1996, Kimura's group (Kimura et al, 1996) measured microvessel red cell ux (RCF) and perivascular PO2 in xenotransplant of R3230Ac mammary carcinomas using intratumoral dorsal ap window chambers. They found that the baseline RCF and PO2 underwent a highly dynamic process, demonstrating that uctuating hypoxia is a common phenomenon within a tumor. More recently, another group (Cárdenas-Navia et al, 2008) used phosphorescence lifetime imaging to detect uctuation of vascular PO2 in rat brosarcomas, 9L gliomas and R3230 mammary adenocarcinomas transplanted in dorsal skin-fold window chambers. By short interval periodic imaging, they found O2 delivery to tumors is constantly instable. In addition, hypoxia, including acute and chronic hypoxia, causes genomic instability. Cyclic hypoxia mostly induces DNA double-strand breaks (DSBs) by generating reactive oxygen species (ROS). Chronic hypoxia causes genomic instability due to the defective HR ability. Thereby, both types of hypoxia facilitate mutagenesis leading to clonal selection with therapyresistant, invasive and metastatic phenotype (see Fig. 2).

Fig. 2. Dynamic heterogeneity of hypoxia in solid tumor. Distinct hypoxia patterns exist in solid tumor. These patterns may overlap and co-exist in the same tumor bulk, which can be reflected though the 3D axis. Any point of this axis represents a combined type of hypoxia pattern in a tumor.

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

renewal.

significant approach to eliminate CSCs.

fittest by the pressure of microenvironment.

**4.2.2 Niche and heterogeneity** 

Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell 223

contacts, cytokines, blood vessels, mesenchymal cells and so on. It serves to protect CSCs from differentiation and apoptosis, and keeps the status of self-renewing (Iwasaki & Suda, 2009). As a matter of fact, the significance of the niche function is far beyond these, as it affects the physiology of CSCs to a far more great extent. As early as 1889, through analysis of 735 cases of breast cancers, British assistant doctor Stephen Paget (Paget, 1989) found that breast cancer cells preferred liver to settle in rather than spleen that has vascular supply as abundant as liver. Nearly a century later, after the quiescence of the " seeds and soils ", Hart and Fidler (Hart & Fidler, 1980) injected melanoma cells into mice implanted with ovary tissue, kidney tissue and lung tissue in muscle or under the skin, and these implanted tissues had previously established vascular supplies of their own. Finally, they demonstrated that melanoma cells just metastasized to grafted ovary and kidney, suggesting that the formation of tumor is not only influenced by the characters of tumor cells but also depends on the niche. Because CSCs are a kind of cells that can selfrenew in malignancy, the niche of CSC must be critical for preserving the property of self-

Recently, many studies provided evidence for the "seeds and soil" even as they further disclosed the relationship between CSC and its niche. A group attenuated the adhesion between CSCs and some components of the niche, such as hyaluronan, through interfering with CD44, thereby, inhibited the neoplasia of myeloid leukemia (Jin et al, 2006; Krause et al, 2006). Calabrese et al (Calabrese et al, 2007) found that most medulloblastoma stem cells grew by adhering to endothelial cells selectively, and these CD133+ cells could give rise to new tumors only when co-transplanted with endothelial cells. Kaplan et al (Kaplan et al, 2005) introduced a concept of "premetastatic niche", which meant the microenvironment of metastasized organs had been induced to transform into a condition better for the formation of secondary tumor. All of the findings above suggest that targeting the niche will be a very

Species are selected by adaptation to environmental changes as proven by the earliest dinosaurs to today's diverse biological species. Such is Darwinian evolution, a possible explanation for survival. The development of tumors might be a process of survival of the

It is known to all that cells in various types of tumors are different from each other in many aspects, such as size, appearance, antigen expression, cell membrane composition and sensitivity to different treatment modalities (Ichim & Wells, 2006; Heppner, 1984; Axelson, 2005). There are two explanatory models to the potential heterogeneity of the tumor cells, the stochastic model and the hierarchical model. Firstly, the stochastic model attributes the tumor development to the "genetic instability", through which the ones best adapting to the microenvironment are selected to obtain the advantage of proliferation (Nowell, 1976; Tysnes & Bjerkvig, 2007). This model shows that the tumor parenchyma contains many types of tumor cells with the ability to form tumors in the microenvironment (Bjerkvig, 2009). Secondly, the hierarchical model supports that the initial tumor and the metastatic

tumor are both evolved from CSCs, which seems contradicting to the first model.

## **3.4.3 Hypoxic duration- and/or degree-related responses**

The existing status of oxygen level in tumor bulk is very heterogeneous, which is reflected by detected PO2 ranging from 0% to 100%, namely, from anoxia to normoxia. It is plausible that paradoxical activation of associated factors under hypoxia is also heterogeneous and dynamic in solid tumor, implying that focusing on one single target factor is insufficient to carry out an effective therapy. To date, although HIF-1α has been comprehensively studied under different hypoxic conditions, it is not the case in the study of short period of severe hypoxia as well as chronic moderate hypoxia. This can be partially explained by the fact that regulation of HIF-1α is actually a negative feedback loop via HIF-1α-dependent induction of prolyl hydroxylase enzymes that promote the von Hippel-Lindau (VHL) tumour suppressor protein-mediated HIF-1α degradation by ubiquitin-proteasome system under moderate hypoxia. On the contrary, the induction of prolyl hydroxylase enzymes is inhibited under severe hypoxia. Activation of eIF2α has been indicated as a transient process during severe hypoxia, which is decreased following the prolonged duration of hypoxic status. Under moderate hypoxia, eIF2α exhibits a gradually elevated activation along with elongation of hypoxic time.

## **4. Role of cancer stem cells (CSCs) in hypoxia-induced therapeutic resistance in HNSCC**

## **4.1 Identification of CSCs in HNSCC**

The theory of CSCs, as a milestone of cancer research, has a history of 150 years. The focus of this theory is that there exists a sub-group of tumor cells, like stem cells of normal tissues, with stem traits characterizing growth stasis and self-renewal with specific cell surface markers. This subset of cells within the tumor bulk is known as "cancer stem cells" (CSCs) or "tumor initiating cells (TICs)". Other tumor cells that are considered as progeny of CSCs would face a final differentiation followed by programmed cell death. In term of CSC theory, tumor bulk only originates from CSCs; therapeutic failures in cancer management are a result of insufficient elimination of these heterogeneous subpopulation. In addition, the subset is believed to play important roles in invasion, metastasis and therapeutic resistance in various malignancies. To date, CSCs or CSC like cells have been identified in different cancer including HNSCC. In 2007, a subpopulation of cells with characterized stemness and CD44 marker in HNSCC was first isolated and identified (Prince et al, 2007). It was also demonstrated that CD133+ cells in Hep-2 human squamous laryngeal carcinoma cells have stem cell-like characters (Zhou et al., 2009). Subsequently, other investigators identified a CD133+ CSC-like subset with chemoresistance in oral squamous carcinoma (Zhang et al., 2009). More recently, CD44+ CSCs have also been isolated and identified from human laryngeal squamous carcinoma. All these data confirm the existence of CSCs in HNSCC, which shed light on a novel area to get further insight into the mechanisms of chemo- and radioresistance in HNSCC with respect to SCCs.

#### **4.2 CSCs and microenvironment**

#### **4.2.1 "Seeds and soil" theory**

The microenvironment of CSCs also called "niche", consists of cellular and non-cellular components surrounding CSCs (Scadden, 2006), including direct cell contacts, cell-matrix contacts, cytokines, blood vessels, mesenchymal cells and so on. It serves to protect CSCs from differentiation and apoptosis, and keeps the status of self-renewing (Iwasaki & Suda, 2009). As a matter of fact, the significance of the niche function is far beyond these, as it affects the physiology of CSCs to a far more great extent. As early as 1889, through analysis of 735 cases of breast cancers, British assistant doctor Stephen Paget (Paget, 1989) found that breast cancer cells preferred liver to settle in rather than spleen that has vascular supply as abundant as liver. Nearly a century later, after the quiescence of the " seeds and soils ", Hart and Fidler (Hart & Fidler, 1980) injected melanoma cells into mice implanted with ovary tissue, kidney tissue and lung tissue in muscle or under the skin, and these implanted tissues had previously established vascular supplies of their own. Finally, they demonstrated that melanoma cells just metastasized to grafted ovary and kidney, suggesting that the formation of tumor is not only influenced by the characters of tumor cells but also depends on the niche. Because CSCs are a kind of cells that can selfrenew in malignancy, the niche of CSC must be critical for preserving the property of selfrenewal.

Recently, many studies provided evidence for the "seeds and soil" even as they further disclosed the relationship between CSC and its niche. A group attenuated the adhesion between CSCs and some components of the niche, such as hyaluronan, through interfering with CD44, thereby, inhibited the neoplasia of myeloid leukemia (Jin et al, 2006; Krause et al, 2006). Calabrese et al (Calabrese et al, 2007) found that most medulloblastoma stem cells grew by adhering to endothelial cells selectively, and these CD133+ cells could give rise to new tumors only when co-transplanted with endothelial cells. Kaplan et al (Kaplan et al, 2005) introduced a concept of "premetastatic niche", which meant the microenvironment of metastasized organs had been induced to transform into a condition better for the formation of secondary tumor. All of the findings above suggest that targeting the niche will be a very significant approach to eliminate CSCs.

## **4.2.2 Niche and heterogeneity**

222 Squamous Cell Carcinoma

The existing status of oxygen level in tumor bulk is very heterogeneous, which is reflected by detected PO2 ranging from 0% to 100%, namely, from anoxia to normoxia. It is plausible that paradoxical activation of associated factors under hypoxia is also heterogeneous and dynamic in solid tumor, implying that focusing on one single target factor is insufficient to carry out an effective therapy. To date, although HIF-1α has been comprehensively studied under different hypoxic conditions, it is not the case in the study of short period of severe hypoxia as well as chronic moderate hypoxia. This can be partially explained by the fact that regulation of HIF-1α is actually a negative feedback loop via HIF-1α-dependent induction of prolyl hydroxylase enzymes that promote the von Hippel-Lindau (VHL) tumour suppressor protein-mediated HIF-1α degradation by ubiquitin-proteasome system under moderate hypoxia. On the contrary, the induction of prolyl hydroxylase enzymes is inhibited under severe hypoxia. Activation of eIF2α has been indicated as a transient process during severe hypoxia, which is decreased following the prolonged duration of hypoxic status. Under moderate hypoxia, eIF2α exhibits a gradually elevated activation along with elongation of

**4. Role of cancer stem cells (CSCs) in hypoxia-induced therapeutic** 

The theory of CSCs, as a milestone of cancer research, has a history of 150 years. The focus of this theory is that there exists a sub-group of tumor cells, like stem cells of normal tissues, with stem traits characterizing growth stasis and self-renewal with specific cell surface markers. This subset of cells within the tumor bulk is known as "cancer stem cells" (CSCs) or "tumor initiating cells (TICs)". Other tumor cells that are considered as progeny of CSCs would face a final differentiation followed by programmed cell death. In term of CSC theory, tumor bulk only originates from CSCs; therapeutic failures in cancer management are a result of insufficient elimination of these heterogeneous subpopulation. In addition, the subset is believed to play important roles in invasion, metastasis and therapeutic resistance in various malignancies. To date, CSCs or CSC like cells have been identified in different cancer including HNSCC. In 2007, a subpopulation of cells with characterized stemness and CD44 marker in HNSCC was first isolated and identified (Prince et al, 2007). It was also demonstrated that CD133+ cells in Hep-2 human squamous laryngeal carcinoma cells have stem cell-like characters (Zhou et al., 2009). Subsequently, other investigators identified a CD133+ CSC-like subset with chemoresistance in oral squamous carcinoma (Zhang et al., 2009). More recently, CD44+ CSCs have also been isolated and identified from human laryngeal squamous carcinoma. All these data confirm the existence of CSCs in HNSCC, which shed light on a novel area to get further insight into the mechanisms of

The microenvironment of CSCs also called "niche", consists of cellular and non-cellular components surrounding CSCs (Scadden, 2006), including direct cell contacts, cell-matrix

**3.4.3 Hypoxic duration- and/or degree-related responses** 

hypoxic time.

**resistance in HNSCC** 

**4.1 Identification of CSCs in HNSCC** 

chemo- and radioresistance in HNSCC with respect to SCCs.

**4.2 CSCs and microenvironment 4.2.1 "Seeds and soil" theory** 

Species are selected by adaptation to environmental changes as proven by the earliest dinosaurs to today's diverse biological species. Such is Darwinian evolution, a possible explanation for survival. The development of tumors might be a process of survival of the fittest by the pressure of microenvironment.

It is known to all that cells in various types of tumors are different from each other in many aspects, such as size, appearance, antigen expression, cell membrane composition and sensitivity to different treatment modalities (Ichim & Wells, 2006; Heppner, 1984; Axelson, 2005). There are two explanatory models to the potential heterogeneity of the tumor cells, the stochastic model and the hierarchical model. Firstly, the stochastic model attributes the tumor development to the "genetic instability", through which the ones best adapting to the microenvironment are selected to obtain the advantage of proliferation (Nowell, 1976; Tysnes & Bjerkvig, 2007). This model shows that the tumor parenchyma contains many types of tumor cells with the ability to form tumors in the microenvironment (Bjerkvig, 2009). Secondly, the hierarchical model supports that the initial tumor and the metastatic tumor are both evolved from CSCs, which seems contradicting to the first model.

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

reside in G0/G1 phase, which reduces efficacy of anti-cancer agents.

**4.2.4.2 Overexpression of protective genes** 

inhibitors, survival of HCC CSCs can be abolished.

status in each individual tumor is evaluated objectively.

**5.1 Targeting genes related with hypoxia** 

**5. How to cope with the therapeutic resistance induced by hypoxia** 

Based on the mentioned above, it is evident that blockage of paradoxical activation of genes by transgenic techniques or improvement of hypoxic status in tumor microenvironment could overcome hypoxia-induced therapeutic resistance and relapse in HNSCC. Gene therapy mostly pointing to some critical target genes and associated gene products in HIF-1α, UPR and mTOR pathways and some activators and regulators of HIF-1α in alternative pathways such as EGFR and STAT3 pathways offers hope in this regard. However, transgenic techniques using either viral or non-viral vectors have limitations for application in human body. As previously described, dynamic hypoxic heterogeneity exists in solid tumors. It is difficult to determine the specificity and effectiveness of a single-gene targeted therapy to hypoxic cells in a huge tumor bulk. Therefore, there should be a long-term exploration before gene therapy can be used as an efficient method of modifying therapeutic resistance in HNSCC. It is likely that a strategy targeting multiple genes would be a potential solution to CSC-associated therapeutic resistance under the condition that hypoxic

categorized as follows.

**4.2.4.1 Quiescence** 

Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell 225

vitro. Currently, the mechanisms of CSCs-related therapeutic resistance have not been well understood. Perhaps, basic principles regarding CSC-caused therapeutic resistance can be

Lots of cytotoxic drugs mostly kill tumor cells with rapid proliferation, which settle in cellular s-phase cycle. However, it is believed that CSCs, like normal stem cells, mostly

Another cause of CSCs-related resistance is that this subset overexpresses some factors that protect CSCs from apoptosis and cytotoxicity. It has been well confirmed that CSCs express high levels of ABC drug transporters. Due to ATP hydrolysis, these proteins function to efflux drugs from tumor cells to protect against cytotoxicity. ABC superfamily includes 7 subfamilies from ABCA to ABCG (ABCB1 is P-gp). Among the superfamily, ABCG2 has been studied most extensively and is believed to be the most critical transporter of drugs. However, in clinic, targeting on ABCG2 alone has a minimal effect in the correction of chemorisistance by cancers, suggesting other ABC components also participate in chemoresistance or CSC's resistance is not determined only by ABC transporters. Liu et al (Liu et al, 2006) isolated CD133-positive tumor cells from glioblastoma and demonstrated that along with ABC transporters, these CSCs overexpressed anti-apoptotic factors, such as BCL-XL, Xiap, Survivin as well as cIAPs and DNA repair protein MGMT, which suggests that powerful repair ability combined with anti-apoptotic features may be partially responsible for CSC's resistance. Through studying CSCs from hepatocellular carcinoma (HCC), Ma et al (Ma et al, 2008) demonstrated that HCC CSCs confer chemorestistance via preferential induction of AKT/PKB and BCL-2 survival pathways. Using specific AKT1

The two controversial models are currently interpreted by some recent investigations. Odoux et al (Odoux et al, 2008) found that a small number of CSCs subsets with the ability of self-renewal and differentiation exist in liver metastases in patients suffering from colon cancer, and this sub-group of cells are more invasive and expanding than the CSCs in the primary tumor. Surprisingly, as a considered decisive element in the evolution of tumor (Cahill et al, 2009), genetic instability was present in this subset. A recent genomics study found that aberrant stem cells significantly express the regulatory protein molecules which function to adapt to microenvironmental stimuli with respect to the gene expression profile of murine embryonic stem cell lines and its malignant counterpart, murine teratocarcinoma cell lines (Heffron, 2007). Campbell and Polyak (Campbell & Polyak, 2007) integrated the results of their research on breast cancer and a number of related reports, and ended up with that the heterogeneity of tumor cells may be due to the combination of some levels of the stochastic model and the hierarchical model. They found that the origin of breast cancer may initially be normal CD44-expression stem cells or progenitor cells, which undergo self-renewal, differentiation and mutation-driven clonal evolution motivated by the environmental changes and gene mutations, resulting in a bunch of different genotypes and diverse stages of development of tumor cells. This indicates that the hierarchical model in cancer stem cell theory is the extension of the stochastic model. Tumor tissues are evolved through genetic alterations, phenotypic changes and the impacts of micro-environment. So there may be more than one type of stem cell subsets with different characteristics in parenchyma, which exhibits different genetic or epigenetic phenotypes and ability to adapt to microenvironment, and the distinct characteristics of epithelial to mesenchymal transition (Werb & Evans, 2004). Therefore, not all CSCs have the ability to survive and metastasize, only those with the ability to adapt to the microenvironment are selected (Odoux, C et al, 2008; Lagasse, 2008) to do so.

#### **4.2.3 Hypoxia and CSCs**

As is known, CSCs are a subpopulation of tumor cells that co-exist with differentiated tumor cells in the same microenvironment, in which hypoxia serves as a necessary component for CSCs growth, self-renewal and differentiation (Keith & Simon, 2007). It has been demonstrated that HIF-1α could induce the expression of some crucial genes related with CSCs' self-renewal and multipotency, including Oct4 and Notch1. Under hypoxia, stable expression of HIF-1α in CSCs can stimulate expressions of Oct4 and Notch1, and activate the associated signaling of critical pathways, promoting specific properties of CSCs and related multipotency. In view of the mentioned above, we can introduce the concept of interaction between tumor hypoxic microenvironment and CSCs. Traditionally, the standard for evaluating the efficacy of a treatment regimen is the sizable contraction of the tumor bulk. However, most tumor relapse after a period of paracmasis, probably because traditional chemo- and radiotherapy only kill tumor cells in rapid proliferation and differentiation rather than CSCs, the latter of which with are in slow divisions and proliferation, conferring therapeutic resistance. The formation of the relapsed tumor is driven by CSCs under proper microenvironment at a certain time after the treatment regimens are completed.

#### **4.2.4 CSC's resistance and related mechanisms**

Lots of convincing data showed that CSCs subset displays powerful resistance to traditional chemo- and/or radio-therapy compared with non-CSCs of in same tumor or parent cells in

vitro. Currently, the mechanisms of CSCs-related therapeutic resistance have not been well understood. Perhaps, basic principles regarding CSC-caused therapeutic resistance can be categorized as follows.

#### **4.2.4.1 Quiescence**

224 Squamous Cell Carcinoma

The two controversial models are currently interpreted by some recent investigations. Odoux et al (Odoux et al, 2008) found that a small number of CSCs subsets with the ability of self-renewal and differentiation exist in liver metastases in patients suffering from colon cancer, and this sub-group of cells are more invasive and expanding than the CSCs in the primary tumor. Surprisingly, as a considered decisive element in the evolution of tumor (Cahill et al, 2009), genetic instability was present in this subset. A recent genomics study found that aberrant stem cells significantly express the regulatory protein molecules which function to adapt to microenvironmental stimuli with respect to the gene expression profile of murine embryonic stem cell lines and its malignant counterpart, murine teratocarcinoma cell lines (Heffron, 2007). Campbell and Polyak (Campbell & Polyak, 2007) integrated the results of their research on breast cancer and a number of related reports, and ended up with that the heterogeneity of tumor cells may be due to the combination of some levels of the stochastic model and the hierarchical model. They found that the origin of breast cancer may initially be normal CD44-expression stem cells or progenitor cells, which undergo self-renewal, differentiation and mutation-driven clonal evolution motivated by the environmental changes and gene mutations, resulting in a bunch of different genotypes and diverse stages of development of tumor cells. This indicates that the hierarchical model in cancer stem cell theory is the extension of the stochastic model. Tumor tissues are evolved through genetic alterations, phenotypic changes and the impacts of micro-environment. So there may be more than one type of stem cell subsets with different characteristics in parenchyma, which exhibits different genetic or epigenetic phenotypes and ability to adapt to microenvironment, and the distinct characteristics of epithelial to mesenchymal transition (Werb & Evans, 2004). Therefore, not all CSCs have the ability to survive and metastasize, only those with the ability to adapt to the microenvironment are selected (Odoux, C et al, 2008; Lagasse, 2008) to do so.

As is known, CSCs are a subpopulation of tumor cells that co-exist with differentiated tumor cells in the same microenvironment, in which hypoxia serves as a necessary component for CSCs growth, self-renewal and differentiation (Keith & Simon, 2007). It has been demonstrated that HIF-1α could induce the expression of some crucial genes related with CSCs' self-renewal and multipotency, including Oct4 and Notch1. Under hypoxia, stable expression of HIF-1α in CSCs can stimulate expressions of Oct4 and Notch1, and activate the associated signaling of critical pathways, promoting specific properties of CSCs and related multipotency. In view of the mentioned above, we can introduce the concept of interaction between tumor hypoxic microenvironment and CSCs. Traditionally, the standard for evaluating the efficacy of a treatment regimen is the sizable contraction of the tumor bulk. However, most tumor relapse after a period of paracmasis, probably because traditional chemo- and radiotherapy only kill tumor cells in rapid proliferation and differentiation rather than CSCs, the latter of which with are in slow divisions and proliferation, conferring therapeutic resistance. The formation of the relapsed tumor is driven by CSCs under proper microenvironment at a certain time after the treatment

Lots of convincing data showed that CSCs subset displays powerful resistance to traditional chemo- and/or radio-therapy compared with non-CSCs of in same tumor or parent cells in

**4.2.3 Hypoxia and CSCs** 

regimens are completed.

**4.2.4 CSC's resistance and related mechanisms** 

Lots of cytotoxic drugs mostly kill tumor cells with rapid proliferation, which settle in cellular s-phase cycle. However, it is believed that CSCs, like normal stem cells, mostly reside in G0/G1 phase, which reduces efficacy of anti-cancer agents.

#### **4.2.4.2 Overexpression of protective genes**

Another cause of CSCs-related resistance is that this subset overexpresses some factors that protect CSCs from apoptosis and cytotoxicity. It has been well confirmed that CSCs express high levels of ABC drug transporters. Due to ATP hydrolysis, these proteins function to efflux drugs from tumor cells to protect against cytotoxicity. ABC superfamily includes 7 subfamilies from ABCA to ABCG (ABCB1 is P-gp). Among the superfamily, ABCG2 has been studied most extensively and is believed to be the most critical transporter of drugs. However, in clinic, targeting on ABCG2 alone has a minimal effect in the correction of chemorisistance by cancers, suggesting other ABC components also participate in chemoresistance or CSC's resistance is not determined only by ABC transporters. Liu et al (Liu et al, 2006) isolated CD133-positive tumor cells from glioblastoma and demonstrated that along with ABC transporters, these CSCs overexpressed anti-apoptotic factors, such as BCL-XL, Xiap, Survivin as well as cIAPs and DNA repair protein MGMT, which suggests that powerful repair ability combined with anti-apoptotic features may be partially responsible for CSC's resistance. Through studying CSCs from hepatocellular carcinoma (HCC), Ma et al (Ma et al, 2008) demonstrated that HCC CSCs confer chemorestistance via preferential induction of AKT/PKB and BCL-2 survival pathways. Using specific AKT1 inhibitors, survival of HCC CSCs can be abolished.

## **5. How to cope with the therapeutic resistance induced by hypoxia**

#### **5.1 Targeting genes related with hypoxia**

Based on the mentioned above, it is evident that blockage of paradoxical activation of genes by transgenic techniques or improvement of hypoxic status in tumor microenvironment could overcome hypoxia-induced therapeutic resistance and relapse in HNSCC. Gene therapy mostly pointing to some critical target genes and associated gene products in HIF-1α, UPR and mTOR pathways and some activators and regulators of HIF-1α in alternative pathways such as EGFR and STAT3 pathways offers hope in this regard. However, transgenic techniques using either viral or non-viral vectors have limitations for application in human body. As previously described, dynamic hypoxic heterogeneity exists in solid tumors. It is difficult to determine the specificity and effectiveness of a single-gene targeted therapy to hypoxic cells in a huge tumor bulk. Therefore, there should be a long-term exploration before gene therapy can be used as an efficient method of modifying therapeutic resistance in HNSCC. It is likely that a strategy targeting multiple genes would be a potential solution to CSC-associated therapeutic resistance under the condition that hypoxic status in each individual tumor is evaluated objectively.

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

inhibition could achieve potential efficiency.

**5.4 Chopping off hypoxia from the "root"** 

fluctuating and continuous hypoxia from the "boot".

susceptible to chemoratiation.

**6. Future directions** 

Cell Carcinoma (HNSCC) – Roles of Hypoxic Microenvironment and Cancer Stem Cell 227

death under severe stress. Induction of UPR pro-death arms may be a promising modality to reverse hypoxia-induced resistance to traditional therapy. At this point, some agents, such as PIs, which can enhance ER overload, represent a promising perspective. PIs inhibit proteasome to reduce ERAD, which can intensify ER-stress caused by accumulation of unfolded protein. Recently, Fels et al (Fels et al, 2008) found that PIs can effectively enhance UPR responses of hypoxic tumor cells and ameliorating ER-stress can reverse PIs effects. They demonstrated that hypoxic tumor cells treated by PIs underwent apoptosis, autophagy and necrosis. Intriguingly, some groups reported that tumor cells can activate STAT3 to resist PIs therapy in HNSCC (C. Li et al, 2009). Therefore, PIs combined with STAT3

For strategies used to improve local hypoxia, some groups have tried using inhalation of Carbogen(95%O2 and 5%CO2)and hyperbaric oxygen chambers to improve local hypoxic condition within tumors, and thus therapeutic resistance, but the results are not as satisfactory. In this regard, it is necessary to modify traditional approaches and to explore a new way of oxygen delivery to rectify the intratumoral hypoxic condition. It is a common sense that vascular structure of tumor is very different from its counterpart of normal tissue, the former of which exhibits architectural distortion, higher permeability and irregular infuse, facilitating fluctuating hypoxia and providing specific target strategy. Vascular disrupting agents (VDAs) serve as a novel type of anti-cancer target agent. In contrast with angiogenesis inhibitors (AIs) that mostly prevent neoformation of vascular structure, VDAs directly block or damage existing blood vessels in tumor bulk to commit necrosis. To date, VDAs have been in phase of clinic trails, and small molecular VDAs have been mostly studied. The mechanisms of VDAs action include: 1) induce TNF-α secretion by tumor cells to cause apoptosis of endothelial cells constituting microvessels; 2) through binding to microtubule protein, VDAs facilitate disaggregation of microtubules to damage cell skeleton of vascular endothelium. VDAs have been believed to cause intratumoral necrosis, leaving the remaining periphery to be oxygenated. Therefore, combination of VDAs may cut both

Although improvement of tumor hypoxia has been achieved, imaging results are not always consistent with changes of endogenous markers of hypoxia, indicating that the improvement of intratumor hypoxia as observed by imaging does not represent the thorough rectification of intracellular hypoxic metabolisms of the cancer cells. Therefore, it is highly likely that there exist a "time gap" between improvement of intratumor hypoxia and thorough rectification of intracellular hypoxic metabolisms. Currently, length of this window phase is unclear. It is of paramount importance for hypoxic cells to gain thorough recovery of the intracellular oxygenation using this compensation time and become more

Up till now, the impact of hypoxia on CSCs in HNSCC and its relation between chemo- and radiotherapeutic resistance is largely unknown. To further elucidate the causes of chemoand radiotherapeutic resistance and post-treatment relapse in HNSCC with respect to effects

#### **5.2 Targeting CSCs and hypoxic microenvironment**

As stated, only CSCs can facilitate tumorigenesis and confer therapeutic resistance, which is the major cause of therapeutic failure. Therefore, successful targeting on CSCs is expected to provide a chance of cure for cancers. To date, targeting on CSCs has been faced with difficulties, because mechanisms underlying CSC-related therapeutic resistance have not been well understood. Although Notch, Oct-4, Wnt, Bmi and other stemness related factors were demonstrated to play a critical role in CSCs physiology, it is difficult to target them specifically in CSCs among the huge population of cancer cells. It is interesting to note that the clinical course of anti-ABCG-2 drugs in cancer treatment mirrors that of anti-bacterial agents in the control of infection. Based on this observation, some scholars believe that CSCs also experience evolutionary processes, and the driving force for these processes, selection stress by microenvironment, should be the target for cancer therapy. Although more CSCs markers have been identified in cancers, CSCs isolated by these markers are in minority, approximately 2-5%. However, increasing evidence has revealed that CSCs are not rare when isolated based on stem traits, which suggests that CSC markers are limited and not all CSCs express the same markers. Therefore, it is possible that CSCs are existing in separate subpopulations with distinct biological features, which are affected by their niche, and what is worse, these features are in constant change. Given that targeting CSCs is a putative approach, it would be much more important to concentrate on the manipulation of niche as the direct target in curing cancers. For example, we can resort to approaches to maintain the homeostasis of the niche by manipulating non-cellular components, especially fluctuating hypoxia. Consistent with this idea, traditional Chinese medicine (TCM) is to cure the disease by rectifying imbalance in body environment and re-establish the homeostasis of the human body, which may offer some hope in this regard. And intriguingly, lots of herbs have been identified as antioxidant compounds. Cai et al (Cai et al, 2004) have demonstrated that 112 traditional chinese medicinal plants used as anti-cancer herbs have a more powerful antioxidant activity compared with common vegetables and fruits which are considered as good natural sources of dietary antioxidants. Tang et al (Tang et al, 2004) also identified the antioxidant function of TCM extracts. These pieces of evidence implicate that TCM is a promising strategy capable of targeting on ROS-induced evolution of CSCs. Indeed, data from several reports have provided direct evidence that some herbs in TCM could target CSCs. Observations made by Jiang et al (Jiang et al, 1983) demonstrated that camptothecin and harringtonin could inhibit the clonal formation of human stem cells. Furthermore, antitumor and therapeutic resistance-reversing effects of some phytochemicals such as Curcumin have been confirmed and proven to be prospective, which exhibit the capability of targeting side population cells (Fong et al, 2010). More recently, high inhibitory effect on breast cancer cells was acquired by combining stealthy liposomes from vinorelbine and parthenolide (Liu et al, 2008). Taken together, chemotherapy combined with TCM may dominate anti-cancer treatment if the niche is properly manipulated to overcome the chemoresisitance of CSCs resulting from genetic instability.

#### **5.3 Inducing UPR pro-death arms**

As is known, UPR is a double-edged sword. On the one side, it can help tumor cells relieve hypoxic stress; On the other hand, UPR can induce apoptosis or autophagy-related cell death under severe stress. Induction of UPR pro-death arms may be a promising modality to reverse hypoxia-induced resistance to traditional therapy. At this point, some agents, such as PIs, which can enhance ER overload, represent a promising perspective. PIs inhibit proteasome to reduce ERAD, which can intensify ER-stress caused by accumulation of unfolded protein. Recently, Fels et al (Fels et al, 2008) found that PIs can effectively enhance UPR responses of hypoxic tumor cells and ameliorating ER-stress can reverse PIs effects. They demonstrated that hypoxic tumor cells treated by PIs underwent apoptosis, autophagy and necrosis. Intriguingly, some groups reported that tumor cells can activate STAT3 to resist PIs therapy in HNSCC (C. Li et al, 2009). Therefore, PIs combined with STAT3 inhibition could achieve potential efficiency.

## **5.4 Chopping off hypoxia from the "root"**

226 Squamous Cell Carcinoma

As stated, only CSCs can facilitate tumorigenesis and confer therapeutic resistance, which is the major cause of therapeutic failure. Therefore, successful targeting on CSCs is expected to provide a chance of cure for cancers. To date, targeting on CSCs has been faced with difficulties, because mechanisms underlying CSC-related therapeutic resistance have not been well understood. Although Notch, Oct-4, Wnt, Bmi and other stemness related factors were demonstrated to play a critical role in CSCs physiology, it is difficult to target them specifically in CSCs among the huge population of cancer cells. It is interesting to note that the clinical course of anti-ABCG-2 drugs in cancer treatment mirrors that of anti-bacterial agents in the control of infection. Based on this observation, some scholars believe that CSCs also experience evolutionary processes, and the driving force for these processes, selection stress by microenvironment, should be the target for cancer therapy. Although more CSCs markers have been identified in cancers, CSCs isolated by these markers are in minority, approximately 2-5%. However, increasing evidence has revealed that CSCs are not rare when isolated based on stem traits, which suggests that CSC markers are limited and not all CSCs express the same markers. Therefore, it is possible that CSCs are existing in separate subpopulations with distinct biological features, which are affected by their niche, and what is worse, these features are in constant change. Given that targeting CSCs is a putative approach, it would be much more important to concentrate on the manipulation of niche as the direct target in curing cancers. For example, we can resort to approaches to maintain the homeostasis of the niche by manipulating non-cellular components, especially fluctuating hypoxia. Consistent with this idea, traditional Chinese medicine (TCM) is to cure the disease by rectifying imbalance in body environment and re-establish the homeostasis of the human body, which may offer some hope in this regard. And intriguingly, lots of herbs have been identified as antioxidant compounds. Cai et al (Cai et al, 2004) have demonstrated that 112 traditional chinese medicinal plants used as anti-cancer herbs have a more powerful antioxidant activity compared with common vegetables and fruits which are considered as good natural sources of dietary antioxidants. Tang et al (Tang et al, 2004) also identified the antioxidant function of TCM extracts. These pieces of evidence implicate that TCM is a promising strategy capable of targeting on ROS-induced evolution of CSCs. Indeed, data from several reports have provided direct evidence that some herbs in TCM could target CSCs. Observations made by Jiang et al (Jiang et al, 1983) demonstrated that camptothecin and harringtonin could inhibit the clonal formation of human stem cells. Furthermore, antitumor and therapeutic resistance-reversing effects of some phytochemicals such as Curcumin have been confirmed and proven to be prospective, which exhibit the capability of targeting side population cells (Fong et al, 2010). More recently, high inhibitory effect on breast cancer cells was acquired by combining stealthy liposomes from vinorelbine and parthenolide (Liu et al, 2008). Taken together, chemotherapy combined with TCM may dominate anti-cancer treatment if the niche is properly manipulated to overcome the

**5.2 Targeting CSCs and hypoxic microenvironment** 

chemoresisitance of CSCs resulting from genetic instability.

As is known, UPR is a double-edged sword. On the one side, it can help tumor cells relieve hypoxic stress; On the other hand, UPR can induce apoptosis or autophagy-related cell

**5.3 Inducing UPR pro-death arms** 

For strategies used to improve local hypoxia, some groups have tried using inhalation of Carbogen(95%O2 and 5%CO2)and hyperbaric oxygen chambers to improve local hypoxic condition within tumors, and thus therapeutic resistance, but the results are not as satisfactory. In this regard, it is necessary to modify traditional approaches and to explore a new way of oxygen delivery to rectify the intratumoral hypoxic condition. It is a common sense that vascular structure of tumor is very different from its counterpart of normal tissue, the former of which exhibits architectural distortion, higher permeability and irregular infuse, facilitating fluctuating hypoxia and providing specific target strategy. Vascular disrupting agents (VDAs) serve as a novel type of anti-cancer target agent. In contrast with angiogenesis inhibitors (AIs) that mostly prevent neoformation of vascular structure, VDAs directly block or damage existing blood vessels in tumor bulk to commit necrosis. To date, VDAs have been in phase of clinic trails, and small molecular VDAs have been mostly studied. The mechanisms of VDAs action include: 1) induce TNF-α secretion by tumor cells to cause apoptosis of endothelial cells constituting microvessels; 2) through binding to microtubule protein, VDAs facilitate disaggregation of microtubules to damage cell skeleton of vascular endothelium. VDAs have been believed to cause intratumoral necrosis, leaving the remaining periphery to be oxygenated. Therefore, combination of VDAs may cut both fluctuating and continuous hypoxia from the "boot".

Although improvement of tumor hypoxia has been achieved, imaging results are not always consistent with changes of endogenous markers of hypoxia, indicating that the improvement of intratumor hypoxia as observed by imaging does not represent the thorough rectification of intracellular hypoxic metabolisms of the cancer cells. Therefore, it is highly likely that there exist a "time gap" between improvement of intratumor hypoxia and thorough rectification of intracellular hypoxic metabolisms. Currently, length of this window phase is unclear. It is of paramount importance for hypoxic cells to gain thorough recovery of the intracellular oxygenation using this compensation time and become more susceptible to chemoratiation.

## **6. Future directions**

Up till now, the impact of hypoxia on CSCs in HNSCC and its relation between chemo- and radiotherapeutic resistance is largely unknown. To further elucidate the causes of chemoand radiotherapeutic resistance and post-treatment relapse in HNSCC with respect to effects

Molecular Mechanisms Involving Therapeutic Resistance in Head and Neck Squamous

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The established concept of interaction between tumor hypoxic microenvironment and CSCs helps us to further understand the mechanisms behind the therapeutic resistance in HNSCC. If CSCs are taken as anti-cancer targets, the strategies by focusing on tumor microenvironment will be promising for purposely intervention of CSCs (Iwasaki & Suda, 2009). It can be inferred that CSCs are the critical element responsible for therapeutic resistance in HNSCC. Improving hypoxic conditions and regulating CSCs-related signaling pathways during chemo- and radiotherapy of HNSCC offers hope for reversion of therapeutic sensitivity in HNSCC and elimination of therapeutic resistance and relapse, aiming at improving outcomes of HNSCC.

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**14** 

*Spain* 

**New Therapeutic Strategies in Small** 

**Cell Lung Cancer: The Stem Cell Target** 

*1Biomedical Research Institute INIBIC. Coruña University Hospital. A Coruña* 

In 1889, Sir S. Paget introduced the *soil and seed* hypothesis of metastasis to medicine and credited the idea to Fuchs. In Paget´s study, he concluded that the distribution of metastases cannot be due to chance alone and that different tissues provide optimal conditions for the growth of specific cancers. In the *soil and seed* metaphor, the *soil* refers to the secondary site of tumour growth and development and perhaps the chemical signals produced in the microenvironment at the sites of metastasis. The *seed* is the ostensible stem cell or tumourinitiating cell from the primary tumour. These tumour-initiating cells are the tumorigenic force behind tumour initiation, growth, metastasis, drug resistance, and relapse. In a variation of this idea, called the *homing* hypothesis, a secondary signal secreted by cells at the future metastatic sites "calls*"* the tumour cells to the site and permits them to proliferate in the new environment. In this hypothesis, the *seed* produces cell surface receptors that are able to recognise the site demarcated by the *soil*. Although the mechanisms that define tissue specificity remain obscure, researchers have focused on small messenger molecules as attractants and larger cell surface receptors that guide the tumour-initiating cells*.* Based on the hypothesis introduced by Paget, other groups have focused on chemokines and their receptors as viable candidates for *soil and seed* signalling and have proposed a "spatial and temporal code" composed of specific combinations of such molecules, while other molecules are responsible for neovascularisation, metastasis, and immunosurveillance avoidance. Lung cancers result from complex genetic and epigenetic changes and are characterised by stepwise malignant progression of cancer cells with an associated accumulation of genetic alterations. This process, referred to as multistep carcinogenesis, develops through the clonal evolution of initiated lung cells. Initiation consists of the acquisition of defined genetic alterations in a small number of genes that confer a proliferative advantage and facilitate progression towards invasive neoplasia. Although many of these genetic changes occur independently of histological type, their frequency and timing of occurrence with respect to cancer progression differ between small cell lung carcinomas (SCLC), which may originate from epithelial cells with neuroendocrine features, and non-SCLCs, which

**1. Introduction** 

Guadalupe Aparicio Gallego1, Vanessa Medina Villaamil1, Guillermo Alonso Curbera2 and L. Antón Aparicio1,2,3

*2Medical Oncology Service. A Coruña University Hospital. A Coruña* 

*3Medicine Department. University of A Coruña* 


## **New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target**

Guadalupe Aparicio Gallego1, Vanessa Medina Villaamil1, Guillermo Alonso Curbera2 and L. Antón Aparicio1,2,3 *1Biomedical Research Institute INIBIC. Coruña University Hospital. A Coruña 2Medical Oncology Service. A Coruña University Hospital. A Coruña 3Medicine Department. University of A Coruña Spain* 

#### **1. Introduction**

232 Squamous Cell Carcinoma

Werb, Z. & Evans, G. (2004). Oncogenes and cell proliferation: maintenance of genomic

Zhou, L., Wei, X., Cheng, L., Tian, J. & Jiang, JJ. (2007). CD133, one of the markers of

in Genetics and Development, Vol. 14 No. (2), pp. 1–3. ISSN 0959-437X Zhang, Q., Shi, S., Yen, Y., Brown, J., Ta, JQ. & Le, AD. (2009). A subpopulation of

pp. 151–160. ISSN 0304-3835

ISSN 0023-852X

integrity, tumor stem cells, and the somatic microenvironment. Current Opinion

CD133(+) cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy. Cancer Letters, Vol. 289 No. (2),

cancer stem cells in Hep-2 cell line. Laryngoscope, Vol. 117 No. (3), pp. 455–460.

In 1889, Sir S. Paget introduced the *soil and seed* hypothesis of metastasis to medicine and credited the idea to Fuchs. In Paget´s study, he concluded that the distribution of metastases cannot be due to chance alone and that different tissues provide optimal conditions for the growth of specific cancers. In the *soil and seed* metaphor, the *soil* refers to the secondary site of tumour growth and development and perhaps the chemical signals produced in the microenvironment at the sites of metastasis. The *seed* is the ostensible stem cell or tumourinitiating cell from the primary tumour. These tumour-initiating cells are the tumorigenic force behind tumour initiation, growth, metastasis, drug resistance, and relapse. In a variation of this idea, called the *homing* hypothesis, a secondary signal secreted by cells at the future metastatic sites "calls*"* the tumour cells to the site and permits them to proliferate in the new environment. In this hypothesis, the *seed* produces cell surface receptors that are able to recognise the site demarcated by the *soil*. Although the mechanisms that define tissue specificity remain obscure, researchers have focused on small messenger molecules as attractants and larger cell surface receptors that guide the tumour-initiating cells*.* Based on the hypothesis introduced by Paget, other groups have focused on chemokines and their receptors as viable candidates for *soil and seed* signalling and have proposed a "spatial and temporal code" composed of specific combinations of such molecules, while other molecules are responsible for neovascularisation, metastasis, and immunosurveillance avoidance. Lung cancers result from complex genetic and epigenetic changes and are characterised by stepwise malignant progression of cancer cells with an associated accumulation of genetic alterations. This process, referred to as multistep carcinogenesis, develops through the clonal evolution of initiated lung cells. Initiation consists of the acquisition of defined genetic alterations in a small number of genes that confer a proliferative advantage and facilitate progression towards invasive neoplasia. Although many of these genetic changes occur independently of histological type, their frequency and timing of occurrence with respect to cancer progression differ between small cell lung carcinomas (SCLC), which may originate from epithelial cells with neuroendocrine features, and non-SCLCs, which

New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target 235

and cytodifferentiation. This mutual interaction is defined as embryonic induction. The morphogenesis and cytodifferentiation of embryonic lung epithelial components are modulated by surrounding mesenchymal components. In embryonic organs that are formed by a process of progressive branching of the epithelium, such as the lung, the mesenchyme plays a determining role in the formation of the characteristic morphology of the organ. Increasing evidence has suggested that the formation of the tracheo-bronchial tree and alveoli results from heterogeneity of the epithelial-mesenchymal interactions along the developing respiratory tract. Genetic data have supported this idea and shown that this heterogeneity is likely the result of activation of distinct networks of signalling molecules along the proximal-distal axis. Among these signals, fibroblast growth factors, retinoids, Sonic hedgehog and transforming growth factors appear to play prominent roles. Variable levels of FGFs, Shh, TGFβ, EGF, retinoid receptors, and other signals that play a role in lung morphogenesis have been reported in the adult lung. Increasing genetic evidence has suggested that the Gli genes play multiple roles during prenatal development, particularly in the lung. All three genes are widely expressed during embryonic development in distinct but sometimes overlapping domains. The extent to which these regulators are expressed during adult life to mediate cellular activities in processes such as post-injury repair and compensatory lung growth is currently unclear. Lung bud initiation has been wellestablished to be regulated by the Sonic hedgehog (Shh) signalling pathway, by fibroblast growth factor (FGF) receptor signalling, and likely by retinoid-related signalling. Branching morphogenesis is a dichotomous branching process that involves defining the proximaldistal structure of the conducting airway prior to the saccular stage and is dependent on the integrated effects of the conducting airway prior to the saccular stage. Several growth factors have been implicated in branching morphogenesis. Epidermal growth factor (EGF) and transforming growth factor (TGFα) are expressed in embryonic murine lung; both factors influence growth and branching morphogenesis. During early lung branching, the EGF protein is present in bronchial epithelial cells, whereas the EGF mRNA is localised to the mesenchyme; this discordance between the location of the protein and mRNA suggests that EGF is produced by the mesenchyme and acts on the epithelium. EGF receptors (EGFR) have been found in epithelial cells and in the mesenchyme surrounding the branching epithelium of the mouse lung. These data are compatible with the notion that EGF acts in an autocrine and paracrine fashion. Retinoic acid (RA) and glucocorticoid signalling pathways have long been appreciated as major contributors to prenatal and postnatal lung maturation, and some evidence exists for their coordination or antagonism during lung development. Retinoic acid also plays an important role in morphogenesis. RA stimulates lung epithelial branching activity via an epithelial-mesenchymal interaction that, in part, involves the upregulation of the expression of EGFR, Insulin-like Growth Factors (IGF), basic Fibroblast

For several years, a consensus has been achieved that various types of stem cells exist, differing according to their position within the pulmonary tree, and that the stem cells often form pools that are ready to proliferate in response to injury and effect local repair. The classical subdivision of the airway tree into regions with individual stem cell harbours was

Growth Factor (bFGF-2), and PDGF.

**3. The airway stem cells** 

originate from bronchial, bronchiolar or alveolar epithelial cells. Furthermore, a number of genetic and epigenetic differences have been identified between squamous cell carcinoma (SCC), which arises from bronchial epithelial cells through a squamous metaplasia/dysplasia process, and adenocarcinoma (ADC), which is derived from alveolar or bronchiolar epithelial cells. Hence, lung tumours have been classified according to tumour morphology, but classification is complicated by the fact that a number of different histologic tumour characteristics frequently exist within the same neoplasm. In the 1990s, SCLC accounted for approximately one-quarter of all lung cancers, but a recent Surveillance Epidemiology and End Results (SEER) database analysis found that the incidence has since decreased to approximately 13%. SCLC now accounts for 15% of all newly diagnosed lung cancers and 60% to 70% of patients present with extensive stage (ES) tumours. For patients with limited-stage (LS)-SCLC, standard treatment has consisted of chemotherapy combined with radiotherapy (RT), while chemotherapy alone has been the standard for ES-SCLC patients. Despite a high initial rate of response to chemotherapy, most patients die from rapid recurrence. The median range of survival time after diagnosis for patients with ES-SCLC is 8 to 10 months, and only 5% to 10% of patients survive for as long as 2 years. Although chemotherapy is an essential component in the treatment of SCLC, improvements in survival in the past two decades have primarily been achieved through the appropriate application of radiotherapy. The standard treatment for patients outside of clinical trials is as follows: LS-SCLC patients receive combination chemotherapy, which generally consists of cisplatin and etoposide, with concurrent thoracic radiotherapy; and ES-SCLC patients receive combination chemotherapy (etoposide and cisplatin or carboplatin). The current standard treatment for most cancers involves some combination of chemotherapy, hormonal therapy, radiation treatment, and a growing list of molecularly targeted therapeutics, depending on the tumour characteristics and stage. Following treatment, tumour regression is normally used as an indicator of therapeutic success. To better treat cancer, the new ideas regarding CSCs must be integrated into our strategies for clinical intervention. One approach to inhibit cancer stem cells is to target the proteins that are essential for the growth and maintenance of stem cells, such as the growth regulatory pathways that function in embryonic cells. One pathway, controlled by the Hedgehog (Hh) signalling molecule, contains several genes that function as either tumour suppressor genes or oncogenes. Other pathways that are critical to embryonic development and are potentially important in cancer have also been described, including the Wnt and Notch pathways. These pathways are also subjects of drug development for the treatment of a number of conditions.

#### **2. Development of the airway**

The respiratory system is an outgrowth of the ventral wall of the foregut, and the epithelium of the larynx, trachea, bronchi, and alveoli originates in the endoderm. The cartilaginous, muscular, and connective tissue components arise in the mesoderm. In the fourth week of development, the tracheo-oesophageal septum separates the trachea from the foregut, dividing the foregut into the lung bud anteriorly and the oesophagus posteriorly. Lungs are composed of two primary tissue layers, namely epithelium and mesenchyme. Previous investigations have demonstrated that mutual interactions between these two tissues are essential for the sequential events of organogenesis, determination, growth, morphogenesis,

originate from bronchial, bronchiolar or alveolar epithelial cells. Furthermore, a number of genetic and epigenetic differences have been identified between squamous cell carcinoma (SCC), which arises from bronchial epithelial cells through a squamous metaplasia/dysplasia process, and adenocarcinoma (ADC), which is derived from alveolar or bronchiolar epithelial cells. Hence, lung tumours have been classified according to tumour morphology, but classification is complicated by the fact that a number of different histologic tumour characteristics frequently exist within the same neoplasm. In the 1990s, SCLC accounted for approximately one-quarter of all lung cancers, but a recent Surveillance Epidemiology and End Results (SEER) database analysis found that the incidence has since decreased to approximately 13%. SCLC now accounts for 15% of all newly diagnosed lung cancers and 60% to 70% of patients present with extensive stage (ES) tumours. For patients with limited-stage (LS)-SCLC, standard treatment has consisted of chemotherapy combined with radiotherapy (RT), while chemotherapy alone has been the standard for ES-SCLC patients. Despite a high initial rate of response to chemotherapy, most patients die from rapid recurrence. The median range of survival time after diagnosis for patients with ES-SCLC is 8 to 10 months, and only 5% to 10% of patients survive for as long as 2 years. Although chemotherapy is an essential component in the treatment of SCLC, improvements in survival in the past two decades have primarily been achieved through the appropriate application of radiotherapy. The standard treatment for patients outside of clinical trials is as follows: LS-SCLC patients receive combination chemotherapy, which generally consists of cisplatin and etoposide, with concurrent thoracic radiotherapy; and ES-SCLC patients receive combination chemotherapy (etoposide and cisplatin or carboplatin). The current standard treatment for most cancers involves some combination of chemotherapy, hormonal therapy, radiation treatment, and a growing list of molecularly targeted therapeutics, depending on the tumour characteristics and stage. Following treatment, tumour regression is normally used as an indicator of therapeutic success. To better treat cancer, the new ideas regarding CSCs must be integrated into our strategies for clinical intervention. One approach to inhibit cancer stem cells is to target the proteins that are essential for the growth and maintenance of stem cells, such as the growth regulatory pathways that function in embryonic cells. One pathway, controlled by the Hedgehog (Hh) signalling molecule, contains several genes that function as either tumour suppressor genes or oncogenes. Other pathways that are critical to embryonic development and are potentially important in cancer have also been described, including the Wnt and Notch pathways. These pathways are also

subjects of drug development for the treatment of a number of conditions.

The respiratory system is an outgrowth of the ventral wall of the foregut, and the epithelium of the larynx, trachea, bronchi, and alveoli originates in the endoderm. The cartilaginous, muscular, and connective tissue components arise in the mesoderm. In the fourth week of development, the tracheo-oesophageal septum separates the trachea from the foregut, dividing the foregut into the lung bud anteriorly and the oesophagus posteriorly. Lungs are composed of two primary tissue layers, namely epithelium and mesenchyme. Previous investigations have demonstrated that mutual interactions between these two tissues are essential for the sequential events of organogenesis, determination, growth, morphogenesis,

**2. Development of the airway** 

and cytodifferentiation. This mutual interaction is defined as embryonic induction. The morphogenesis and cytodifferentiation of embryonic lung epithelial components are modulated by surrounding mesenchymal components. In embryonic organs that are formed by a process of progressive branching of the epithelium, such as the lung, the mesenchyme plays a determining role in the formation of the characteristic morphology of the organ. Increasing evidence has suggested that the formation of the tracheo-bronchial tree and alveoli results from heterogeneity of the epithelial-mesenchymal interactions along the developing respiratory tract. Genetic data have supported this idea and shown that this heterogeneity is likely the result of activation of distinct networks of signalling molecules along the proximal-distal axis. Among these signals, fibroblast growth factors, retinoids, Sonic hedgehog and transforming growth factors appear to play prominent roles. Variable levels of FGFs, Shh, TGFβ, EGF, retinoid receptors, and other signals that play a role in lung morphogenesis have been reported in the adult lung. Increasing genetic evidence has suggested that the Gli genes play multiple roles during prenatal development, particularly in the lung. All three genes are widely expressed during embryonic development in distinct but sometimes overlapping domains. The extent to which these regulators are expressed during adult life to mediate cellular activities in processes such as post-injury repair and compensatory lung growth is currently unclear. Lung bud initiation has been wellestablished to be regulated by the Sonic hedgehog (Shh) signalling pathway, by fibroblast growth factor (FGF) receptor signalling, and likely by retinoid-related signalling. Branching morphogenesis is a dichotomous branching process that involves defining the proximaldistal structure of the conducting airway prior to the saccular stage and is dependent on the integrated effects of the conducting airway prior to the saccular stage. Several growth factors have been implicated in branching morphogenesis. Epidermal growth factor (EGF) and transforming growth factor (TGFα) are expressed in embryonic murine lung; both factors influence growth and branching morphogenesis. During early lung branching, the EGF protein is present in bronchial epithelial cells, whereas the EGF mRNA is localised to the mesenchyme; this discordance between the location of the protein and mRNA suggests that EGF is produced by the mesenchyme and acts on the epithelium. EGF receptors (EGFR) have been found in epithelial cells and in the mesenchyme surrounding the branching epithelium of the mouse lung. These data are compatible with the notion that EGF acts in an autocrine and paracrine fashion. Retinoic acid (RA) and glucocorticoid signalling pathways have long been appreciated as major contributors to prenatal and postnatal lung maturation, and some evidence exists for their coordination or antagonism during lung development. Retinoic acid also plays an important role in morphogenesis. RA stimulates lung epithelial branching activity via an epithelial-mesenchymal interaction that, in part, involves the upregulation of the expression of EGFR, Insulin-like Growth Factors (IGF), basic Fibroblast Growth Factor (bFGF-2), and PDGF.

#### **3. The airway stem cells**

For several years, a consensus has been achieved that various types of stem cells exist, differing according to their position within the pulmonary tree, and that the stem cells often form pools that are ready to proliferate in response to injury and effect local repair. The classical subdivision of the airway tree into regions with individual stem cell harbours was

New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target 237

the SC. Pluripotency is the ability of a SC to differentiate into the heterogeneous population of cells that comprise a tissue or, in the case of cancer stem cells (CSCs), a tumour. There is growing evidence that some, if not all, tumours are derived from cells with the stem cell properties of self-renewal, multilineage potential, and proliferative capacity. Stem cells are candidates as the "cell of origin" for cancer because they have a pre-existing capacity for self-renewal and unlimited replication. In addition, stem cells are relatively long-lived compared to other cells within tissues. They therefore have a greater opportunity to accumulate the multiple additional mutations that may be required to increase the rate of cell proliferation and produce clinically significant cancers. Recent work has suggested that a subpopulation of cancer cells with stem-cell-like properties may be critical for triggering tumour development. Insights into the function and characteristics of CSCs offer a novel approach to understanding the progression of metastasis. Given that a single cancer cell can drive the formation of a metastatic tumour, CSCs are likely responsible for distant tumourigenesis and primary tumour formation. Thus, research focussed on the role of CSCs in primary lesions has led to discovery that CSCs can drive tumour formation in leukaemia and various solid tumours. While little work has been done to elucidate the role of CSCs in metastasis, properties of CSCs, such as self-renewal and differentiation, make them logical candidates as metastatic colonisers. To facilitate the discussion of CSCs with different metastatic ability, a distinction should be made when referring to two potential subtypes of CSCs: primary tumour cancer stem cells (pCSCs) and metastatic cancer stem cells (mCSCs). The first, pCSCs, constitute the original population of tumorigenic cells that initiate the formation of haematopoietic and solid tumours and are the centre of most CSC. The second group, mCSCs, represent a distinct population of cells with the intrinsic properties to disseminate from the primary site and generate the distant metastases. Although other cell subpopulations may break free of the primary tumour and invade the blood stream, mCSCs, like their pCSCs counterparts, are solely responsible for the initiation of tumours. mCSCs are related to pCSCs in the essential properties of self-renewal and differentiation that are needed for the propagation of the bulk of the tumour, but the two cell types differ in key ways. Unlike pCSCs, mCSCs disseminate from the tumour, colonise foreign tissue, and likely have additional alterations (whether mutational, epigenetic, or adaptive) that allow survival and propagation in secondary sites. The key to developing effective future therapies thus seems to be the identification and characterisation of these cancer stem cells and the development of drugs that specifically target these cells. Classically, the stem/progenitor cells of the pulmonary epithelium have been considered the basal cells in the proximal airways, Clara cells in the bronchioles and type II pneumocytes in the alveoli. There is evidence that the basal and parabasal cells are stem cells in the human lung. Clara cells have been shown to be the progenitors of themselves and of ciliated cells in the bronchioles. Recent research has established that a subset of Clara cells fulfils the criteria of adult, niche-specific stem cells. Pools of stem cells have been discovered that express Clara cell secretory protein (CCSP) but are not typical Clara cells. These variant CCSP-expressing (or vCE) cells show multipotent differentiation. The vCE cells are located in discrete pools in neuroepithelial bodies and at the broncho-alveolar duct junction. In the trachea and bronchi, the basal cells are widely believed to be stem cells. The basal cells and the parabasal cells that lie just above them certainly form a pluripotential reserve cell that, unlike the surrounding epithelium, usually survives injury. Procedures that involve denuding the trachea have demonstrated the capacity of basal cells to produce all of the major cell phenotypes found in the trachea, including basal, ciliated, goblet and granular secretory

accepted many years ago. Thus, the local repopulating cells of the trachea (basal, mucous secretory), bronchus (basal, mucous secretory), bronchiole (Clara) and alveolus (type II peneumocytes) remain, for the most part, the first reserve of airway stem cells. Stem cell research in the lung has progressed rather slowly due to the anatomical and functional complexities associated with the numerous distinct cell types. This organ must be divided into various anatomical regions when considering multipotent progenitor or stem cells. Evidence has clearly suggested that multipotent progenitors of the conducting airway epithelium and gas-exchange alveolar regions are derived from different populations of stem cells that are anatomically separated in the lung. Stem cell niches in the conducting airways must also be uniquely divided between the proximal and distal regions. Bronchial airways harbour at least two distinct progenitor cell populations. Both basal and nonciliated secretory cell types of the bronchial airways have been shown to exhibit proliferative capacity. The disparity between bronchial and bronchiolar airways is consistent with a mechanism in which the activity of distinct progenitor cell pools accounts for the regional differences both in lineage specifications during lung development and in the cellular composition of tracheo-bronchial and bronchiolar airways (Table 1).


Table 1. Stem or progenitor cell characteristics in the airway

Epithelial cell composition and zone boundaries depend on both the species and the individual animal history. In normal mice, a renewing cell system encompassing a glandcontaining, pseudostratified epithelium with Clara cells and few goblet cells is present in the upper trachea. In rats, a similar system, but with more goblet cells and no Clara cells, is present in the entire trachea, whereas this zone in humans penetrates many bronchial generations. Distally, the airway epithelium becomes glandless and cuboidal. This region is dominated by a Clara cell based lineage system before its transformation into a type II cellbased system in the alveoli. Stem cell niches in the airway have been characterised through experiments with rodent models. Stem cells in the proximal mouse trachea reside in the submucous gland duct, whereas those from the bronchi and bronchioles come from a subset of cells expressing a Clara-cell-specific protein located near neuroendocrine bodies and bronchoalveolar-duct junctions.

#### **4. Stem cells and lung cancer**

Stem cells give rise to a number of different cell types that can be classified into three groups: fully differentiated cells, transit-amplifying cells, and stem cells. The fully differentiated cells are mitotically inactive cells. These cells are at the end stages of cellular differentiation and will never re-enter the active cell cycle. The transit-amplifying (TA) cells are fast growing cells that are not fully differentiated. TA cells are able to proliferate for several generations, but they eventually terminally differentiate and must be replenished by

accepted many years ago. Thus, the local repopulating cells of the trachea (basal, mucous secretory), bronchus (basal, mucous secretory), bronchiole (Clara) and alveolus (type II peneumocytes) remain, for the most part, the first reserve of airway stem cells. Stem cell research in the lung has progressed rather slowly due to the anatomical and functional complexities associated with the numerous distinct cell types. This organ must be divided into various anatomical regions when considering multipotent progenitor or stem cells. Evidence has clearly suggested that multipotent progenitors of the conducting airway epithelium and gas-exchange alveolar regions are derived from different populations of stem cells that are anatomically separated in the lung. Stem cell niches in the conducting airways must also be uniquely divided between the proximal and distal regions. Bronchial airways harbour at least two distinct progenitor cell populations. Both basal and nonciliated secretory cell types of the bronchial airways have been shown to exhibit proliferative capacity. The disparity between bronchial and bronchiolar airways is consistent with a mechanism in which the activity of distinct progenitor cell pools accounts for the regional differences both in lineage specifications during lung development and in the

cellular composition of tracheo-bronchial and bronchiolar airways (Table 1).

Epithelial cell composition and zone boundaries depend on both the species and the individual animal history. In normal mice, a renewing cell system encompassing a glandcontaining, pseudostratified epithelium with Clara cells and few goblet cells is present in the upper trachea. In rats, a similar system, but with more goblet cells and no Clara cells, is present in the entire trachea, whereas this zone in humans penetrates many bronchial generations. Distally, the airway epithelium becomes glandless and cuboidal. This region is dominated by a Clara cell based lineage system before its transformation into a type II cellbased system in the alveoli. Stem cell niches in the airway have been characterised through experiments with rodent models. Stem cells in the proximal mouse trachea reside in the submucous gland duct, whereas those from the bronchi and bronchioles come from a subset of cells expressing a Clara-cell-specific protein located near neuroendocrine bodies and

Stem cells give rise to a number of different cell types that can be classified into three groups: fully differentiated cells, transit-amplifying cells, and stem cells. The fully differentiated cells are mitotically inactive cells. These cells are at the end stages of cellular differentiation and will never re-enter the active cell cycle. The transit-amplifying (TA) cells are fast growing cells that are not fully differentiated. TA cells are able to proliferate for several generations, but they eventually terminally differentiate and must be replenished by

Mucous, ciliated, neuroendocrine Mucous, ciliated, neuroendocrine Mucous, ciliated, neuroendocrine Mucous, ciliated (Type I/II pneumocyte) Type I and II pneumocytes (Clara cells),

PNEC (and Clara cells)

Tissue Epithelial stem cell niche Daughter cells

Tracheal mucus-gland duct cell

Tracheal basal cell

Neuroendocrine

bronchoalveolar-duct junctions.

**4. Stem cells and lung cancer** 

Tracheal secretory cell Bronchiolar Clara cell Alveolar type II pneumocyte

Table 1. Stem or progenitor cell characteristics in the airway

Lung proximal

Distal

the SC. Pluripotency is the ability of a SC to differentiate into the heterogeneous population of cells that comprise a tissue or, in the case of cancer stem cells (CSCs), a tumour. There is growing evidence that some, if not all, tumours are derived from cells with the stem cell properties of self-renewal, multilineage potential, and proliferative capacity. Stem cells are candidates as the "cell of origin" for cancer because they have a pre-existing capacity for self-renewal and unlimited replication. In addition, stem cells are relatively long-lived compared to other cells within tissues. They therefore have a greater opportunity to accumulate the multiple additional mutations that may be required to increase the rate of cell proliferation and produce clinically significant cancers. Recent work has suggested that a subpopulation of cancer cells with stem-cell-like properties may be critical for triggering tumour development. Insights into the function and characteristics of CSCs offer a novel approach to understanding the progression of metastasis. Given that a single cancer cell can drive the formation of a metastatic tumour, CSCs are likely responsible for distant tumourigenesis and primary tumour formation. Thus, research focussed on the role of CSCs in primary lesions has led to discovery that CSCs can drive tumour formation in leukaemia and various solid tumours. While little work has been done to elucidate the role of CSCs in metastasis, properties of CSCs, such as self-renewal and differentiation, make them logical candidates as metastatic colonisers. To facilitate the discussion of CSCs with different metastatic ability, a distinction should be made when referring to two potential subtypes of CSCs: primary tumour cancer stem cells (pCSCs) and metastatic cancer stem cells (mCSCs). The first, pCSCs, constitute the original population of tumorigenic cells that initiate the formation of haematopoietic and solid tumours and are the centre of most CSC. The second group, mCSCs, represent a distinct population of cells with the intrinsic properties to disseminate from the primary site and generate the distant metastases. Although other cell subpopulations may break free of the primary tumour and invade the blood stream, mCSCs, like their pCSCs counterparts, are solely responsible for the initiation of tumours. mCSCs are related to pCSCs in the essential properties of self-renewal and differentiation that are needed for the propagation of the bulk of the tumour, but the two cell types differ in key ways. Unlike pCSCs, mCSCs disseminate from the tumour, colonise foreign tissue, and likely have additional alterations (whether mutational, epigenetic, or adaptive) that allow survival and propagation in secondary sites. The key to developing effective future therapies thus seems to be the identification and characterisation of these cancer stem cells and the development of drugs that specifically target these cells. Classically, the stem/progenitor cells of the pulmonary epithelium have been considered the basal cells in the proximal airways, Clara cells in the bronchioles and type II pneumocytes in the alveoli. There is evidence that the basal and parabasal cells are stem cells in the human lung. Clara cells have been shown to be the progenitors of themselves and of ciliated cells in the bronchioles. Recent research has established that a subset of Clara cells fulfils the criteria of adult, niche-specific stem cells. Pools of stem cells have been discovered that express Clara cell secretory protein (CCSP) but are not typical Clara cells. These variant CCSP-expressing (or vCE) cells show multipotent differentiation. The vCE cells are located in discrete pools in neuroepithelial bodies and at the broncho-alveolar duct junction. In the trachea and bronchi, the basal cells are widely believed to be stem cells. The basal cells and the parabasal cells that lie just above them certainly form a pluripotential reserve cell that, unlike the surrounding epithelium, usually survives injury. Procedures that involve denuding the trachea have demonstrated the capacity of basal cells to produce all of the major cell phenotypes found in the trachea, including basal, ciliated, goblet and granular secretory

New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target 239

squamous cell carcinoma, large cell carcinoma, or spindle cell or giant cell carcinoma). Although various synonyms are in the current clinical terminology (anaplastic small-cell carcinoma, small-cell undifferentiated carcinoma, small-cell neuroendocrine carcinoma, oat cell carcinoma, and mixed small-cell/large-cell carcinoma), the use of these terms is discouraged to avoid confusion. Although the precise cell of origin is not known for SCLC, there is probably a pluripotent bronchial precursor cell that can differentiate into each of the major histologic types of lung cancer. However, within the spectrum of neuroendocrine tumours, a closer morphologic and genetic similarity exists between large cell neuroendocrine carcinoma and small cell carcinoma than either typical or atypical carcinoid. Although classified as a neuroendocrine (NE) tumour, the biological origins of this cancer have remained a matter of conjecture. Recently, SCLC has been shown to be dependent on the activation of Hedgehog signalling, an embryonic pathway implicated in the regulation of stem cell fates. This finding sheds new light on the potential histogenesis of SCLC. SCLC and carcinoid tumours both show high-level expression of neuroendocrine genes. Only a few markers are shared between SCLC and carcinoids, whereas a distinct group of genes defines carcinoid tumours, suggesting that carcinoids are highly divergent from malignant lung tumours, as has been reported. Recent studies have shown that the most useful neuroendocrine markers for SCLC in formalin-fixed, paraffin-embedded tissue sections are chromogramin A, synaptophysin, Leu-7, and certain neural cell adhesion molecules (NCAMs). Bombesin or gastrin-related peptide (GRP), keratin (AE1/AE3) and membrane antigen (EMA). DNA analysis of SCLC reveals a high percentage of aneuploidy in up to 85% of cases. Finally, the expression of proliferative markers, such as PCNA, thymidylate synthase, MCM2 and MCM6,

is highest in SCLC, which is known to be the most rapidly dividing lung tumour.

Various chemotherapy schemes have been evaluated for SCLC, but the combination of cisplatin and etoposide is widely considered the standard, with observed response rates of 80-85% and approximately 25% of patients obtaining a complete response. However, most patients experience disease relapse, and neither maintenance chemotherapy nor dose-

A topoisomerase I inhibitor, Topotecan, has shown response rates of 14% to 38% in chemosensitive patients, but the response rates in patients with chemorefractory disease are lower. Irinotecan, another topoisomerase I inhibitor, has demonstrated 10% partial response and 22% stable disease in refractory or relapsed SCLC. Etoposide-containing regimens currently remain the standard first line therapy in North America, while irinotecancontaining regimens are used in Japan. Thus, the combination of carboplatin and irinotecan may be a viable alternative to etoposide-containing regimens. Novel topoisomerase I and II inhibitors appear to continue to exhibit activity in patients with SCLC and warrant further investigation in this disease (particularly in non-Asian populations). However, whether

**6. Targeted agents that have been evaluated in SCLC** 

intensive chemotherapy regimens have led to improved outcomes.

these agents will be more active than etoposide remains to be determined.

The results are similar to those seen with other regimens.

**6.1 Topoisomerase I and II inhibitors** 

**6.2 Alkylating agents** 

cells. Controversially, pulmonary neuroendocrine cell (PNEC) populations have been suggested to be able to proliferate and serve as a reservoir of progenitor/stem cells that are capable of epithelial regeneration.


Table 2. Possible lung cell lineages. Adapted from Otto WRJ. Pathol. 2002.

## **5. Small cell lung cancer**

SCLC is the most common lung tumour in the spectrum of pulmonary neuroendocrine malignancies, which include typical carcinoid (TC), atypical carcinoid (AC), large-cell neuroendocrine carcinoma (LCNEC), and small-cell lung carcinoma (SCLC). The histological classification of SCLC has evolved substantially over the past several decades


WHO: World Health Organization

IALSC: International Association for the Study of Lung Cancer

Table 3. Classification of small-cell lung carcinoma

Interestingly, a large proportion of SCLC contains a component of NSCLC. Approximately 5% to 10% of patients diagnosed with SCLL will have mixed tumours, meaning that other pathologies, such as adenocarcinoma or squamous cell carcinoma, can be found within the pathologic specimen. The WHO classification of SCLC includes only one variant, combined small cell carcinoma*,* an SCLC with a mixed non-small-cell component (adenocarcinoma,

cells. Controversially, pulmonary neuroendocrine cell (PNEC) populations have been suggested to be able to proliferate and serve as a reservoir of progenitor/stem cells that are

> Ciliated Ciliated

Stem/progenitor Daughter Lineage progression

Mucous Secretory PNEC

Ciliated

Ciliated PNEC Type II?

Type I PNEC Clara

Table 2. Possible lung cell lineages. Adapted from Otto WRJ. Pathol. 2002.

classification of SCLC has evolved substantially over the past several decades

Other Combined oat cell

IALSC: International Association for the Study of Lung Cancer

Table 3. Classification of small-cell lung carcinoma

SCLC is the most common lung tumour in the spectrum of pulmonary neuroendocrine malignancies, which include typical carcinoid (TC), atypical carcinoid (AC), large-cell neuroendocrine carcinoma (LCNEC), and small-cell lung carcinoma (SCLC). The histological

WHO (1967) WHO (1981) IASLC (1988)

Fusiform Mixed small-cell/large-

cell carcinoma

carcinoma

Combine small-cell

Oat cell Lymphocyte-like Oat cell Small-cell carcinoma Polygonal Polygonal Intermediate Small-cell carcinoma

carcinoma

Interestingly, a large proportion of SCLC contains a component of NSCLC. Approximately 5% to 10% of patients diagnosed with SCLL will have mixed tumours, meaning that other pathologies, such as adenocarcinoma or squamous cell carcinoma, can be found within the pathologic specimen. The WHO classification of SCLC includes only one variant, combined small cell carcinoma*,* an SCLC with a mixed non-small-cell component (adenocarcinoma,

capable of epithelial regeneration.

Basal Basal

Tracheal Basal Gland duct Mucous

Clara Clara

Type II Type II

PNEC Clara

**5. Small cell lung cancer** 

WHO: World Health Organization

squamous cell carcinoma, large cell carcinoma, or spindle cell or giant cell carcinoma). Although various synonyms are in the current clinical terminology (anaplastic small-cell carcinoma, small-cell undifferentiated carcinoma, small-cell neuroendocrine carcinoma, oat cell carcinoma, and mixed small-cell/large-cell carcinoma), the use of these terms is discouraged to avoid confusion. Although the precise cell of origin is not known for SCLC, there is probably a pluripotent bronchial precursor cell that can differentiate into each of the major histologic types of lung cancer. However, within the spectrum of neuroendocrine tumours, a closer morphologic and genetic similarity exists between large cell neuroendocrine carcinoma and small cell carcinoma than either typical or atypical carcinoid. Although classified as a neuroendocrine (NE) tumour, the biological origins of this cancer have remained a matter of conjecture. Recently, SCLC has been shown to be dependent on the activation of Hedgehog signalling, an embryonic pathway implicated in the regulation of stem cell fates. This finding sheds new light on the potential histogenesis of SCLC. SCLC and carcinoid tumours both show high-level expression of neuroendocrine genes. Only a few markers are shared between SCLC and carcinoids, whereas a distinct group of genes defines carcinoid tumours, suggesting that carcinoids are highly divergent from malignant lung tumours, as has been reported. Recent studies have shown that the most useful neuroendocrine markers for SCLC in formalin-fixed, paraffin-embedded tissue sections are chromogramin A, synaptophysin, Leu-7, and certain neural cell adhesion molecules (NCAMs). Bombesin or gastrin-related peptide (GRP), keratin (AE1/AE3) and membrane antigen (EMA). DNA analysis of SCLC reveals a high percentage of aneuploidy in up to 85% of cases. Finally, the expression of proliferative markers, such as PCNA, thymidylate synthase, MCM2 and MCM6, is highest in SCLC, which is known to be the most rapidly dividing lung tumour.

## **6. Targeted agents that have been evaluated in SCLC**

Various chemotherapy schemes have been evaluated for SCLC, but the combination of cisplatin and etoposide is widely considered the standard, with observed response rates of 80-85% and approximately 25% of patients obtaining a complete response. However, most patients experience disease relapse, and neither maintenance chemotherapy nor doseintensive chemotherapy regimens have led to improved outcomes.

#### **6.1 Topoisomerase I and II inhibitors**

A topoisomerase I inhibitor, Topotecan, has shown response rates of 14% to 38% in chemosensitive patients, but the response rates in patients with chemorefractory disease are lower. Irinotecan, another topoisomerase I inhibitor, has demonstrated 10% partial response and 22% stable disease in refractory or relapsed SCLC. Etoposide-containing regimens currently remain the standard first line therapy in North America, while irinotecancontaining regimens are used in Japan. Thus, the combination of carboplatin and irinotecan may be a viable alternative to etoposide-containing regimens. Novel topoisomerase I and II inhibitors appear to continue to exhibit activity in patients with SCLC and warrant further investigation in this disease (particularly in non-Asian populations). However, whether these agents will be more active than etoposide remains to be determined.

#### **6.2 Alkylating agents**

The results are similar to those seen with other regimens.

New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target 241

At this time, mTOR inhibitors do not appear to be beneficial in the treatment of patients

Imatinib appears not to be beneficial in SCLC, even in patients with known c-kit mutations.

Despite these discouraging results, a new class of oral BCL-2 antagonists is currently being

In cancer tissues, homeostasis is tightly regulated to ensure the generation of mature cancer cells throughout life without a depletion of the cancer stem cell pools. Each tissue is composed of a cellular hierarchy including stem cells able to generate all progeny, committed progenitors, and terminally differentiated cells. The stem cells in each tissue are believed to communicate with their microenvironment or surrounding stroma to maintain their homeostasis. Thus, the pathways that control stem cell self-renewal and the microenvironment in which the cancer stem cells (CSCs) reside may both play roles in

The Hh gene family encodes several secreted glycoproteins, including Indian Hedgehog (Ihh), Desert Hedgehog (Dhh), and Sonic Hedgehog (Shh). These proteins mediate signalling in embryogenesis and development through activation of the Gli family transcription factors. The Hh pathway is somewhat unique in that the signals serve to relieve a series of repressive interactions. The receptor for Hh, the transmembrane protein Patched 1 (Ptch), normally binds and inhibits smoothened (Smoh), a G-protein-coupled receptor that is related to Frizzled (Frz). When secreted Hh binds both Ptch and Hedgehoginteracting protein (Hip), Smoh initiates a transcriptional response. Specifically, Smoh activates the serine/threonine kinase Fused (Fu) to release Gli from sequestration by Suppressor of Fused (SuFu). Subsequently Gli proteins are able to translocate to the nucleus and regulate transcription of cyclin D and E, c-myc, and other genes involved in cell proliferation and differentiation. Shh is one among several important factors derived from the lung endoderm and is required for proliferation, differentiation, and patterning of the mesenchyme. Shh regulates pattern formation of a variety of developing structures, including the formation of the primary lung bunds. However, Shh is expressed in the ventral foregut endoderm. Shh is subsequently expressed in a gradient fashion (in the developing lung epithelium) with the highest levels in cells at the tips. In turn, most components of the Shh pathway, including Shh target genes and its receptor Ptch1, are found in the mesenchyme. Shh signalling is initiated upon binding to Ptch1 and results in activation of Shh target genes by Gli transcription factors. Ptch expression in the lung follows the proximal-distal gradient of Shh. Gli1, 2, and 3 are expressed in overlapping but

**6.8 B cell leukaemia/lymphoma-associated gene 2 (Bcl-2)** 

**7. Signalling pathways that drive cancer stem cells** 

developed and evaluated in patients with SCLC.

**6.6.1 mTOR inhibitors** 

with SCLC.

**6.7 Kit inhibition** 

targeted therapies

**7.1 Hedgehog (Hh)** 

## **6.3 Picoplatin**

The role of picoplatin in SCLC is still not well defined and should be further explored in the future.

## **6.4 Antimetabolites**

Pemetrexed has been shown to have minimal activity as a second-line agent in the treatment of patients with SCLC. Elevated thymidylate synthase expression in SCLC tumours has been proposed as one of the reasons for the observed lack of efficacy.

### **6.5 Antiangiogenic agents**

Bevacizumab combined with standard first line therapy of cisplatin plus etoposide has shown a 64% response rate (RR), 4.7 months of progression-free survival (PFS), 30% of PFS at 6 months and 10.9 months of overall survival (OS). Upon employing bevacizumab to cisplatin plus irinotecan, the RR, PFS and OS were similar to those in the study conducted by ECOG. Another trial has reported an 84% overall RR, with PFS of 9.1 months and OS of 12.1 months. The importance of maintenance bevacizumab following combined modality treatment in patients with LD-SCLC is questionable; the response rate and OS are similar to what is seen with traditional chemotherapy with cisplatin, etoposide and radiation alone. Cediranib, a potent inhibitor of both VEGFR-1 and VEGFR-2, also has activity against c-kit, platelet derived growth factor beta (PDGFR-β), and FMS-like tyrosine kinase 4 (Flt-4). The response rate for Cediranib in recurrent SCLC that had progressed following platinumbased chemotherapy did not meet the predefined target. Vandetanib is an oral inhibitor of angiogenesis that targets VEGFR-2 and VEGFR-3 and inhibits tumour growth through activity against RET and EGFR/HER1. No difference in PFS or OS exists in vadetanibtreated patients compared with placebo-treated patients. Sorafenib, an oral multi-kinase inhibitor that targets both tumour proliferation via inhibition of Raf, stem cell factor receptor (KIT), and Flt-3 and angiogenesis by targeting VEGFR-2, VEGFR-3, and PDGFR-β, has been recommended for further evaluation in SCLC. Sunitinib is a novel, multi-targeted, smallmolecule inhibitor of VEGFR-1, -2, and -3, PDGFR-α and –β, Flt-3, c-kit, the receptor encoded by the rearranged during transfection (*ret*) proto-oncogene, and Flt3. Thalidomide initially appeared to be a promising drug, but inclusion of this drug has ultimately failed to show any benefit in OS. Thalidomide in combination with chemotherapy in patients with SCLC shows, contrary to the results of the prior study, no significant difference between the thalidomide-treated patients and placebo-treated patients in OS. Based on the results of these trails, the role of anti-angiogenic therapy in the treatment of patients with SCLC remains to be determined. All agents studied to date appear to produce similar response rates and OS that are similar to the results achieved with chemotherapy alone (in most cases). Maintenance therapy with these agents does not appear to be beneficial in patients with SCLC.

#### **6.6 MMP inhibitors**

Many trials with MMPIs in SCLC have been equally disappointing. Of the multiple MMPs elevated in SCLC, marimistat targets MMP-1, MMP-2, MMP-9 and MMP-12 at low concentrations, while BAY 12-9566 targets MMP-2 at low concentrations.

## **6.6.1 mTOR inhibitors**

At this time, mTOR inhibitors do not appear to be beneficial in the treatment of patients with SCLC.

## **6.7 Kit inhibition**

240 Squamous Cell Carcinoma

The role of picoplatin in SCLC is still not well defined and should be further explored in the

Pemetrexed has been shown to have minimal activity as a second-line agent in the treatment of patients with SCLC. Elevated thymidylate synthase expression in SCLC tumours has been

Bevacizumab combined with standard first line therapy of cisplatin plus etoposide has shown a 64% response rate (RR), 4.7 months of progression-free survival (PFS), 30% of PFS at 6 months and 10.9 months of overall survival (OS). Upon employing bevacizumab to cisplatin plus irinotecan, the RR, PFS and OS were similar to those in the study conducted by ECOG. Another trial has reported an 84% overall RR, with PFS of 9.1 months and OS of 12.1 months. The importance of maintenance bevacizumab following combined modality treatment in patients with LD-SCLC is questionable; the response rate and OS are similar to what is seen with traditional chemotherapy with cisplatin, etoposide and radiation alone. Cediranib, a potent inhibitor of both VEGFR-1 and VEGFR-2, also has activity against c-kit, platelet derived growth factor beta (PDGFR-β), and FMS-like tyrosine kinase 4 (Flt-4). The response rate for Cediranib in recurrent SCLC that had progressed following platinumbased chemotherapy did not meet the predefined target. Vandetanib is an oral inhibitor of angiogenesis that targets VEGFR-2 and VEGFR-3 and inhibits tumour growth through activity against RET and EGFR/HER1. No difference in PFS or OS exists in vadetanibtreated patients compared with placebo-treated patients. Sorafenib, an oral multi-kinase inhibitor that targets both tumour proliferation via inhibition of Raf, stem cell factor receptor (KIT), and Flt-3 and angiogenesis by targeting VEGFR-2, VEGFR-3, and PDGFR-β, has been recommended for further evaluation in SCLC. Sunitinib is a novel, multi-targeted, smallmolecule inhibitor of VEGFR-1, -2, and -3, PDGFR-α and –β, Flt-3, c-kit, the receptor encoded by the rearranged during transfection (*ret*) proto-oncogene, and Flt3. Thalidomide initially appeared to be a promising drug, but inclusion of this drug has ultimately failed to show any benefit in OS. Thalidomide in combination with chemotherapy in patients with SCLC shows, contrary to the results of the prior study, no significant difference between the thalidomide-treated patients and placebo-treated patients in OS. Based on the results of these trails, the role of anti-angiogenic therapy in the treatment of patients with SCLC remains to be determined. All agents studied to date appear to produce similar response rates and OS that are similar to the results achieved with chemotherapy alone (in most cases). Maintenance

therapy with these agents does not appear to be beneficial in patients with SCLC.

concentrations, while BAY 12-9566 targets MMP-2 at low concentrations.

Many trials with MMPIs in SCLC have been equally disappointing. Of the multiple MMPs elevated in SCLC, marimistat targets MMP-1, MMP-2, MMP-9 and MMP-12 at low

proposed as one of the reasons for the observed lack of efficacy.

**6.3 Picoplatin** 

**6.4 Antimetabolites** 

**6.6 MMP inhibitors** 

**6.5 Antiangiogenic agents** 

future.

Imatinib appears not to be beneficial in SCLC, even in patients with known c-kit mutations.

## **6.8 B cell leukaemia/lymphoma-associated gene 2 (Bcl-2)**

Despite these discouraging results, a new class of oral BCL-2 antagonists is currently being developed and evaluated in patients with SCLC.

## **7. Signalling pathways that drive cancer stem cells**

In cancer tissues, homeostasis is tightly regulated to ensure the generation of mature cancer cells throughout life without a depletion of the cancer stem cell pools. Each tissue is composed of a cellular hierarchy including stem cells able to generate all progeny, committed progenitors, and terminally differentiated cells. The stem cells in each tissue are believed to communicate with their microenvironment or surrounding stroma to maintain their homeostasis. Thus, the pathways that control stem cell self-renewal and the microenvironment in which the cancer stem cells (CSCs) reside may both play roles in targeted therapies

#### **7.1 Hedgehog (Hh)**

The Hh gene family encodes several secreted glycoproteins, including Indian Hedgehog (Ihh), Desert Hedgehog (Dhh), and Sonic Hedgehog (Shh). These proteins mediate signalling in embryogenesis and development through activation of the Gli family transcription factors. The Hh pathway is somewhat unique in that the signals serve to relieve a series of repressive interactions. The receptor for Hh, the transmembrane protein Patched 1 (Ptch), normally binds and inhibits smoothened (Smoh), a G-protein-coupled receptor that is related to Frizzled (Frz). When secreted Hh binds both Ptch and Hedgehoginteracting protein (Hip), Smoh initiates a transcriptional response. Specifically, Smoh activates the serine/threonine kinase Fused (Fu) to release Gli from sequestration by Suppressor of Fused (SuFu). Subsequently Gli proteins are able to translocate to the nucleus and regulate transcription of cyclin D and E, c-myc, and other genes involved in cell proliferation and differentiation. Shh is one among several important factors derived from the lung endoderm and is required for proliferation, differentiation, and patterning of the mesenchyme. Shh regulates pattern formation of a variety of developing structures, including the formation of the primary lung bunds. However, Shh is expressed in the ventral foregut endoderm. Shh is subsequently expressed in a gradient fashion (in the developing lung epithelium) with the highest levels in cells at the tips. In turn, most components of the Shh pathway, including Shh target genes and its receptor Ptch1, are found in the mesenchyme. Shh signalling is initiated upon binding to Ptch1 and results in activation of Shh target genes by Gli transcription factors. Ptch expression in the lung follows the proximal-distal gradient of Shh. Gli1, 2, and 3 are expressed in overlapping but

New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target 243

stem cell properties, i.e., self-renewal and differentiation. If the malignancy results from a blocked ontogeny, the treatment of cancer by inducing differentiation should be possible. These strategies have had variable success. In addition to inducing differentiation, a number of stem cell self-renewal pathways have been targeted for the treatment of various human tumours. If most solid tumours are composed of a minor population of self-renewing (stem) cells and a large fraction of non-renewing cells, cancer therapy failure following radiation and chemotherapy treatment is not the result of a rare cell evolving from within the tumour but the result of regrowth of the cancer stem cells. Of course, tumour stem cells could accumulate genetic changes that render them even more drug resistant, radiation resistant, or aneuploid. Because cures are achieved for many types of cancer, the cancer stem cells must be eliminated by a given therapeutic strategy. Regardless of which therapeutic paradigm turns out to be most effective, SCLC will clearly have to be treated with a "targeted medicine" approach if chemotherapy is to be widely successful in the clinic. This approach requires that each patient be segregated into a specific treatment group according to the constellation of molecular alterations that define his or her disease. The remarkable variation in genetic profiles across patients suggests that each tumour represents a distinct disease state that can only be effectively treated with precision therapy that targets the specific signalling pathway that is unique to each tumour. An important molecular mechanism that promotes cell differentiation is signal transduction. Signal transduction pathways ensure the reception of the concentration gradients of morphogens and their transformation into the differentiation of cells within tissues and organs. Hence, the key molecular rearrangements at the molecular level may be assumed to be related to changes in genes that participate in signal transduction pathways. In some contexts, these signals may be independently responsible for distinct aspects of tissue self-renewal, such as survival, proliferation and inhibition of differentiation. In other cases, the various signalling cascades may act in a hierarchy and regulate each other. Studies in which pathways are antagonised by treatment with pharmacological agent antagonists and/or agonists of Hh pathway signalling further demonstrate an ongoing requirement for pathway activity in the growth of additional cancer types. As a specific Smo antagonist, cyclopamine may be generally useful in the treatment of such cancers and represents a therapeutic strategy that may be further supported by the absence of observable toxicity in cyclopamine-treated animals. Cyclopamine inhibits Hh pathway activation by binding directly to Smo. This binding interaction is localised in the heptahelical bundle. Moreover, the binding influences the Smo protein conformation. Cyclopamine binding is also sensitive to Ptch function and provides biochemical evidence for an effect of Ptch on the structure of Smo. Cyclopamine appears to interfere with these signalling events by influencing Smo function; cyclopamine antagonises Hh pathway activity in a Ptch-independent manner and exhibits attenuated potency toward an oncogenic, constitutively active form of Smo. Pharmacologic inhibition of the Hh pathway has been necessary as a research tool to understand Hh pathway biology and is an attractive mechanism to evaluate antitumour activity. The first evidence that Smo could be antagonised came with the isolation of compounds called cyclopamine and jervine from corn lilies, which caused teratogenic effects (including cyclopia) in lambs. Significant new therapeutic strategies in SCLC will result from a deep understanding of the biology of response and resistance to targeted therapy. These approaches are in development to block embryonic pathways that

play a role in cancer stem cells, including the Notch, Hh, and Wnt pathways.

distinct domains in the lung mesenchyme. The proximal-distal gradient is evident in Gli1, which together with Ptch, is transcriptionally upregulated by Shh and is expressed in the subepithelial mesenchyme. All three Gli genes are expressed in the lung mesenchyme during the pseudoglandular stage of development, and mutations in the Gli genes give rise to various lung and foregut defects. Shh signalling has been implicated in the regulation of Gli genes, notably in Gli1 and Gli3 transcription in the lung. Gli2 has also been implicated in the regulation of Ptch1 and Gli1 components of the Shh signalling cascade in the lung. Thus, Shh is part of an epithelial network of regulators that restricts fibroblast growth factor 10 (FGF-10) expression. Shh-FGF-10 interaction supports a model in which the growing epithelial bud, which expresses high levels of Shh, interacts with a chemotactic source (FGF-10) in the distal mesenchyme for its elimination. This model supports the idea that not only the presence of FGF-10, but also its correct spatial distribution, is necessary for patterning. If FGF-10 signals are diffuse rather than localised, direct clues are lost and branching is disrupted. Importantly, the data suggest that under normal conditions, Shh plays a role in controlling FGF-10 expression in the distal lung. Expression of Shh and Ptch does not seem to be influenced by FGF-10; however, both genes are down-regulated by FGF-7 in lung explant cultures.

#### **7.2 Gli genes**

The vertebrate Gli gene family currently consists of three members, Gli1, 2 and 3, which are orthologous to Drosophila cubitus interruptus and encode DNA-binding proteins with five zinc fingers.

#### **7.3 BMP-4**

Bone Morphogenetic Protein (BMP) belongs to the TGFβ superfamily of growth factors, and at least three members (BMP-4, -5 and -7) are present in the developing lung. BMP-4 is an important regulator of epithelial proliferation and proximal-distal cell fate during lung morphogenesis. During branching morphogenesis, BMP-4 is dynamically expressed in the distal epithelium of branching airways. BMP-4 stimulates distal lung formation but might preferentially induce alveolar type I cell fate.

## **7.4 TGFβ-1**

TGFβ-1 is a member of a sub-family of peptides having at least two other members, all expressed in the developing lung. TGFβ signalling is mediated by serine-threonine kinase receptors (type I and II) and Smad transcription factors. TGFβ-1 transcripts are uniformly expressed in the sub-epithelial mesenchyme. TGFβ-1 protein accumulates later at sites of cleft formation and along proximal airways. TGFβ-1 promotes the synthesis of the extracellular matrix, which, when deposited in the epithelial-mesenchymal interface, is thought to prevent local branching.

#### **8. Perspectives and future directions in therapy for SCLC**

The recurrence of tumours after initial tumour regression by conventional therapies is also frequent. One potential reason for this recurrence is the failure of current therapies to target CSCs. The design and development of new cancer treatments is therefore necessary to target

distinct domains in the lung mesenchyme. The proximal-distal gradient is evident in Gli1, which together with Ptch, is transcriptionally upregulated by Shh and is expressed in the subepithelial mesenchyme. All three Gli genes are expressed in the lung mesenchyme during the pseudoglandular stage of development, and mutations in the Gli genes give rise to various lung and foregut defects. Shh signalling has been implicated in the regulation of Gli genes, notably in Gli1 and Gli3 transcription in the lung. Gli2 has also been implicated in the regulation of Ptch1 and Gli1 components of the Shh signalling cascade in the lung. Thus, Shh is part of an epithelial network of regulators that restricts fibroblast growth factor 10 (FGF-10) expression. Shh-FGF-10 interaction supports a model in which the growing epithelial bud, which expresses high levels of Shh, interacts with a chemotactic source (FGF-10) in the distal mesenchyme for its elimination. This model supports the idea that not only the presence of FGF-10, but also its correct spatial distribution, is necessary for patterning. If FGF-10 signals are diffuse rather than localised, direct clues are lost and branching is disrupted. Importantly, the data suggest that under normal conditions, Shh plays a role in controlling FGF-10 expression in the distal lung. Expression of Shh and Ptch does not seem to be influenced by

FGF-10; however, both genes are down-regulated by FGF-7 in lung explant cultures.

The vertebrate Gli gene family currently consists of three members, Gli1, 2 and 3, which are orthologous to Drosophila cubitus interruptus and encode DNA-binding proteins with five

Bone Morphogenetic Protein (BMP) belongs to the TGFβ superfamily of growth factors, and at least three members (BMP-4, -5 and -7) are present in the developing lung. BMP-4 is an important regulator of epithelial proliferation and proximal-distal cell fate during lung morphogenesis. During branching morphogenesis, BMP-4 is dynamically expressed in the distal epithelium of branching airways. BMP-4 stimulates distal lung formation but might

TGFβ-1 is a member of a sub-family of peptides having at least two other members, all expressed in the developing lung. TGFβ signalling is mediated by serine-threonine kinase receptors (type I and II) and Smad transcription factors. TGFβ-1 transcripts are uniformly expressed in the sub-epithelial mesenchyme. TGFβ-1 protein accumulates later at sites of cleft formation and along proximal airways. TGFβ-1 promotes the synthesis of the extracellular matrix, which, when deposited in the epithelial-mesenchymal interface, is thought

The recurrence of tumours after initial tumour regression by conventional therapies is also frequent. One potential reason for this recurrence is the failure of current therapies to target CSCs. The design and development of new cancer treatments is therefore necessary to target

**8. Perspectives and future directions in therapy for SCLC** 

**7.2 Gli genes** 

zinc fingers.

**7.3 BMP-4** 

**7.4 TGFβ-1** 

to prevent local branching.

preferentially induce alveolar type I cell fate.

stem cell properties, i.e., self-renewal and differentiation. If the malignancy results from a

blocked ontogeny, the treatment of cancer by inducing differentiation should be possible. These strategies have had variable success. In addition to inducing differentiation, a number of stem cell self-renewal pathways have been targeted for the treatment of various human tumours. If most solid tumours are composed of a minor population of self-renewing (stem) cells and a large fraction of non-renewing cells, cancer therapy failure following radiation and chemotherapy treatment is not the result of a rare cell evolving from within the tumour but the result of regrowth of the cancer stem cells. Of course, tumour stem cells could accumulate genetic changes that render them even more drug resistant, radiation resistant, or aneuploid. Because cures are achieved for many types of cancer, the cancer stem cells must be eliminated by a given therapeutic strategy. Regardless of which therapeutic paradigm turns out to be most effective, SCLC will clearly have to be treated with a "targeted medicine" approach if chemotherapy is to be widely successful in the clinic. This approach requires that each patient be segregated into a specific treatment group according to the constellation of molecular alterations that define his or her disease. The remarkable variation in genetic profiles across patients suggests that each tumour represents a distinct disease state that can only be effectively treated with precision therapy that targets the specific signalling pathway that is unique to each tumour. An important molecular mechanism that promotes cell differentiation is signal transduction. Signal transduction pathways ensure the reception of the concentration gradients of morphogens and their transformation into the differentiation of cells within tissues and organs. Hence, the key molecular rearrangements at the molecular level may be assumed to be related to changes in genes that participate in signal transduction pathways. In some contexts, these signals may be independently responsible for distinct aspects of tissue self-renewal, such as survival, proliferation and inhibition of differentiation. In other cases, the various signalling cascades may act in a hierarchy and regulate each other. Studies in which pathways are antagonised by treatment with pharmacological agent antagonists and/or agonists of Hh pathway signalling further demonstrate an ongoing requirement for pathway activity in the growth of additional cancer types. As a specific Smo antagonist, cyclopamine may be generally useful in the treatment of such cancers and represents a therapeutic strategy that may be further supported by the absence of observable toxicity in cyclopamine-treated animals. Cyclopamine inhibits Hh pathway activation by binding directly to Smo. This binding interaction is localised in the heptahelical bundle. Moreover, the binding influences the Smo protein conformation. Cyclopamine binding is also sensitive to Ptch function and provides biochemical evidence for an effect of Ptch on the structure of Smo. Cyclopamine appears to interfere with these signalling events by influencing Smo function; cyclopamine antagonises Hh pathway activity in a Ptch-independent manner and exhibits attenuated potency toward an oncogenic, constitutively active form of Smo. Pharmacologic inhibition of the Hh pathway has been necessary as a research tool to understand Hh pathway biology and is an attractive mechanism to evaluate antitumour activity. The first evidence that Smo could be antagonised came with the isolation of compounds called cyclopamine and jervine from corn lilies, which caused teratogenic effects (including cyclopia) in lambs. Significant new therapeutic strategies in SCLC will result from a deep understanding of the biology of response and resistance to targeted therapy. These approaches are in development to block embryonic pathways that play a role in cancer stem cells, including the Notch, Hh, and Wnt pathways.

New Therapeutic Strategies in Small Cell Lung Cancer: The Stem Cell Target 245

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## **9. Conclusions**

The introduction of effective targeted agents for SCLC has lagged behind that for non-smallcell lung cancer. However, the number of agents now being tested has increased and includes agents that have shown some anti-tumour activity against other types of cancer, such as inhibitors of the Hh signalling pathway. This activity has prompted the development of agents that can inhibit Hh signalling. If the cancer stem cells that are responsible for driving the growth of cancer types associated with Hh pathway activation indeed come from stem cells trapped in a state of active renewal by pathway activities, then a logical therapeutic approach for these cancers would be to impose a state of pathway blockade. As we look towards the future, an important area of investigation will clearly involve analysing how the Hh pathway exerts its effect and whether shared molecular targets are involved in influencing self-renewal in the context of stem cells and cancer. Additionally, Hh probably integrates with other niche-derived signals, such as BMP (Bone Morphogenic Protein), Wnt and Notch. By understanding the molecular events governing CSCs, the development of therapeutics aimed at targeting these cells will become possible. The development of such therapeutics is of paramount importance because CSCs may mediate the resistance to current treatment and the relapse of the most aggressive tumours. This resistance may in part result in the reactivation of several signalling cascades, such Hh, Wnt, Notch, and EGF, in the CSCs combined with an increase in DNA repair mechanisms and ABC transporter-mediated multi-drug resistance.

## **10. References**

#### **10.1 Introduction**


#### **10.2 Development of airway**

244 Squamous Cell Carcinoma

The introduction of effective targeted agents for SCLC has lagged behind that for non-smallcell lung cancer. However, the number of agents now being tested has increased and includes agents that have shown some anti-tumour activity against other types of cancer, such as inhibitors of the Hh signalling pathway. This activity has prompted the development of agents that can inhibit Hh signalling. If the cancer stem cells that are responsible for driving the growth of cancer types associated with Hh pathway activation indeed come from stem cells trapped in a state of active renewal by pathway activities, then a logical therapeutic approach for these cancers would be to impose a state of pathway blockade. As we look towards the future, an important area of investigation will clearly involve analysing how the Hh pathway exerts its effect and whether shared molecular targets are involved in influencing self-renewal in the context of stem cells and cancer. Additionally, Hh probably integrates with other niche-derived signals, such as BMP (Bone Morphogenic Protein), Wnt and Notch. By understanding the molecular events governing CSCs, the development of therapeutics aimed at targeting these cells will become possible. The development of such therapeutics is of paramount importance because CSCs may mediate the resistance to current treatment and the relapse of the most aggressive tumours. This resistance may in part result in the reactivation of several signalling cascades, such Hh, Wnt, Notch, and EGF, in the CSCs combined with an increase in DNA repair mechanisms

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#### **10.7 Signaling pathways driving cancer stem cells**


#### **10.8 Perspective and future directions in therapy for SCLC**

Antón Aparicio LM, García Campelo R, Alonso Curbera G. Small-cell lung carcinoma: What is new in therapy? Cancer & Chemotherapy 2007;2:168-174.

**Part 6** 

**Genetic Manipulation and Its Possible Clinical** 

**Implications for Squamous Cell Carcinoma** 

