INTRODUCTION

Cancer is a major health problem both in humans and animals and its percentage of mortality shows inequality being more in developing countries (poor people) compared to developed countries. The percentage of deaths due to cancer have shown an increasing trend in developing countries and by 2025 almost 80% deaths will be due to different type of cancers (Stewart and Wild, 2014). The cancer is also a leading cause of deaths in America being second only to cardiac diseases in human (Anderson and Smith, 2005). Advances in the biomedical research along with the dietary modifications have significantly reduced deaths (>60%) due to cardiac disease during last 50 years of the 20th century but during the same period only 5% reduction in mortality rate due to cancer was witnessed (http://www.cancer.org). Presently, annually about 4.2 million people die prematurely (aged 30-69 years) across the world (World Global Health Observatory). Among the animals, pets (canine) that receive more attention have an annual incidence rate of 99.3/100000 in males and 272.1/100000 in female dogs. The higher female to male incidence has been attributed to the higher risk of the mammary tumors in the latter (Merlo et al., 2008).

Cancer though caused by a wide variety of causes but its higher rate in developing countries seems to arise from infections such as Papillomavirus. Besides, adoption of the western lifestyle like smoking, drinking and lack of physical activity makes people more prone to the cancer (Stewart and Wild, 2014). Cancer in humans and animals are highly comparable but the preferences in two fields vary. In the former, main focus is to treat or expand the life span by employing either a single or the combinations of therapy viz. radiation, chemotherapy or surgery.

In veterinary field the cancer therapy normally ends up in euthanasia (Breen and Modiano, 2008). However, since American college of veterinary internal medicine/oncology certified radiation oncology as a separate discipline two decades ago, there has been an increased awareness about the role of veterinary oncology in understanding the cancer further besides changing the therapeutic perspective in animal cancer (Mack, 2006; Khanna et al., 2006).

The conventional therapeutic options for cancer treatment include radiotherapy, chemotherapy, surgery and/or the combinations thereof. Radiotherapy is used based upon the principle that cells replicating faster have more chances of DNA damage upon exposure to radiations. Though the tumor cells are affected primarily but normal cells do also get affected. A number of constraints are associated with the radiation therapy including localized tumor efficacy, suppression of normal stem cells besides the inflammatory effects, damage to the nerve and other tissue upon prolonged use (Hall, 2000). Chemotherapy kills cancer cells by interfering with DNA synthesis or cell functioning. The added advantage over the radiation therapy is its widespread efficacy for tumors at multiple locations and whether superficial or deep. However, the drawbacks in the form of toxic effects, development of drug resistance and need for supplemntary treatment options demand research at molecular level in order to achieve the desired results (Ross, 2000). Surgery is used both as a diagnostic as well as the therapeutic procedure and works by removing the tumor mass as a whole. Its disadvantages like usefulness only against the localized tumors, impossibility of removal of the whole tumor without any spillage and inefficiency against the tumors involving the critical organs like heart, brain, etc. demand further research in order to hold complete control over cancer treatment (Gilson and Stone, 1990; Enneking and Maale, 1988).

The main reason behind the failure of all the above mentioned therapies is actually not encountering the source of tumor, the cancer stem cells. Tumors in general are composed of different kinds of cells including neoplastic cells, inflammatory cells, supporting vascular cells and fibroblasts. Barring the neoplastic cells and fibroblasts, all cells have restricted self-renewal capacity and are mostly differentiated and short lived (Rich and Bao, 2007; Han et al., 2013). In contrast neoplastic cells are long lived have self renewal potential and are able to differentiate and form tumor. Such cells are popularly known as Cancer Stem Cells (CSCs) or Cancer Initiating Cells (CICs) or Tumor Initiating Cells (TICs) (Wu et al., 2012; Dou and Gu, 2010; Han et al., 2013). By employing or developing the therapies that could control or destroy the tumor initiating cells, the pathological features of tumors can be controlled and thus, tumor treatment can be achieved. The current review thrusts upon the cancer stem cell model, concepts and its possible therapeutic implications.

DEVELOPMENT OF CANCER STEM CELL HYPOTHESIS

The concept of cancer stem cells may be ascribed to the research of Cohnheim and Durante, who observed comparable proliferation and differentiation potential in embryonic tissues and cancers, about 150 year back. The unique research made the basis for the possibility of resident stem cells present in adult tissues which upon activation may lead to cancer (Gugjoo et al., 2013). Further, impetus to the concept came from the work of Furth et al. (1937) who reported AML in mouse upon single undifferentiated leukemia cell transplantation (Al-Hajj et al., 2003). It was for the first time that self-renewal properties of stem cells were shown. Three decades later, in vitro colony assay showed that AML can self-renew, proliferate and can give rise to new tumors (Sell, 2004). Later the concept of blocked ontogeny, maturation arrest of stem cells was formally tested under in vitro conditions in 1990’s by John Dick and colleagues (Castor et al., 2005) and was also supported by others (Caussinus and Gonzalez, 2005; Chen and McKearin, 2003). However, all the above studies were conducted on leukemia or liquid phase tumors. The first report of cancer stem cell in solid tumors came from Al-Hajj and colleagues in 2003 who reported it from breast cancer (Al-Hajj et al., 2003). Subsequently CSCs were also observed in a wide variety of solid tumors viz. lung cancer (Kim et al., 2005), colon cancer (O’Brien et al., 2006), prostate cancer (Collins et al., 2005), ovarian cancer (Szotek et al., 2006), brain cancer (Piccirillo et al., 2006), melanoma (Fang et al., 2005) and others. The stem cell hypothesis is thus, different from earlier stochastic model, wherein all the tumor cells are considered to have potential for tumor development (Gugjoo et al., 2013).

TUMOR GROWTH AND METASTASIS

The CSCs are being recognised as distinct cell population in cancers that are capable of developing the whole tumor and thus, represent its characteristic features viz. tumor growth (size), lymph node involvement and metastasis (Han et al., 2013; Gugjoo et al., 2013). Stem cells under normal circumstances are regulated by the microenvironment/niche. The niche is composed of; group of cells at a special location, an anchoring site for stem cells via adhesion molecules, growth factors and signalling molecules like fibroblast growth factors, Wnt, Hedgehog, etc. that regulate stem cell self-renewal property, multiplication and differentiation (Li and Neaves, 2006; Gugjoo et al., 2015). The microenvironment/niche facilitates either proliferation or inhibition of the stem cell properties depending upon the need (Potten et al., 2009; Fuchs et al., 2004). The notion was based upon the observation that stem cells are lost upon loss in niche (Xie and Spradling, 2000). The other question that arises is how does tumor develops from CSCs. The mechanisms that may be involved include the mutations leading to self-sufficient cell proliferation or alteration in niche leading to dominance in proliferation-differentiation signals over the inhibition signals. The finding was supported by a study in which it was found that neuroblastoma requires the coordination in mutations involving both the Schwann cells and supporting cells (Zhu et al., 2012). The long life span of stem cell makes it more prone to mutations. Thus, cancer stem cells like other stem cells have the potential to differentiate into different lineages and accounts for the heterogeneous cell population in tumors (Mannelli and Gallo, 2012). Continuous proliferation following mutation or altered niche thus, may account for the tumor growth (increase in size). Besides, the normal stem cells have characteristic homing (arrest of stem cells within the vasculature of a tissue followed by transmigration across the endothelium) property, which may still be intact in CSCs leading to tissue invasion to nearby lymph nodes or to other tissue far or near (metastasis) (Li and Neaves, 2006).

The CSCs may be defined by the following properties:

ISOLATION AND IDENTIFICATION OF CANCER STEM CELLS

Isolation and identification of CSCs from tumor mass can be done easily based upon the properties of stem cells as both the CSCs and normal stem cells have a number of common features. These features include ability to form sphere in non-adherent medium, drug exclusion by efflux transport over activity, presence of different cell surface markers, enzyme activity or signalling molecules (Alison et al., 2011; Ghani et al., 2011; Zhang et al., 2011). However, the above mentioned in vitro tests do not differentiate between normal stem cells, progenitor cells and cancer stem cells. The differentiation between normal and cancer stem cells can be done by in vivo tests and serial transplantation of such cells in animal models is considered as gold standard (Clarke et al., 2006).

THERAPEUTIC IMPLICATIONS BASED ON CSC MODEL

The conventional therapeutic options for cancer treatment include chemotherapy, radiotherapy or surgical excision. However, these options stand without the desired results besides producing lot of side effects including local or systemic toxicity, drug resistance or loss of function (Ajani et al., 2009; Rich and Bao, 2007). The CSCs being the cardinal reason behind tumor development account for failure of conventional therapies in cancer patients. The main reasons behind the therapy failure due to involvement of CSCs include gradual cell cycle kinetics, better DNA repair capability, over expression of multi-drug resistance membrane transporters and anti-apoptosis proteins and finally micro-environment (acidosis, etc) (Rich and Bao, 2007; Raguz and Yague, 2008; Signore et al., 2013; Gugjoo et al., 2013). Therefore, the need of the hour may be to combine conventional therapies with the options that aim at the CSCs.

CSC BASED THERAPEUTIC OPTIONS

Targeting cancer stem cell marker and metabolic pathways: Cancer stem cell markers, though also possessed by normal stem cells, may be targeted to eliminate tumour source. The cytotoxic drugs specific to CSCs markers and also the inhibitors targeting drug detoxifying enzymes, efflux pumps and or transcription factors may yield desired results.

Different tumors express different surface markers like CD44+, CD24+ and ESA+ in pancreatic CSCs and CD133 in hepatocellular and gastric CSCs (Smith et al., 2008), which may be targeted to destroy CSCs. The CD133 is considered as an important marker and upon its down regulation, cell growth, motility, ability to form spheroids and above all capacity to metastasize would reduce (Rappa et al., 2008). Such a down regulation may be achieved either by direct monoclonal antibody application (Rappa et al., 2008) or by antibody drug conjugate like monomethyl auristatin F (Smith et al., 2008).

Drug detoxifying enzymes like aldehyde dehydrogenase act as a marker for CSCs and normal stem cells. It plays a significant functional role in self-protection, differentiation and expansion of stem cells (Marcato et al., 2011). In human breast cancer cell lines positive for ALDHhiCD99+ standard cancer therapy was ineffective but cells were sensitive upon inhibition of ALDH activity (Croker and Allan, 2012). This explains the role of ALDH towards resistance against the conventional cancer therapies and offers a therapeutic option against CSCs.

Drug efflux pumps are normally over expressed in CSCs or normal stem cells and give protection against the noxious agents/drugs. Among the efflux pumps ABCG2 is a potent CSC marker. Besides it also is expressed by normal stem cells and the blood brain barrier. Phytochemical agent, curcumin has been found to inhibit the drug efflux mechanism of CSCs in rat C6 glioma cell line (Fong and Chan, 2012). However, the negative effect upon the normal stem cell and the blood brain barrier need to be taken care.

Cancer stem cell niche target: Niche form the basis for maintaining the activity of CSCs as well as normal stem cells. Targeting the niche could help target the CSCs by exhausting the nutritional source and the essential signals required for normal functioning of CSCs (Han et al., 2013). However, the niche targeting may affect the normal stem cells also, demanding further detailed research.

Among the microenvironment/niche factors, microvascularization has been found to be important for CSCs maintenance and thus could be a target for therapeutic approaches (Calabrese et al., 2007). Hypoxia can control CSCs and may be considered for control of tumor progression. Antiangiogenic therapy induces CSCs niche hypoxia and thus, radioresistance to CSCs (Morrison et al., 2011). The anti-angiogenic agents like sinitinib, may be combined with CSCs targeted drugs to achieve better results (Conley et al., 2012).

Targeting stem cells signaling pathways: Normal stem cells maintain their characteristic features viz. self-renewal, multiplication or differentiation through signaling pathways. The same signaling pathways when dysregulated, normal stem cells turn into the CSCs. Although, a number of signaling pathways are involved, the well studied pathways are Hedgehog (Hh), Notch, Wnt/â-catenin and are believed to play an important role in CSCs formation and thus may be effective targets to control CSCs and thereby tumor formation (Han et al., 2013).

Hedgehog (Hh) is an essential signaling pathway for CSCs maintenance in different cancers including pancreatic cancer, colorectal or gastric cancers (Song et al., 2011; Merchant and Matsui, 2010; Chen et al., 2007), besides its significant role in therapeutic resistance (Chen et al., 2007). Thus, inhibiting the Hh pathway would control CSCs and tumor formation. Smoothened signaling molecule (SMO) may be inhibited with inhibitors like cyclopamine, however, tumors that harbour lesions beyond the SMO, cyclopamine remains ineffective (Xia et al., 2012; Singh et al., 2011). To overcome the problem, glioma-associated oncogene homolog (Gli) proteins are blocked with the agents like arsenic trioxide together with the SMO blockade (Ng and Curran, 2011).

Notch pathway has an important role in cell-cell communication and in multiple cell fate decisions both during embryonic as well as adult phase (Soltanian and Matin, 2011; Wang et al., 2008). It is involved in stem cell proliferation, differentiation and also apoptosis and may act as oncogenic in human tissues/organs like cervix, lung, head, prostate, etc and oncosuppressive in skin and hepatocellular carcinoma (Wang et al., 2008). The ã-secretase inhibitors (GSIs) that block Notch pathway in glioblastoma have been found to result in reduced neurosphere growth and clonogenicity in vitro, reduced CSCs markers expression and reduced tumor growth in vivo. Thus GSI may be useful in glioblastoma by inhibiting notch pathways but may be toxic as they block all the four notch ligands and also ã-secretase substrates (Wang et al., 2011).

Wnt, the other important signalling pathway is involved in multiple biological processes like embryogenesis, development, cell proliferation, survival and differentiation (Klaus and Birchmeier, 2008). It plays an essential role in self-renewal and mantainance of normal stem cells as well as CSCs in tissues (Wend et al., 2010; Katoh and Katoh, 2007). Upon oncogenic mutations, Wnt dysregulation leads to CSCs formation and subsequent tumor development (Reya and Clevers, 2005; Reya et al., 2001). There are two types of Wnt signalling pathway inhibitors viz. small molecule inhibitors (NSAIDs) and biologic inhibitors including Mab SiRNAs (Wend et al., 2010; Katoh and Katoh, 2007). However, the signaling pathway that may be blocked have potential to interfere with the essential pathways of normal stem cells.

Induction of apoptosis: Dysregulation in apoptotic mechanism also contribute to the cancer development, progression as well as resistance to conventional therapy. The mechanism that might interplay includes increased DNA repair after conventional radiotherapy/chemotherapy, alteration in cell cycle check point and MDR protein upregulation (Signore et al., 2013). Thus, drugs like bicyclic cyclohexamines that induce apoptosis may offer good therapeutic option (Hexum et al., 2012).

Differentiation of CSCs: By differentiation, CSCs population will deplete and thus, will have no self-renewal property (Soltanian and Matin, 2011). The CSCs differentiation may be achieved with retinoic acid and drugs targeting tumor epigenetic changes (Massard et al., 2006). To eliminate the cancer, thus, combined use of CSCs differentiation agents and chemotherapy may prove to be a good therapeutic approach (Han et al., 2013).