Apart from identification of cervical cancer stem cells, there is no information on the impact of HPV infection or its oncoproteins on putative CSCs proportions or on the stem cell signaling and downstream functions. It is hypothesized that HPV infects the basal cells residing within transformation zone where columnar cells of endocervix meet the squamous cells of ectocervix (Martens et al. 2004; Cid Arregui et al. 2012). This region which is known as squamocolumnar junction (SCJ) is suggested to generate stem cells that maintain columnar and stratified epithelial cells of the cervix (Martens et al. 2009). SCJ cells are known to express high levels of cytokeratin CK17. Infection of these slow-cycling epithelial cells is believed to be necessary for the infection to persist over a long period, which can eventually integrate to induce tumorigenic transformation and cancer. However, till date there is no experimental evidence to validate the hypothesis. A recent study demonstrates that there exists a discrete population of cells at the SCJ of the cervix with a unique gene expression profile that could be responsible for most HR-HPV-associated cervical carcinomas (Herfs et al. 2012). The selected junctional biomarkers proposed, i.e., keratin Krt7, anterior gradient AGR2, cluster differentiation marker CD63, matrix metalloproteinase MMP7, and guanine deaminase (GDA), were expressed by SCJ cells. These markers were found to be expressed by a high percentage of high-grade CIN and cervical cancers associated with carcinogenic HPVs but rarely in ectocervical/transformation zone CINs. Interestingly, these markers displayed on these SCJ are also displayed by some most common cervical cancer cell lines SiHa, HeLa, and CaSki, suggesting that these cell lines do serve as suitable in vitro models for the SCJ cells to be used in experimental procedures. These markers, however, do not confirm stemness and were not associated particularly with CSC as majority of cervical precancer or cancer cells expressed them. Stemness and differentiation being the two extremes of a gradient, it is likely that studying the quantitative gradient of stemness-related markers would be helpful in finding CSC in situ in a tumor tissue. Nevertheless, these studies provide essential leads demonstrating a potential tropicity of HPV towards a set of cells with probable stem cell characteristics that reside in SCJ region of transformation zone. If SCJ cells or the resident stem cells are the true targets of HPV, it will be quite interesting to determine how HPV modulates the stem cell signaling in these cells. Recent advancement in technology has made it possible to study the life cycle of HPV in vitro by organotypic raft culture that has been an important tool to study HPV replication and its interactions with the host cell. An immortalized human foreskin keratinocyte (HFK) cell line, BC-1-Ep/SL, when stably transfected with HPV16 can sustain productive stages of HPV16 life cycle in organotypic raft culture system (Flores et al. 1999). Using these in vitro models, the progression of low-grade intraepithelial lesions (LSIL) to high-grade intraepithelial lesions (HSIL) can be studied (Isaacson Wechsler et al. 2012). Such organotypic raft culture system can open up an avenue to understand the target cells of HPV infection and the mechanism of generation of cervical CSC. These experiments can also reveal the contribution of HPV oncoproteins in regulation of stem cell signaling pathway and other stemness-related transcription factors, reported to be expressed in cervical cancer.

Stem cell property is highly dynamic in nature and conserved even in tumor tissues. A homeostasis that determines the proportion of CSC or non-CSC is believed to be maintained in tumors despite tumorigenic transformation (Reya et al. 2001). Both normal stem cells and CSC resemble in their stemness properties and are proposed to be controlled by the same/similar self-renewal pathways that control stemness in normal stem cells (Fig. 24.1). These stemness-related signaling pathways are broadly classified as Notch, Hh, and Wnt signaling pathways (Pardal et al. 2003). These pathways primarily govern stem cell fate determination, self-renewal, and differentiation. The signaling events act in tandem along with a set of transcription factors like Sox2, Oct4, Nanog, and STAT3 that have been implicated with stemness, self-renewal, pluripotency, and nuclear reprogramming (Tam and Lim 2008). The balance between Wnt, Notch, and Hh signaling networks is important for the maintenance of homeostasis among stem and progenitor cells. Aberrant activation of these pathways has been frequently implicated in progression of many tumor types (Dreesen and Brivanlou 2007; Takebe et al. 2011) including cervical cancer (Table 24.2). Reports indicate activation of Notch, Hh, and Wnt pathways to be necessary for the maintenance of normal cervix and subsequent tumor progression (Zagouras et al. 1995; Daniel et al. 1997; Gray et al. 1999; Shinohara et al. 2001; Okino et al. 2003; Xuan et al. 2006). In independent studies, expression of these pathways have been highly correlated with the progression of cervical cancer with their tendency to increase with the tumor progression from normal to CIN and cervical cancer, indicating gross disturbance in these signaling pathways during cancer progression. However, the orchestration of these pathways, their hierarchy and cross-talk between each other in regulation of cervical CSCs and cellular homeostasis, is not well defined. Cell fate of proliferating stratum spinosum and reserve cells of squamous epithelium that differentiate into squamous and columnar cells respectively in normal cervix have been hypothesized to be determined by Notch signaling via differential expression of Notch1/2 (Zagouras et al. 1995). Overexpression of Notch in these cells might be responsible for generating CSC in cancer as the overexpression of Notch1, Notch2, and Notch intracellular domain (NICD), along with their ligands Jagged1 and Jagged2, have been implicated in progression of cervical cancer (Daniel et al. 1997; Gray et al. 1999; Weijzen et al. 2003). Likewise, Hes1 and Hes5, the downstream target of Notch signaling, are suggested to inhibit differentiation and promote cell proliferation in cervical cancer (Liu et al. 2007, 2010) but their role/status in cervical CSC is yet to be ascertained. Apart from Notch pathway, the overexpression of nuclear β-catenin, a central mediator of Wnt pathway, has been correlated with the progression of cervical cancer (Shinohara et al. 2001). Later, it was shown that overexpression of DVL-1, an upstream regulator that prevents sequestration of β-catenin, resulted in constitutively active Wnt signaling and promoted tumorigenesis (Okino et al. 2003). High nuclear expression of GLI, the downstream mediator of Hh, and other components of this pathway are overexpressed in cervical cancer and have been implicated in tumor recurrence (Xuan et al. 2006; Chaudary et al. 2012). These studies represent individual contribution of signaling pathways in cervical cancer. Further studies are needed to understand the extent of contribution of each signaling pathway in regulation of CSC in an in vivo situation.

Similar to majority of the signaling pathways, there is an evidence of the activation of stemness-related transcription factors c-myc, Sox2, Stat3, and Oct4 during cervical cancer progression (Abba et al. 2004; Ji and Zheng 2010; Liu et al. 2010; Shukla et al. 2010; Wang et al. 2013b) (Table 24.3). c-myc, well known for its capabilities of cellular reprogramming, was found amplified in HPV positive precancer and cancer lesions (Abba et al. 2004). Sox2 was found localized in nucleus of highly tumorigenic primary cervical cancer cells (Ji and Zheng 2010). Active STAT3 required for early embryonic stem cell potency and self-renewal (Raz et al. 1999) was found increased with increasing severity of the cervical lesions (Shukla et al. 2010, 2013). Similarly, Oct4, a key regulator that maintains the pluripotency and self-renewal properties of embryonic stem cells, was overexpressed in cervical lesions and was associated with lymphatic metastasis (Wang et al. 2013b). Experimental or intrinsic overexpression of transcription factors such as Sox2 or Oct4 in cervical cells was found to increase cells with CSC phenotype (Liu et al. 2014; Yang et al. 2014). Interestingly, stemlike cells isolated on CD44/CK17 marker from primary cervical carcinoma tissues also showed elevated levels of c-myc, Sox2, STAT3, and Oct4 transcript in the spheroid cells, thus suggesting their role in cancer stem cell signaling (Feng et al. 2009). Collectively, these studies clearly demonstrate the presence of CSC as well as an active CSC signaling in cervical cancer tissues.