Notch signalling in cervical cancer

Journal Pre-proof Notch signalling in cervical cancer Calvin Rodrigues, Leanna R. Joy, Sasikala P. Sachithanandan, Sudhir Krishna PII:

S0014-4827(19)30556-7

DOI:

https://doi.org/10.1016/j.yexcr.2019.111682

Reference:

YEXCR 111682

To appear in:

Experimental Cell Research

Received Date: 6 August 2019 Revised Date:

12 October 2019

Accepted Date: 16 October 2019

Please cite this article as: C. Rodrigues, L.R. Joy, S.P. Sachithanandan, S. Krishna, Notch signalling in cervical cancer, Experimental Cell Research (2019), doi: https://doi.org/10.1016/j.yexcr.2019.111682. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Notch signalling in cervical cancer

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Calvin Rodrigues1, Leanna R Joy1, *Sasikala P Sachithanandan1, and *Sudhir Krishna1

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Author Affiliation: Cellular Organization and Signalling Group, National Centre for Biological Sciences, Tata Institute of Fundamental Research (NCBS-TIFR), GKVK, Bellary Road, Bangalore 560065, India.

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*Correspondence may be addressed to Sudhir Krishna, Phone: +91-80-2366-6071. Email: [email protected]; and to Sasikala P Sachithanandan, Email: [email protected]. National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore 560065, India.

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Abstract

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The initial discovery of key developmental signalling pathways, largely using classical genetic approaches in model organisms, was followed by an intense burst of characterisation of the molecular components. Studies also began demonstrating a role for these pathways in oncogenesis. Patterns of mutations in Notch pathway components, such as those reported in sub-sets of hematological malignancies, have been easier to study, and the cumulative information is leading to potentially new therapies. However, it has been more challenging to clearly define the role of the Notch pathway in human solid tumours, given the absence of widespread specific activating or repressive mutations in key components of the pathway. In this review, we trace more than two decades of work looking at the role of Notch signalling in human cervical cancer progression. We document the contrasting reports on a tumour suppressive role and pro-oncogenic role in cervical cancers. However, an analysis of recent genomic data strikingly shows both widespread features of Notch expression and genetic changes that largely amplify positive regulators and delete negative controllers of the Notch pathway. This analysis reinforces a largely pro-oncogenic role for Notch signalling and lays the foundation for a nuanced exploration of synergistic and targeted therapies. Lastly, we further trace some of the complex challenges in advanced cervical cancer progression, including issues of cancer stem cells and metastasis.

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Introduction Cervical cancer associated mortality remains high in the developing world, due to a deficiency in the implementation of vaccinations and screening. Further, patients in these settings often present in clinics with advanced progression stages. Thus, the development of effective treatment approaches remains vital and the understanding of molecular drivers of cervical cancer progression will be important to these efforts.

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Notably, while E6 and E7 are sufficient to immortalize keratinocytes and drive cell proliferation, an additional “hit” is required to transform these cells. Activated Ras signalling is one suggested mechanism, playing central event in transformation and cancer progression [6,7], acting largely through MAPK, PI3K pathway activation. However, Ras mutations have not been commonly observed in cervical cancers. Instead, one mechanism which phenocopies activated Ras in cervical cancers is activated Notch signalling [8]. In this review, we describe the roles of Notch signalling in cervical cancer progression, and mechanisms by which its activity is regulated.

Cervical cancers are driven by Human Papillomaviruses of the high-risk (HR) variety. The HR HPV E6 and E7 oncogenes hijack a range of cellular mechanisms, including cell proliferation, to carry out the viral life cycle [1–3] (For reviews, refer to [4,5]). The biology of the viral life cycle is complex, and is linked to epithelial differentiation. Viruses infect the basal layer of the cervix, cause deregulated cellular proliferation in progenitors, and then complete assembly of mature virions in the upper, differentiated layer of the epithelium.

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Canonical Notch signalling in cervical cancer

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Notch signalling can function in diverse ways across cell types and cellular contexts including cell division, differentiation, and survival [9–11]. Notch pathway deregulation has been shown to play central roles in cancer progression [9]. In canonical Notch signalling, binding of the transmembrane Notch receptors (Notch 1-4 in mammals) to cell bound ligands (Jagged 1,2 and Delta-like 1,3 and 4) leads to sequential cleavage in the receptor, releasing active Notch intracellular domain (NICD). Subsequently, NICD is transported to the nucleus, where it acts as a transcriptional co-activator and activates transcription of target genes (Fig.1.; for further detail on Notch signalling, refer to [10,12]. Within human cancers, more than 70% of all T-cell leukemia (T-ALL) cases showed activated mutation in Notch receptor [13], leading to constitutively active Notch signalling. On the other hand, such high rates of mutations of the Notch receptor have not been reported in solid cancers, including cervical cancer. This raises some important questions: 2

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1. Is Notch pathway activation observed in clinical solid tumours?

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2. What are the consequences of the activation of the Notch pathway in solid tumours?

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3. What mechanisms activate Notch signalling?

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I. Pro-oncogenic roles of Notch Signalling 1.Transformation

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Oncogenic HPV16 is known to cooperate with Ras to mediate transformation in multiple cell types, including baby rat kidney cells, baby mouse kindey cells, rat embryo fibroblasts [14–16]. It was shown that like Ras, activated intracellular Notch1 and 2 could complement adenoviral E1A oncogene during transformation [17]. Rangarajan et al. showed that activated intracellular Notch1 may phenocopy Ras in transforming immortalized HaCaT keratinocytes, together with HPV16 E6 and E7 [8].

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Notably, HPV16 has also been shown to cooperate with c-Myc to immortalize and transform cells [18]. HPV16 E6 activates the human telomerase reverse transcriptase (hTERT) promoter via a c-Myc-dependent mechanism, influencing cell immortalization [19] Additionally, c-Myc was found to be a key component in E6/E7 - Notch mediated transformation [20]. Building on this, it was also shown that activated Notch1 could also similarly cooperate with HPV16 oncogenes in these cells to form tumours in nude mice[21]. Lastly, Lathion et al. have also extended these findings using primary keratinocytes [22].

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In addition to the complementation analysis of papillomavirus oncogenes with activated Notch signalling, recent experiments have reported similar results using more genetically tractable model organisms like Drosophila[23]. For example, tumourigenesis can be induced in this system by the expression of E6+UBE3A in conjunction with activated/oncogenic forms of Ras or Notch. These model systems offer an opportunity to further refine these molecular interactions and potentially develop genetic/ drug screens.

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2. Anoikis resistance

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Activated Notch pathway has been shown to aid transformation and anoikis resistance in HaCaT cells transfected with E6/E7, and this is dependent on Akt signalling. Expression of constitutive active Akt phenocopied activated Notch1 (AcN1) expression, and knockdown of Akt along with AcN1 abrogated the effects of AcN1 overexpression[8]. 3

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3. Cell migration and EMT

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In addition to transformation and anoikis, Akt signalling has been shown to be important in driving Notch-mediated enhancement of cell migration and EMT [24]. In addition to acting via the Notch pathway, HPV16 E7 is also known to directly activate Akt signalling. E7 has been shown to increase the level of Akt phosphorylation, by binding and sequestering PP2A, which otherwise dephosphorylates Akt [25]. Further, E7 is also known to enhance cell migration in keratinocytes, and this ability is also dependent on Akt [26]. The phosphorylation of E7 itself plays a direct role in the ability of E7 to activate Akt signalling, which also impacts cell invasion through the regulation of MMP secretion[27]

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II. Tumour suppressor roles of Notch pathway

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The role of Notch in cervical cancer is complex, with its differentiation-promoting role in normal cervical epithelium [29]. Additionally, high expression of Notch leads to growth arrest of cervical tumour-derived cells [30–32]. Further, Lefort et al., also demonstrated that Notch suppression (using MAM51, a peptide that suppresses MAML activity) in keratinocytes infected with oncogenic Ras produce more aggressive carcinomas as compared to control treated cells[33]. In addition, Sun et al. showed that transient overexpression of Intracellular Notch (ICN1,2,3,4) lead to a decrease in cell proliferation in HeLa cells [34].

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Regulation of Notch signalling

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1.Genetic regulation: Point mutations and/or copy number variations in Notch signalling components in cervical cancer

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It is well documented that in T-ALL (T cell acute lymphoblastic leukemia), increased Notch activity was highly correlated to the occurrence of mutations in Notch receptors [13] and its component, FBXW7. Though Notch pathway components are overexpressed in primary cervical cancer cells, high frequency mutations in Notch pathway components have not been reported so far in cervical cancer. In the absence of a specific kind of activating mutations reported in leukemias as in the case of T-

In addition, Notch1 signalling was found to modulate tumourigenic properties in cervical cancer through RhoC. Both knockdown of Notch1 receptor or treatment with a Notch inhibitor (DAPT -gamma secretase inhibitor) decreased RhoC activity and subsequently led to a decrease in cell migration and invasion in cervical cancer cell line. Lastly, coexpression of Notch1, and RhoC was observed in primary cervical cancer biopsies[28].

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ALL[13], it becomes important to explore for cytogenetic abnormalities (copy number variations, including amplification, deletion) that might lead to the activation of the Notch pathway.

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Recent genome-wide studies from The Cancer Genome Atlas (TCGA) project have described gene mutation spectra across several primary cervical cancers, including EP300, FBXW7, NFE2L2, TP53, PIK3CA, PTEN and ERBB2 [35,36]. Among them, FBXW7, a negative regulator of Notch pathway was altered (truncating mutation, deep deletion, missense mutation) in 15% of the primary squamous cell cervical carcinoma[35]. This data led us to explore genetic alterations in other Notch pathway components (NOTCH1-4, MYC, HES1, JAG1-2, DLL1,3,4, NUMB, ADAM17, DELTEX1) in cervical cancer tissues. It is known that Notch signalling pathway can be activated by several genetic lesions including, activating mutations in the NOTCH receptors (NOTCH 1, 2, 3, 4), amplification/overexpression of Notch ligands and inactivation of E3 ligases (mutations and deletions/epigenetic changes). Here we questioned if such changes are observed in the Notch pathway players in cervical cancer tissues. Using the TCGA cervical cancer database [36,37], we then investigated for the gene mutation; copy number variation (amplification, deletion) and the gene expression pattern of the Notch pathway components. Our meta-analysis of TCGA data showed that alteration expression of Notch pathway components (mostly overexpression, z-score threshold ± 1.5 fold change) was observed in > 80% of primary cervical cancer tumours (Fig. 4).

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Also, some of these components were perturbed by genetic changes (point mutations, amplification, and deletion) in half of the primary cervical cancer tissues. Some of the observed genetic lesions including amplification in positive regulators (Notch receptor, ligand, Hes1, c-Myc) and deletion in negative regulators (FBW7, CBL) may lead to an increased expression of the Notch pathway components (Fig. 5). In particular, we found that Hes1 gene was amplified in 15% of the CC cells. Using the UALCAN portal[38] we also observed that Hes1 transcript was overexpressed significantly when compared to that of control tissue (Fig. 6). In addition, it is possible that gene amplifications observed in some of the negative regulators (ITCH, Numb) may control the activation of Notch pathway. For example, ITCH transcript was altered in 29% of the CC cases. Further study is warranted to explore the role of ITCH in the context of Notch signalling pathway in CC. Together, these data hint that genetic lesions might be one of the reasons for heterogeneity observed in the expression and the activity of the Notch pathway in different primary cervical cancer tissues. This also raises an important question about the cellular consequence of this activation and its molecular intermediates.

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In addition, mutations in PI3K-Akt pathway components are also noted in CC tissues[35,36]. On the other hand, PI3K-Akt signalling has been shown to act

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downstream of activated Notch [8]. Together PI3K- Akt mutations may either mimic downstream activation of Notch, or maybe independent of Notch signalling.

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2. Non-genetic regulation of Notch signalling

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Since the genetic perturbation of the Notch pathway is observed only in a subset of cervical cancer tissues, the regulatory processes in other cases are not well understood. It is likely that in addition to genetic perturbation, other mechanisms that contribute to the regulation of notch signalling, including ligand-dependent Notch activation, and regulation by long noncoding RNAs has to be evaluated in great detail.

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a. Ligand dependent Notch activation

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Since the canonical Notch pathway is activated by different ligands in a tissue-specific manner, ligand-dependent activation of the Notch pathway can occur in cervical cancer. Higher expression of Jagged1, and downregulation of Manic Fringe[39] was reported during progression stages of W12 cell line, including in organotypic raft cultures. Here, a similar pattern of expression was also observed in biopsy sections, examining transitions from CIN III to SCC.

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b. Regulation of Notch signalling by noncoding RNAs

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Another mode of regulation of the Notch pathway occurs via noncoding RNAs. Pang et al. showed that miR34a binds to the 3’ UTR of Notch1 and Jagged1 mRNA, downregulated their protein expression and resulted in decreased cell migration in HeLa cell line[40]. In another study, overexpression of HOTAIR, (a long noncoding RNA) upregulates Hes1 and Notch1 (mRNA and protein) and showed an increase in cell migration and invasion in SiHa and CaSki cell lines[41]. Together these data suggest that Notch signalling pathway can be regulated by non coding RNA independent of activating Notch mutation in cervical cancer cells.

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c. Modulation of Notch signalling by HR HPVs Notch signalling has been shown to be associated with the epithelial differentiation process, where high Notch signalling causes differentiation of the basal cell [29]. Given that HPV lifecycle is dependent on keratinocyte differentiation, it is important to ask if HPV oncoproteins regulate the Notch pathway in the HPV life cycle.

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Here, the relation between HPV proteins and Notch signalling depends on the subtype of HPV involved. HPVs can be roughly classified on the basis of the epithelia that they 6

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affect. Cutaneous HPVs include the genus beta of HPVs, and these target skin. Whereas Mucosal HPVs comprise the genus alpha of HPVs, and infect mucosal epithelium (e.g. cervical epithelium)[42]. Mucosal HPVs are further classified on their capacity to initiate carcinomas - High risk HPVs (e.g. HPV16, HPV18, HPV31) are known to induce carcinomas, and low risk HPVs (e.g. HPV5, HPV8) cause benign lesions, but not induce carcinomas[43].

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High Risk HPV E6 proteins (e.g. HPV16, HPV18) are capable of binding to P53 in a trimeric complex with together with Ubiquitin ligase E6AP, resulting in a degradation of p53[44–47]. The degradation of P53 in turn downregulates Notch1, resulting in repression of differentiation markers in keratinocytes [48]. Immortalized human keratinocyte cell line (NIKS) with HPV16 showed deregulated differentiation, loss of NICD and impaired differentiation in organotypic raft assays. Low Risk HPV E6 proteins (e.g. HPV11 and HPV6) bind to E6AP, but fail to degrade P53 [49–51]. In contrast, cutaneous HPV E6 proteins do not bind E6AP, but instead bind MAML1, a transcriptional coactivator in the notch complex, and thus inhibit Notch activity[50,52,53]. Further, this inhibition of Notch signalling is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes[54,55].

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On the other hand, other studies show HPV-induced enhancement of Notch signalling. HPV16 E6 is also known to interact with the host cellular protein NFX-123, and together they upregulate Notch1 expression[56]. Further, Weijzen et al. showed that both E6 and E7 increase Notch1 expression and activity [57]Further, Pattabiraman et al. showed that ectopic expression of the HPV31 genome into human foreskin keratinocytes (HFKs) resulted in an increase in abundance in the activated form of Notch1[58].

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Most of the studies summarized here address the regulation of the Notch pathway by HPV oncoproteins in Keratinocyte models. It is plausible that these studies better address aspects of normal replication of HPVs in Keratinocytes. On the other hand, the regulation of Notch signalling by HPV oncoproteins in cervical cancers is less explored. It would be interesting to assess the regulation of Notch signalling by the HPV oncoproteins in cervical cancer cell lines, since these proteins still play important and distinct roles in advanced cervical cancers. For example, Wang et al. show that the deletion of HPV16 E6 and E7 using an oncolytic adenovirus results in reduced invasiveness and increased radiosensitivity using SiHa cells in vitro and in vivo[59]. Further, Kennedy et al. have also shown that HPV 18 E6 and E7 deletion using CRISPR/Cas in HeLa result in cell death [60]. These studies suggest that HPV induced Notch pathway may have a different role in cervical cancer, which needs to be evaluated in detail.

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Expression of Notch players during progression

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Expression of Notch players during clinical cervical cancer progression

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Given that Notch can play as a tumour suppressor and oncogenic role, it becomes important to comprehensively examine the expression of Notch and its downstream players, during cervical cancer progression. A number of studies have examined the expression of these players during cervical cancer progression. These also report a complex pattern - a majority of these see an upregulation of Notch with progression, but a few studies also report downregulation during progression.

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1. Upregulation of Notch1 components during progression:

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Notch1 pathway components expression was found to be increased during cervical cancer progression, from normal, through CINI-III to cervical cancer [61]. Campos-Parra et al. showed an upregulation of Notch pathway components in cervical cancers compared to control tissues[62]. Also, Daniel et al. showed that there are greater levels of intracellular Notch in invasive cervical cancers compared to high grade lesions[63]. Greater levels of nuclear Notch1 was observed in CIN sections compared to invasive cervical cancers. Most of the invasive cervical cancers showed cytoplasmic localisation of these proteins and was associated with cervical cancer progression. It was also observed that Nuclear Notch1 in tumours was associated with poorer clinical outcomes compared to tumours showing cytoplasmic Notch1 [64].

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In addition to the Notch expression and its localisation, Rong et al. also showed that splicing of Numb (a negative regulator of Notch), was perturbed in cervical cancer[61]. Splice variants with or without exon 12 (NUMB-L and NUMB-S) were observed, and the expression of NUMB-L variant was increased in cervical cancer tissues. While overexpression of NUMB-L variant increased Hes1 and Hey1 expressed and cell proliferation in Hela cells, NUMB-S variant decreased it.

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2. Downregulation of Notch1 components during progression:

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Tripathi et al. showed that nuclear and cytoplasmic Notch1 expression was downregulated during progression, from non-neoplastic cervix tissues to precancer to tumours. They noted that Notch3 showed an increase in expression with progressive stages [65]. In another study, examination of a small number of biopsy sections showed a reduction in the number of Notch1 positive cells with progression from normal to CIN I , CIN II, CIN II (n values= 1 to 3 for different stages) [66]. Here, examination of a small number of sections showed a reduction in the number of Notch1 positive cells with progression from Normal to CIN I, CIN II, CIN II (n values= 1 to 3 for different stages).

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Together these studies show that the activation and the roles of the Notch pathway in cervical cancer tissues is heterogeneous. One possible explanation for these differing roles is due to the difference observed in the dosage and the stability of the Notch pathway activation. Another is intratumoural heterogeneity. In the subsequent sections, we will discuss these, and other possible causes for this complexity in the role of Notch.

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Notch and tumourigenic subpopulations in cervical cancers

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Models of understanding stemness in the context of solid tumour progression have struggled with the direction of differentiation and the stage specific roles.

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One aspect, which may contribute to differing patterns of Notch signalling, is intratumoural heterogeneity. We have previously reported the presence of one such cellular subpopulation in cervical cancers, marked by the surface expression of CEACAM/ CD66 [67]. CD66 is encoded by the CEACAMs1,3,5,6 and 8 genes. Interestingly, an analysis of HPV integration sites in multiple cervical cancer studies revealed CEACAM5 gene as a recurrent point of HPV integration[37,68,69], and tumours in which this integration is observed show higher expression of CEACAM5[37].

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Additionally, we described that the CD66+ve cells (cell lines, spheroids and clinical samples) show enrichment of several Notch pathway components and enhanced migration, clonogenic potential, tumour formation in mice. In addition, these cells were more sensitive to Gamma Secretase inhibition (DAPT) and showed increased sensitivity to chemotherapeutic agent Cisplatin in combination with DAPT. Notch inhibition either by DAPT or by overexpression of a negative regulator of Notch, Manic fringe, resulted in a downregulation of CD66[67]. In addition, an orthotopic mouse model also showed increased expression of Notch1. Thus, CD66 high populations show features of enhanced tumourigenicity, show elevated Notch signalling, and are dependent on Notch signalling.

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In an attempt to further resolve the heterogeneity in the CD66 subset, suggested in our previous work (clinical outcome to CD66 levels in tumours), we looked at a second marker, CD49f, known to mark the basal layer in the normal cervix. A comprehensive study of these subpopulations revealed that CD49f marks a subset of cells that are basal and proliferative while CD66 expressing cells are more differentiated and migratory[70]. The conventional model of progression assumes a linear cascade of events, starting with local invasion, followed by intravasation, extravasation, and colonization of secondary site leading to distant metastasis. In contradiction, our data suggests that local invasion and distant metastasis need not be sequential events, but may be independently driven by these two populations with CD49f subset driving local 9

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invasion and CD66 subset driving distant metastasis. It will be interesting to investigate the role of Notch pathway in these subsets in better detail. Notably, these subpopulations (CD66 and CD49f) also show phenotypic plasticity between migratory and proliferative states.

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Such phenotypic plasticity may be required during metastasis, to allow metastasized cells to revert to a proliferative, non-migratory state, allowing the formation of macroscopic tumours at metastatic sites. Mechanisms regulating migratory plasticity are currently poorly understood. CD66 and migration-linked plasticity may be at least partially regulated by chromatin regulation [71]. Chromatin regulation may also help maintain phenotype changes induced by signalling.

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Consistent with the idea that local invasion and metastasis are not sequential events we show that CD66 high populations arise in pre-cancer stages, and these populations show features of partial differentiation and a link with the HPV lifecycle. In addition, CD66 populations in pre-cancer cell line CIN612, show increased levels of cleaved Notch1 and keratinocyte stemness regulator p63[58]. Further, the transfection of HPV31 into HFKs increased the expression of cleaved Notch. Under differentiating conditions, the level of cleaved Notch1 decreased in normal and HPV31 transfected HFKs. In CIN612, differentiation in methylcellulose decreased the expression of both cleaved as well as full-length Notch1. CD66+ve cells in CIN612 also supported a higher abundance of HPV31 DNA and expression of E1^E4. These populations also showed greater clonogenic potential, and strong enrichment for migratory features. Thus, these populations in CIN612 (as well as in CaSki) show features of both stemness as well as differentiation, marking a progenitor population. Notably, completion of the HPV life cycle requires both differentiation as well as proliferation components [58] These populations may thus represent an important intermediate stage during cervical cancer progression.

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Summary and future directions:

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In summary, we describe the current understanding of Notch signalling in cervical cancers- in terms of links with the HPV oncogenes, intratumoural heterogeneity, roles in migration and metastasis, and mutations. These findings shed further light on Notch signalling in cervical cancers and also highlight the complexity and several unknowns and future directions. Further understanding could help reassess and re-devise treatment strategies targeting Notch components – as earlier attempts to broadly target Notch signalling resulted in extensive gut toxicity [72]. In subsequent studies, it will be important to address the following:

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1. Stage specificity and dose-dependency in Notch signalling:

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Notch signalling can have different roles in the normal cervix, and in early and advanced stages of cervical cancer progression [73]. Such findings have been reported in other signalling pathways such as TGFβ signalling, where TGFβ plays a tumour suppressor role in early stages of progression, and a pro-oncogenic role during the later stages of progression [74]. Additionally, in this review, we have highlighted associations between Notch signalling and cell migration, an early stage in cancer metastasis. Links between Notch and metastasis need to be further developed.

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In normal differentiation in epithelial tissues, Notch1 signalling plays a central role in initiating epithelial differentiation. Notch1 signalling occurs in an asymmetric manner to initiate differentiation in the basal layer - leading to a switch from a basal to a progenitor state ([29,75]. However, during cervical cancer progression, it is likely that the partially differentiated progenitors are the cells of origin. These progenitors show a greater rate of proliferation than the basal cells, and the expression of the E6 and E7 oncogenes (which deregulate the cell cycle) is highest in them. In support of this notion, we showed that CD66 populations show features of partial differentiation and stemness, and show higher levels of HPV31 proliferation. Suggesting that these cell populations are important intermediates, showing links with the viral life cycle. Importantly, these cells also show elevated Notch signalling. They also simultaneously show high levels of P63, a keratinocyte stemness marker.

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The function of Notch may also depend on the level of expression of Notch. A dose dependency has been reported, where moderate Notch activation increased viability and anchorage independent growth, whereas high level Notch activation decreased anchorage independent growth. This dose dependency is linked to AP1 alterations[76]. Another study of HPV induced oncogenesis in oral cancers showed that both overexpression or knockdown of Notch could promote tumourigenesis, although via different pathways [77].

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2. Links between Notch and intra-tumoural subpopulations.

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Given our findings with CD66+ve populations, we note that active Notch signalling is maintained in a subpopulation of cells, where it drives migration/ other tumourigenic phenotypes. Some of the complexities in Notch signalling may be resolved by understanding if there are distinct patterns of signalling across cell populations in tumours. It would be important to assess intratumoural heterogeneity in Notch signalling, using single cell approaches- including imaging and single cell genomics. Additionally, while mouse models have been useful in understanding early stages of cervical cancer progression, these models do not show the extent of heterogeneity observed in human patients, and do not show metastatic progression[78]. There is a 11

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requirement for animal models which provide higher degree of inter and intra-tumoural heterogeneity, and which show metastatic progression.

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3. Plasticity and Signalling epigenetics.

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Chromatin regulation changes associated with Notch activation are currently poorly understood. The study of chromatin alterations in cervical cancer cell migration/ metastasis is especially interesting [79], since such chromatin changes would allow prolonged activated signalling, even in the absence of the initial metastasis-inducing cues.

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Funding:

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This work was supported by grants from Department of Biotechnology (DBT) and National Center for Biological Sciences, Tata Institute for Fundamental Research (NCBS-TIFR).

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Figure legends

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Figure 1. The Notch signalling pathway. The notch signalling pathway is initiated by the binding of Notch ligands such as Delta, Jagged, to the Notch receptor. Binding initiates cleavage of the Notch receptor, generating activated intracellular Notch. This activation in turn may activate pathways such as PI3K-Akt. Additionally, intracellular Notch translocates to the nucleus. Here, it initiates transcriptional activation of Notch1 target genes, including Hes1, Cyclin D1, C-Myc, etc. The Notch receptor can be targeted for ubiquitination by ligases such as FBXW7, which targets it for proteolytic degradation.

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Figure 2. Roles for Notch signalling in cervical cancers. Notch signalling has been shown to play both tumour suppressor and oncogenic roles in cervical cancers. This may be linked with the dosage of Notch activation. Constitutive high expression of Notch is linked with differentiation and induction of apoptosis. However, Notch signalling has also been shown to cooperate with the HPV oncogenes E6 and E7 to immortalise and transform epithelial cells. Notch signalling has also been shown to promote anoikis resistance. Lastly, it has been shown in the induction of migration and EMT, through PI3K-Akt and RhoC.

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Figure 3. Expression of Notch and its targets during cervical cancer progression. In the normal, uninfected cervical epithelium, Notch initiates differentiation, regulating the basal-spinous switch. Upon HPV infection, there is an increase in Notch1 levels in keratinocytes. In CIN lesions, which correspond to the subsequent stage of progression, CD66 high populations show high Notch1. These CD66high cells also show features of partial differentiation, stemness, and migratory traits. Similarly, in cervical squamous cell carcinomas (SCCs) and in the metastasis-derived cell line CaSki, CD66high populations are Notch high and Notch dependent. These CD66high populations also show features of stemness and enhanced migration, and correlations with metastasis. Additionally, genome wide cervical cancer studies from TCGA have reported Notch- linked mutations, showing copy amplifications of notch targets including HES1, CCND1, and Myc.

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Figure 4. Expression pattern of Notch pathway components in primary squamous cell carcinomas. Using c-bioportal[80,81] we studied the expression pattern of Notch pathway components (as indicated in the figure) in TCGA primary cervical cancer cervical squamous cell carcinomas [35,36]. Percentage correspond to percentage of cases showing altered regulation.

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Figure 5. Point mutation and copy number variation spectra across cervical squamous cell carcinoma. Using c-bio portal[80,81] we studied the point mutation and 23

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copy number variation in the Notch pathway components (as indicated in the figure) in TCGA primary cervical squamous cell carcinoma dataset [35,36].

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Figure 6. Expression pattern of Hes1 in primary cervical cancer cell. Using the UALCAN portal (ualcan.path.uab.edu)[38] we studied the expression pattern of Hes1 transcripts in primary cervical cancer (n=305) and normal tissues (n=3).

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Author’s name Calvin Rodrigues Leanna Rose Joy Sudhir Krishna Sasikala P Sachithanandan

Affiliation NCBS, Bangalore NCBS, Bangalore NCBS, Bangalore NCBS, Bangalore