Potential role of cancer stem cells as biomarkers and therapeutic targets in cervical cancer

Abstract

Background

Eradicating cancer stem cells (CSCs) that are termed as the “beating heart” of various malignant tumors, including cervical cancer, holds great importance in cancer therapeutics. CSCs not only confer chemo‐radio resistance but also play an important role in tumor metastasis and thereby pose a potential barrier for the cure of cervical cancer. Cervical cancer, a common malignancy among females, is associated with high morbidity and mortality rates, and the study on CSCs residing in the niche is promising.

Recent findings

Biomarker approach to screen the cervical CSCs has gained impetus since the past decade. Progress in identification and characterization of the stem cell biomarkers has led to many insights. For the diagnostic purpose, several biomarkers like viral (HPV16), stem cell markers, transcription factors (viz, SOX2, OCT 4, and c‐Myc), and CSC surface markers (viz, ALDH1 and CD44) have been identified. The research so far has been directed to study the CSC stemness and demonstrates various gene expression signatures in cervical CSCs. Such studies hold a potential to improve diagnostic accuracy and predict therapeutic response and clinical outcome in patients.

Conclusions

Stem cell biomarkers have been validated and their therapeutic targets are being developed as “strategies to improve therapeutic ratio in personalized medicine.” This review gives a brief overview of the cervical CSC biomarkers, their current and future diagnostic, prognostic, and therapeutic potential.

List of abbreviations

AGR2

anterior gradient protein 2

AkT

a RAC‐alpha serine/threonine‐protein kinase

ALDH1

aldehyde dehydrogenase 1

AP endonuclease

apurinic/apyrimidinic endonuclease

Ape‐1/Ref‐1

apurinic/apyrimidinic endonuclease 1/redox factor1

BMI1

polycomb complex protein (also known as polycomb group ring finger 4)

CCSCs

cervical cancer stem cells

CD117

proto‐oncogene c‐Kit, also known as Mast/stem cell growth factor receptor (SCFR)

CD133

cluster of differentiation 133 (prominin‐1)

CD24

cluster of differentiation 24 (heat stable antigen‐HSA)

CD44

cluster of differentiation 44, a cell surface glycoprotein

CD49F

Integrin alpha‐6

CD63

cluster of differentiation 63antigen coded by CD63 gene

CDC6

cell division cycle 6

CDH1

cadherin 1

CIN

cervical intraepithelial neoplasia

CK17

cytokeratin 17

Claudin1

claudin 1

c‐Myc

a proto‐oncogene

CNS

central nervous system

CRIPTO (CR‐1)

epidermal growth factor, also known as teratocarcinoma‐derived growth factor 1 (TDGF1)

CSC

cancer stem cell

DAPK1

death‐associated protein kinase 1

EMT

epithelial–mesenchymal transition

ESC

embryonic stem cells

FACS

fluorescence activated cell sorting

GDA

guanine deaminase

hProgC

hematopoietic progenitor cell

HPV

16 human papilloma virus 16 (high risk)

HPV

human papilloma virus

HS3ST2

heparan sulfate‐glucosamine 3‐sulfotransferase 2

HSC

hematopoietic stem cells

IARC

International Agency for Research on Cancer

iPSC

induced pluripotent stem cells

Ki67

cellular marker for proliferation (also known as MKI67)

KRT7

keratin 7

MIB‐I

mindbomb E3 ubiquitin protein ligase 1

MMP7

matrix metalloproteinase

Msi1

Musashi1

mTOR

mammalian target of rapamycin

NF‐κB

nuclear factor‐kappa beta

NS

nucleostemin

OCT (3/4) (Pou5fl)

octamer‐binding protein ¾ (POU class 5 homeobox 1)

OCT4

octamer‐binding transcription factor 4

p16 (INK4a)

a cyclin dependent kinase inhibitor

p53

tumor protein, also known as TP53

PAP Smear test

papanicolaou test

PAR C

secreted protein acidic and rich in cysteine

PI3K

phosphatidylinositol‐4,5‐bisphosphate 3‐kinase

PIWIL1

Piwi‐like RNA‐mediated gene silencing 1

PSCA

prostate stem cell antigen

RARB

retinoic acid receptor beta S

ROS

reactive oxygen species

SC

squamocolumnar Junction

SOX2

sex‐determining region Y‐box 2

STAT3

signal transducer and activator of transcription 3

TBX2

T‐box transcription factor 2

TFP12

tissue factor pathway inhibitor 12

TGF‐β

transforming growth factor beta‐1

UTF‐1

undifferentiated embryonic cell transcription factor 1

VIA

visual inspection test with acetic acid

1. INCIDENCE AND BURDEN OF CERVICAL CANCER

Cervical cancer, the second most common gynecological malignancies in India, and third worldwide, has shown a 5.6% annual increase.1 As per the International Agency for Research on Cancer (IARC), the gynecological cancer statistics for the Indian subcontinent accounts for 30% of the 1.6 million new cases and 90 000 cancer deaths. Five‐year estimates show that cervical cancer alone accounts for 123 000 new cases and 67 000 deaths.2, 3, 4

Currently, the standard screening programs across the world use Papanicolaou test (Pap smear test) for screening precancerous and cancerous cells in the cervix. However, International Federation of Gynecology and Obstetrics (FIGO) now reports the use of visual inspection test with acetic acid (VIA), a low‐cost and efficient technology, for cervical cancer screening.5 As far as cervical cancer diagnosis and staging is concerned, histopathology of the tissue sections and clinico‐radiological estimates are still the standard of care in the therapeutic management of these tumors. However, recent technical innovations in the field of molecular biology have set the platform for digging deeper into the molecular pathogenesis of cervical cancer at cellular and subcellular level and prompted researchers to identify and validate new biomarkers for detection, therapy response, and long term clinical outcome.6 Cervical cancer if localized, in its early stages, is treated by surgery, while in advanced cases, use of radiation therapy along with chemotherapy is done, and palliative care is resorted to in case of terminal malignancies.5, 7

Despite the advances in cervical cancer management, metastasis and relapse of tumor are common in advance stage disease.8 Recent analysis shows a 40% probability of relapse and metastasis over 5 years.9 The underlying reason associated with metastasis is multifold and is a complex mix of hypoxia, DNA repair, and neoangiogenesis, which confers resistance to chemotherapy and/or radiotherapy. A crucial factor affecting the treatment and prognosis is the presence of slow‐cycling, cervical cancer stem cells (CCSCs), residing in the niche areas of the tumors, known to initiate and maintain neoplastic growth.10 The CSC undergoes asymmetric division and self‐renewal, which enable it to maintain its pool. Moreover, tumors contain both CSC and differentiated daughter cells that contribute to the heterogeneity and are difficult to eradicate.11 Therefore, these cells are more resistant to conventional therapies like radiation and chemotherapy and may be the possible route for genesis of cervical cancer and distant metastasis.12, 13 If this theory holds good, then therapeutic targeting of CCSC may reduce the tumor burden by preventing the generation of new tumor clones. This approach would be critical for not only preventing resistance to conventional therapies but also prevent distant metastasis and locoregional relapse.13

Certain strains of human papillomavirus (HPV) are considered to have causal relationship with cervical cancer.14 These viruses have a predilection towards the cells located in the squamocolumnar (SC) junction of the cervical epithelium. These SC junction cells express unique gene profiles, similar to HPV‐associated cervical intraepithelial neoplasia (CIN) and carcinomas, indicating that cervical cancers are derived from the SC junction cells.15 Therefore, it is hypothesized that cervical carcinoma develops from stem‐like cells in the transition zone of the cervical opening that are infected with carcinogenic HPV and that the transition area may be a possible niche for CCSCs.

The current hot spots in the study of cervical cancer are to investigate the relapse patterns after the chemoradiation and regulatory factors in metastasis and identifying new targets/markers for treatment. Overall, optimum management of cervical cancer would involve a dual approach of conventional therapy in combination with CCSC targeted therapy. To achieve this objective, an in‐depth study on identifying specific CCSC biomarkers and targeting these CSCs is required to achieve therapeutic gain.9, 16

2. SEARCH STRATEGY AND SELECTION CRITERIA

Data for this review article was identified by PubMed searches using the search terms: “cervical cancer,” “cancer stem cells,” “cervical cancer stem cells,” “biomarkers,” “HPV and CCSC,” “prognostic potential,” “therapeutic potential,” “targeting CCSC,” “functional assays,” and “discovery of CSC.” Additional references from a few selected articles were also considered. Articles published in English language only were selected.

3. DISCOVERY OF CCSCs AND THEIR IDENTIFICATION USING FUNCTIONAL ASSAYS

The concept that a “rare and specific population of cells” with stem cell (SC) properties give rise to cancer was proposed about over 150 years ago,17, 18 and around 40 years ago, it was hypothesized that the “maturation arrest of the stem cells” caused cancer.17, 18, 19, 20 The cancer stem cells (CSCs) were isolated, and their carcinogenic role was identified a little over the past decade by many studies in the human breast cancer,21, 22 alongside the CSC identification in multiple myelomas,19, 23 and in cancers of the ovary, prostrate, lung, endometrium, and brain.20, 24, 25, 26, 27 Several experimental strategies have been used to identify the presence of SCs in the tissue, combined sorting of tumor cell populations and identification of the CSC based on their surface marker expression.19, 20, 28 Many studies have validated several in vitro and in vivo functional assays that demonstrate the presence of CSCs. An in vivo method using serial transplantation technique as described by Podberezin et al. identifies many immature CSCs at a single‐cell level, and despite being the current gold standard to identify CSCs, these assays are not as cost‐effective and are time consuming.29, 30 “Colony forming cell assays” are the in vitro assays, useful in quantitating the CSCs based on their morphology and measure their proliferative potential.30 Microsphere assay (first performed in the 1960s as “neurosphere‐assay”) is a relatively simple technique, to identify CSC and their markers, that uses flow cytometry analysis of the cells extracted from tumor tissue by enzymatic digestion.29 Although this is a simple and relatively cheap technique, it heavily depends on cell density and cannot detect the quiescent CSCs.29, 30

Current advancements have made it possible to sort tumor cell populations (Fluorescence‐Activated Cell Sorting‐(FACS) or magnetic bead isolation) that express specific surface markers, which are differentially expressed in subpopulations possessing CSC‐like properties. Functional validation assays based on stemness properties of CCSC are the current gold standards. These include measuring clonogenic activity on soft agar, sphere‐forming efficiency, characteristic of self‐renewal (activated PTEN/Akt/β‐catenin pathway) and differentiation, tumorigenic capacity, and asymmetric cell division.28 Another candidate method is side population (SP) assay using the flow cytometry to detect CCSCs based on the ATP‐binding cassette (ABC) family of transporter proteins' dye efflux properties. For example, the expression of the ABCG2 protein can be used to identify CSCs,30 but this method is not specific to CCSCs and therefore needs optimization but is a simple and an effective method.31 However, SPs are not exclusive to SCs and is not universal in all cancer types.32 As opposed to the studies using expression of a single functional marker, recent studies have demonstrated the use of “sequential gating and intermittent of CCSCs,” which helps in high yield of CSCs.33, 34 Methods like optical imaging of CCSCs can be useful to monitor CCSC real time, in vitro and in vivo. Recently, an in vitro optical imaging of CSCs was reported in human cervical cancer in Zs Green‐ODC positive cells using a retroviral vector. The chemo‐radio resistance and cancer stemness of the Zs positive cells reported resistance to cisplatin, paclitaxel, and to ionizing radiation. This indicated that Zs positive cells shared features with CSCs, and this study is suggestive that using a fluorescent protein system, cervical CSC‐like cells can be visualized.31 Till date, majority of the CSC specific functional assays have been carried out in controlled in vitro systems, however it is important to validate these functional assays in in vivo systems.

4. CERVICAL CANCER BIOMARKERS: ROLE OF CCSCs

Until recently, proliferative and nontumorigenic tumor cells were known to have a “non‐stem cells” component that was identified to be “all cancer cells” component. Now, many clinical and experimental observations confirm the presence of tumorigenic CSCs in cervical cancers, and it is possible that these immortal, quiescent CSCs and the non‐CSCs can “stochastically transit between states” and generate an equilibrium, even after these were eliminated.28, 35 As a direct clinical consequence, a single CSC that survives can lead to relapse or metastasis owing to the its properties (Figure 1).16, 36

Figure 1.

Properties of cancer stem cells. The figure shows that the cancer stem cells (CSCs) possess properties of self‐renewal, multilineage differentiation, and tumorigenesis. The small subpopulations of tumour cells selectively differentiate into cancer progenitors and expand the CSC pool.29, 30 Multidrug resistance, quiescence, DNA repair, and antiapoptotic mechanisms render the CSCs resistant to conventional therapy6, 31

HPV infection in the distinct group of cuboidal epithelial cells at the transformation zone (TZ) leads to squamous and columnar cervical neoplasms.7, 15 These group of cells express unique markers, eg, guanine deaminase (GDA), keratin 7 (KRT7), cluster differentiation 63 (CD63), anterior gradient 2 (AGR2), and matrix metalloproteinase 7 (MMP7) among many other. Evidences suggest that SCs from the TZ (niche for cells with embryonic characteristics) in the cervical epithelium serve as targets of malignancy15 (Table 1).

Table 1.

Biomarkers in cervical cancer

Biomarkers	Type	Expression	Ref
GDA	Cancer cell markers	Cuboidal epithelium of transition zone in cervical cancer	Michael et al15
AGR2
KRT7
CD63
MMP7
HPV 16	Expression of genes in CCSC as biomarkers	Tumor tissues/cervical cancer cell lines	Schmitt et al9
p16 (INK4a)			Boulet, Horvath, Depuydt, and Bogers37
p53, MIB‐I			Krkavcová, Jancárková, Janashia, Freitag, and Dusková38
Ki67, CDC6, claudin1			Steinau et al39
SPAR C, TFP12			Sova et al40
CDH1, HS3ST2			Shivapurkar et al41
DAPK1, RARB			Murty and Narayan42

Abbreviations: AGR2, anterior gradient protein 2; CCSC, cervical cancer stem cell; CD63, cluster of differentiation 63 antigen coded by CD63 gene; CDC6, cell division cycle 6; CDH1, cadherin 1; Claudin1, claudin 1;DAPK1, death‐associated protein kinase 1; GDA, guanine deaminase; HPV16, human papilloma virus 16 (high risk); HS3ST2, heparan sulfate‐glucosamine 3‐sulfotransferase 2; Ki67, cellular marker for proliferation (also known as MKI67); KRT7, keratin 7; MIB‐I, Mindbomb E3 ubiquitin protein ligase 1; MMP7, matrix metalloproteinase; p16 (INK4a), a cyclin dependent kinase inhibitor; p53, a tumor protein; RARB, retinoic acid receptor beta; SPAR C, secreted protein acidic and rich in cysteine; TFP12, tissue factor pathway inhibitor 12.

Recent studies speculate that cervical cancer develops from “stem‐like cells” in the transition zone of the cervical opening that is infected with HPV. This junction could be a possible CCSC niche.43, 44, 45 PSCA, PIWIL1, HPV16 E7, and transcriptional factor TBX2 are stem–cell‐associated genes found to be expressed in CCSC. Comparison between normal cervix cells and CCSC showed higher expression of PSCA (60%), PIWIL1 (70%), and TBX2 (50%).43, 45 PSCA and PIWIL1 showed elevated expression, and this was linked to CCSC invasions. On the other hand, the expression of TBX2 was upregulated and had a positive association with metastasis of lymph node. HPV16 showed a positive correlation with PIWIL1, PSCA, and TBX2. On the basis of this finding, one can argue whether HPV16 viral oncoproteins have any role in upregulating the expression of CCSC‐associated genes. Recently, a Piwil2‐like (PL2L) protein was identified that is expressed in human primary and metastatic cervical cancers in association with nuclear factor kappa beta (NF‐κB) known to promote tumorigenesis.46 In vitro or in vivo models have been used to demonstrate a definite role for HPV16 in the over expression of PL2L, PSCA, PIWIL1, and TBX2 (Table 2).

Table 2.

Stem cell markers in cervical cancer

Markers	Expression		Ref
	On CSC	Progenitor Cell/Normal Stem Cell
Nanog	Cytoplasmic	ESC	Ye, Zhou, Cheng, and Shen47
Nucleostemin	Nuclear
Musashi1	Cytoplasmic	ESC, neural stem/progenitor, astroglial and astrocytes in CNS
ALDH1	Cell surface	Adult stem cell (breast)	Gu, Yeo, McMillan, and Yu8, 48
CD44		HSC, MSC, hProgC, PSC
CD49f		ESC	Haraguchi, Ishii, Mimori, Ohta, Uemura, and Nishimura49
SOX2	Nuclear		Ji and Zheng50
Oct3/4 (Pou5fl)	Nuclear (diffused pattern)	ESC, iPSC	Liu et al51
Podoplanin	Nuclear and cell surface	ESC
CRIPTO (CR‐1)	Receptor of TGF‐β pathway		Ertoy et al52
BMI‐1	Nuclear	HSC, ESC, adult stem cell (intestine, breast, prostrate)	Zhang et al,53 Tong et al54
PSCA	Cell surface	ESC	Sasaki, Shiohama, Minoshima, and Shimizu,44 Liu, Jiang, and Zhang45
TBX2	Nuclear	…
PIWIL1
STAT3		ESC	Chen et al,55 Shukla et al56
CD24	Cell surface		Kwon et al,57 Sung58
UTF‐1	Nuclear		Guenin et al59
C‐Myc			Dingqing et al60
p63		…	Mighty and Laimins61

Abbreviations: ALDH1, aldehyde dehydrogenase 1; BMI1, polycomb complex protein (also known as polycomb group ring finger 4); CCSCs, cervical cancer stem cells; CD133, cluster of differentiation 133 (Prominin‐1); CD24, cluster of differentiation 24 (heat stable antigen‐HSA); CD44, cluster of differentiation 44, a cell surface glycoprotein; CD49f, integrin alpha‐6; CIN, cervical intraepithelial neoplasia; CK17, cytokeratin 17; CNS, central nervous system; c‐Myc, a proto‐oncogene; CRIPTO (CR‐1), epidermal growth factor, also known as teratocarcinoma‐derived growth factor 1 (TDGF1); CSC, cancer stem cell; ESC, embryonic stem cells; hProgC, hematopoietic progenitor cell; HSC, haemetopoetic stem cells; iPSC, induced pluripotent stem cells; OCT (3/4; Pou5fl) octamer‐binding protein ¾ (POU class 5 homeobox 1); PIWIL1, piwi like RNA‐mediated gene silencing 1; PSCA, prostate stem cell antigen; SOX2, sex‐determining region Y‐box 2; STAT3, signal transducer and activator of transcription 3; TBX2, T‐box transcription factor 2; UTF‐1, undifferentiated embryonic cell transcription factor 1.

SCs have a hallmark of cancer self‐renewability, and therefore fewer mutations for malignant transformation are necessary than their mature counterparts. SCs are stubborn to survive for longer period than the highly proliferative mature cells that succumb faster. This suggests that, in contrast to mature cells, there is a greater chance that mutations would occur in the SCs,53 and certain evidences point toward the existence of “stem‐like cells” in cervical cancer.

4.1. Nanog, nucleostemin, Musashi1, and p63

The undifferentiated embryonic SCs express SC proteins, like, Nanog, nucleostemin (NS), and Musashi1 (Msi1) in high quantities. These proteins are known to regulate SC differentiation and proliferation in a negative feedback mechanism. Immunohistochemical studies have shown very high expression of Nanog in tissue specimens of invasive cervical cancer and cervical dysplasia as opposed to normal cervical epithelium supporting its role in cervical cancer carcinogenesis and disease progression.47SC populations were identified using antibodies against p63 and cytokeratin 17 (CK17) in normal endo‐cervical, ecto‐cervical, and CIN (I to III). Expression of p63 (nuclear) and CK17 (cytoplasmic) was observed in reserve cell hyperplasia and in CIN lesions (I to III).62 Some other studies substantiate the expression of p63 in the metaplastic squamous cells and in subcolumnar TZ reserve cells.63, 64 p63 expression inversely correlates with maturation of squamous cells and differentiation of nonsquamous cells. This in turn induces cancer cell proliferation, anchorage‐dependent growth, and clonogenicity, indicating that p63 (a probable tumor‐initiating factor in the cancer cell) is a CSC marker in the epithelial tissue.61, 65, 66

4.2. Aldehyde dehydrogenase

Aldehyde dehydrogenase (ALDH1), an enzyme involved in the retinol oxidation and in the early stages of SC differentiation, is a useful CCSC marker. Its expression was studied using immunohistochemistry (IHC) on tissues of invasive squamous carcinoma and adenocarcinoma. The results showed reactivity to ALDH1 in SiHa, HeLa, and CaSki cells.7 Cervical cancer cells that express ALDH1 is associated with a high rate of cellular proliferation and tumorigenesis,67 moreover, in vivo studies have shown that cells expressing high levels of ALDH have high‐tumorigenicity potential indicating that it acts as a stemness factor in cervical cancer.67 Clinical studies have shown ALDH1 to be associated with poorer clinical outcomes.68ALDH1 is defined as a marker for identifying CSC suggesting that cervical carcinoma has a small group of cells that exhibit high ALDH levels and share characteristics with CSCs.8, 69

4.3. SOX2 and OCT4

SOX2, a member of the SOX gene family, encodes for transcription factors expressed in embryonic development, affecting cell fate and differentiation. SOX2 expression in normal and pathological tissues of the cervix when compared in “tumor spheres,” and the differentiated cells confirmed its role in tumorigenesis, suggesting it to be a possible therapeutic target molecule.50 OCT4 (POU5fl), a POU‐domain transcription factor, is yet another marker related to the pluripotency of the inner cell mass in the preimplant embryos, which is highly expressed in CCSC. OCT4 inhibits apoptosis and induces tumorigenesis in vitro.70 High expression of OCT4 in cervical cancer cells relates with the low differentiation of cervical cancer cells and positive lymph node metastasis. High OCT4 expression is linked with chemo‐radio resistance, and it is an independent risk factor for survival of cervical cancer patients.70

4.4. ABCG2

Another marker ABCG2 is an ATP‐binding cassette (ABC) subfamily G member 2. It acts as a drug efflux membrane transporter of the ABC family and plays a major role in multidrug resistance in various cancers due to its ability to pump out a variety of chemicals from the cell.71 In cervical cancer, Nrf2, a redox sensing factor, has an important role in the transcriptional regulation of ABCG2. The upregulation of Nrf2 and ABCG2 is found in cells that have stem‐like characters, like infinite cell proliferation, longevity, and prevention of apoptosis. In a recent study, when the cell lines‐SiHa and C‐33A were sorted using FACS to obtain SP and non‐SP (NSP) cells, SP showed high ABCG2 expression and a potential to generate colonies. This is suggestive that ABCG2 plays a pivotal role in stemness maintenance.72

4.5. CD44 and CD117

IHC study is suggestive that there is high expression of CD44 in normal and cervical cancer cervix. Sorted SP and NSP HeLa cells did not have significant differences in their CD44 expression. Thus, the evidence for CD44 as a marker for CCSC remains incomprehensive.72 CD117/C‐Kit is surface transmembrane cytokine receptor expressed in hematopoietic SCs that is activated by binding to the KIT ligand, an SC factor. In HPV‐associated cervical cancer, IL‐2 receptor‐bγ signaling is activated by c‐Kit that supports T cell proliferation. Also, CD117 expression was found in cervical cancer, which had a high DNA methylation ratio. Although this is used as a biomarker in other types of cancers, its use in cervical cancer is under research.73

4.6. CD49F

CD49F, a cell surface protein, is expressed in mesenchymal, embryonic, and hemopoietic SCs.74 Studies have shown that CD49f is not only upregulated in breast cancer but also in other cancer types like gastric, colon, and prostate cancers and is responsible for maintaining the stemness of these cancer cells.49, 75, 76 Furthermore, tumor spheroids of cervical CSCs expressing high levels of CD49F have been shown to be resistant to radiation therapy.7 Table 2 lists the several SC markers identified for screening of cervical cancer. These findings suggest that CCSCs participate in carcinogenesis, and these cells may be potential molecules that can be used as therapeutic target; however, it, being a new area under investigation, have many questions that remain elusive.69

5. TARGETING CCSC FOR THERAPEUTIC GAIN

Absolute elimination of cancer can presumably be possible provided anticancer therapy targets differentiated cancer cells along with the potential CCSC niche. Relapse or proliferation of resistant and more aggressive tumor cells is often common when therapies are exclusively targeted for differentiated cancer cells. The reason is that such therapies fail to eradicate the CSC pool leading to relapse, causing the death of the patient. The challenge is to have a cancer therapy that targets both the CSCs and the mature cancer cells (by slowing down the differentiated cancer cell proliferation) and increases apoptosis. But, in case of invasive tumors, conventional tumor therapy can prove to be ineffective. Also, treatments that target mature cancer cells to reduce tumor mass without targeting the CSC niche can be proven to be ineffective.28 Moreover, therapeutic intervention can increase the CSC pool by selecting for CSCs that are radio and chemo resistant. These CSCs will have a greater ability for repairing DNA damage caused because of radiotherapy. Such CSCs may overexpress ABC transmembrane pumps, leading to a quick efflux of certain chemotherapeutics. The CSC therapy targets the self‐replicating potential of CSCs and breaks the crosstalk in the SC niche.28 Limited number of mature cancer cells shows increase in CCSC apoptosis as successfully achieved by conventional therapies for cervical cancer.

Exposure to ionizing radiation or platinum compounds causes DNA damage that leads to formation of reactive oxygen species (ROS), which stimulate AP endonuclease activity. This leads to an increased expression of Ape1/Ref‐1.77, 78, 79 It was observed that the cancer cells with primary or secondary resistance to the DNA damaging agents have an overexpression of Ape1/Ref‐1. Such factors help in conferring treatment resistance and suggest the association of Ape1/Ref‐1 in CSC activities. Low ROS levels are found in the CSCs because of the ROS elimination by activated scavenging system. The Ape1/Ref‐1 over expression protects CSCs from ROS damage. Hence, Ape1/Ref‐1 can be responsible for primary resistance in patients. Studies show that p53‐regulated Ape1/Ref‐1 and ROS are crucial in the self‐renewal, differentiation, and survival of CSCs (Figure 2).60, 72, 78, 79, 80

Figure 2.

Specific inhibition of Ape‐1/Ref‐1 and antitumour effects. In cervical cancer cells, exposure to ionizing radiation and/or other agents causes DNA damage, stimulate reactive oxygen species (ROS) production and aid in DNA repair, ultimately leading to relapse. The damaged DNA leads to increased activity of PI3/Akt/mTOR pathway, which promotes cell proliferation and supports cancer cell survival. It also leads to increased cancer stem cell (CSC) formation due to expression of ATP‐binding cassette (ABC) transporters. Alongside, the ROS formation activates c‐Myc and upregulates Ape‐1/Ref‐1(ROS scavenging agents) expression, which favors CSC formation. This Ref‐1 domain decreases ROS levels in cervical cancer cells and result in the increased stemness and self‐renewal potential of the cancer cells resulting in metastasis. As opposed to this, specific inhibition of Ape‐1/Ref‐1 domain leads to increase in intracellular ROS levels, p53 activation, and promotion of cell differentiation. This ultimately leads to cell death caused by repression of the self‐renewal property. Hence, Ape‐1/Ref‐1 blockers have a potential to be considered in combination with DNA‐damaging agents to enhance their antitumor effects. (adapted and modified from Semin Cancer Biol, 31, Skvortsov S, Debbage P, Lukas P, Skvortsova I, Crosstalk between DNA repair and cancer stem cell [CSC] associated intracellular pathways, 36–42, Copyright 2015 with permission from Elsevier)

Targeting CCSC for cervical cancer therapy is relatively new, and CSC targeting therapies are still evolving, the detailed mechanistic of which remains elusive. Considering the nature and functional role of CCSCs in treatment resistance, relapse, an ideal therapy would reach up to the level of eradiating even the minority, resistant CCSC population to achieve disease control.10 RNA interference (RNAi)–based therapies have a great potential for the treatment of cervical cancer. In vitro E6/E7 silencing with RNA interference has shown to influence TGF‐β expression in CD44 high/CD24low cells.48 This is suggestive that RNAi‐based therapy holds a potential for CCSC targeted therapy. Many miRNAs are also responsible in the regulation CCSC stemness and are attractive therapeutic agents for therapy. A certain miR‐302–367 cluster, that was identified in the tumor initiating glioma cells, is known to suppress the growth of CCSCs by negatively regulating cyclin D1 and the AKT1 pathway.81

Although targeting the SCs is an attractive and futuristic approach, it is associated with certain undeniable limitations. CSCs are typically present at very low levels in tumors, accounting for only approximately less than 10% of tumor cells.82 Moreover, the present strategies to target the SCs may not distinctly discriminate between normal and CSCs due to overlapping surface markers. As a result, CSC‐targeted therapy may have deleterious influence on normal SCs and limit the tissue regenerative capacity, thereby interfering with normal tissue functions.

6. DESIGN OF CURRENT AND FUTURE TREATMENT STRATEGIES IN CERVICAL CANCER

Post radiotherapy, the prognostic, and predictive investigations on biomarkers characterize only bulk of tumors and fail to distinguish CSC with non‐CSC. The predictive potential of such assays would be limited if the CSC and non‐CSC responses were completely different. Radiotherapy by CCSC inactivation has shown to have a significant potential in solid tumors, but the tumor recurrences demand the use of combined approaches.83 Even the combining of irradiation with novel targeted drugs has not led to permanent and improved tumor control, indicating a lack of cytotoxic or radio sensitizing effects on CSCs. Biological differences in CSCs and non‐CSCs are reported in many studies, eg, higher hypoxia tolerance, higher radiotherapy resistance, and differential gene expression.84, 85 It is suggestive that CSCs might be protected in their microenvironmental niches. By validating such biological differences, CSC‐specific biomarkers for cancer response prediction can considerably improve treatment.

Still, there are major challenges in the CSC field (Table 3), (Figure 3), and more specific markers are required to understand the physiological roles of CCSCs. This will help to study the process of transition from pluripotency and apply this knowledge to novel therapeutic targeting strategies. Research on the selective targeting of CSCs is still very new, and there is scope for the understanding the mechanistic of CCSC expressions. The selective targeting of specific subpopulations of CSCs can be the central focus for developing drugs. It has become critically important to have gene mapping done for the mutations of Rb, p53, and other major oncogenes3, 12 to identify the posttranslational modifications in the SC markers. This approach will not only expedite the detection methods that are more sophisticated but also help in developing novel therapies. When progress on CCSC markers is made in terms of having enough number of markers, CSC‐specific therapies can be developed that do not affect healthy SCs. This will reduce the adverse effects and contribute to novel regenerative medicine‐based treatment strategies—a new hope for cervical cancer patients (Figure 3).

Table 3.

Challenges in using cervical cancer stem cells (CCSCs)

Figure 3.

Targeting cervical cancer stem cell (CCSC) for therapy. The figure shows a possible model for using CCSCs in therapy. The CCSCs are known to become resistant to conventional treatments and cause metastasis of the tumor (as shown on the top‐right). Targeting CCSCs for therapeutic gain would require eradication of CCSCs by using a selective CSC target treatment from the combination therapy to achieve metastasis‐free survival3, 11

7. CCSCs AS MARKERS FOR PROGNOSTICATING TUMORS: ARE WE THERE YET?

The factors helping clinical oncologists in their first patient interaction are the “diagnostic and prognostic biomarkers” that can be measured. For instance, CD24 was reported as an independent marker for prognostic purpose and in a study of 140 patients with cervical cancer, reported 59 CD24+ patients, showing a high statistical significance with metastasis free survival.58 IHC expression of Nanog, NS, and Msi1 in cervical intraepithelial neoplasia (CIN)‐I to III lesions confirm the presence of these proteins in cervical cancer, however, no correlation was found with the clinical prognostic factors.47 IHC expression of Nanog, NS, and Msi1 in cervical intraepithelial neoplasia (CIN)‐I to III lesions confirm the presence of these proteins in cervical cancer, however, no correlation was found with the clinical prognostic factors.86 IHC on 32 normal cervices, 30 CIN‐III, and 40 cervical carcinoma tissues showed SOX2 expression in 8 normal cervices, 25 CIN‐III and 31 cervical carcinomas, but failed to provide clinical prognostic correlation. OCT 4, expressed in the 91.9% of cervical cancer tissues, is another independent prognostic factor70 (Table 4).

Table 4.

Diagnostic, predictive, and prognostic potential of CSC markers in cervical cancer

Marker	Target	Diagnostic, Prognostic, and Predictive Potential		Ref
p63	Normal cervix and CIN (I, II, III)	+/−	Possibility of metastasis free survival	Martens et al,66 Kim, Lee, Lee, Kim, and Lee87
CK17
Nucleostemin	Normal cervix and CCSC	+/−
Mushashi1				Ye, Zhou, Cheng, and Shen47
Nanog
OCT4	CCSC and normal cervix, CIN III	+	Metastasis free survival	Yang, Wang, Chunxia, and Xiuying70
SOX2	Normal cervix, CIN III	+	Metastasis free survival	Ji and Zheng50
ALDH1	Invasive carcinoma	+	CD133 expression	Yao, Wu, Liu, Rao, and Lin68
CD24	Normal cervix and invasive carcinoma	+	Metastatic free survival of patients	Sung et al58

Abbreviations: ALDH1, aldehyde dehydrogenase 1; CCSCs, cervical cancer stem cells; CD133, cluster of differentiation 133(Prominin‐1); CD24, cluster of differentiation 24 (heat stable antigen‐HSA); CIN, cervical intraepithelial neoplasia; CK17, cytokeratin 17; OCT4, octamer‐binding transcription factor 4; PIWIL1, piwi‐like RNA‐mediated gene silencing 1; PSCA, prostate stem cell antigen; SOX2, sex‐determining region–Y‐box 2.

Such diagnostic and prognostic biomarkers help in identifying who is at risk and therefore aid in early diagnosis. This translates in selecting the best suited treatment and monitor response for the patients. Treatment resistance is known to be conferred by the residual CSCs and this relates with poor prognosis88 as there the circulating tumor cells are not eradicated and could lead to relapse. Such circulating tumor cells although sometimes nonproliferative can express stem–cell‐like characteristics (including expression of specific biomarkers).10, 88, 89, 90 Radiological techniques and circulating levels of tumor‐specific antigens are useful in assessing traditionally known biomarkers. Technologies like mass spectrometry, microarrays, high‐throughput DNA sequencing, complete human genome sequence, and the information on potential cancer biomarkers are useful to know the DNA, RNA, and protein expressions.37, 39, 40, 59 Advances in imaging technologies will give way to noninvasively monitor molecular biomarkers (eg, those responding to therapy) in cancer patients.7, 90

The current understanding of the concepts of cervical CSC biomarkers and their evaluation in clinical scenario is suggestive that most of the markers indicate progression of lesions that are already initiated. Also, these markers may not be very sensitive to identify all initiated lesions, however, very limited data is available that validates and concludes the clinical diagnostic value of these biomarkers. Large, prospective cohort studies can be conducted, where conventional histological analysis of disease outcomes is measured with biomarker evaluation.3, 16 In cervical cancer screening, the use of multiple markers remains the current trend among the investigators. The use of multiple markers in combination increases the sensitivity and specificity of the assay, and using these well‐characterized markers and highly accurate screening for cervical cancer and CIN can be made possible feasible in the future.

8. CONCLUSION

Recent research to detect and characterize CSC has important implications for cervical cancer treatment and therapy. The area of research is dynamically emerging, and the biomarker development for CCSC pool has a promising avenue despite the challenges related to the CCSC markers and the recent finding of marked genetic and functional CCSC heterogeneity. Important and useful information is expected to come from radiobiological characterization of CCSCs and their validation studies performed in vitro and in vivo, eg, tumor control assays. Perhaps, in clinical studies, it is indicative to use appropriate confounders by multivariate analysis or unbiased prospective design to achieve the “bench‐to‐bed side” translation of new treatment strategies. This may form the basis for treatments that target CCSC in cervical cancer. The CSCs are more resistant to conventional therapies like radiation and chemotherapy and are the possible route for genesis of cervical cancer and distant metastasis. The research on cervical CSCs is still at an initial stage, but the existence of CSCs is becoming increasingly convincing due to the evolution in molecular technology that helps researchers to track tumor cells.

CONFLICTS OF INTEREST

None.

FUNDING INFORMATION

None.

AUTHORS' CONTRIBUTION

All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization, J.S.G., N.S.; Methodology, N.S., J.S.G., N.P.R.; Investigation, S.C., J.S.G., S.K.S.; Formal Analysis, N.P.R., H.S., A.M.; Resources, S.C., J.S.G., S.K.S.; Writing ‐ Original Draft, N.S.; Writing ‐ Review & Editing, J.S.G., N.S.; Visualization, N.S., S.C., J.S.G.; Supervision, J.S.G., S.C.; Funding Acquisition, S.C., J.S.G.

Sudhalkar N, Rathod NP, Mathews A, et al. Potential role of cancer stem cells as biomarkers and therapeutic targets in cervical cancer. Cancer Reports. 2019;2:e1144. 10.1002/cnr2.1144