Cervical Cancer Stem-like Cells Research Articles

SF1: A Standardised Fraction of Clinacanthus nutans That Inhibits the Stemness Properties of Cancer Stem-Like Cells Derived from Cervical Cancer

Cancer stem cells (CSCs) are a small population of tumour cells that are responsible for tumour initiation, metastases, recurrence, and resistance to conventional therapy. Hence, targeting CSCs is crucial in the fight against cancer. SF1, a standardised fraction from Clinacanthus nutans leaf extract, has been reported to exhibit potent and selective antineoplastic effects against cervical cancer cells. In this study, the potential of SF1 to inhibit the stemness of cervical cancer stem-like cells has been evaluated. SF1 extraction was carried out using the dry column vacuum chromatography technique. SiHa cell lines were cultured as spheres in CSC-conditioned medium (cervospheres), and the IC50 of SF1 against cervospheres was determined using the OZBlue Cell Viability Kit. The effects of SF1 on the cervosphere’s stemness markers, including CD49f, CK17, SOX2, OCT4, and NANOG, were assessed using a flow cytometry assay. Self-renewal inhibition and anti-tumorigenesis effects of SF1 in cervospheres were evaluated using a sphere formation assay and a xenograft mouse model. The present study shows that SF1 treatment at an IC50 of 17.07 µg/mL inhibited the proliferation, self-renewal, and tumorigenic capacity of SiHa cervospheres in vitro and in vivo. A decrease in the expressions of CK17, SOX2, CD49f, and OCT4 in cervical CSCs indicated that SF1’s inhibitory effects were also associated with the suppression of stemness markers. These results suggest that SF1 possesses an antitumor effect against cervical CSCs and may be regarded as a promising approach to the development of targeted anticancer agents for cervical cancer.

Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1.

To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism. Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels, self-renewal capability (Nanog and Sox2), and cancer stem cell markers (CD133 and CD44), were detected by a colorimetric DNMT activity/inhibition assay kit, quantitative real-time reverse transcription-polymerase chain reaction, sphere and colony formation assays, and immunoblot, respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA, respectively, were performed to explore the mechanism for action of CAS (0, 10, 30, and 100 nmol/L). DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (P<0.05). In addition, HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities, and lower CD133, CD44, Nanog and Sox2 protein expressions (P<0.05). Conversely, overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore, CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (P<0.05). Interestingly, DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected, DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells. CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation, suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.

Cytotoxic effect of disulfiram/copper on human cervical cancer cell lines and LGR5-positive cancer stem-like cells

BackgroundTumor resistance is a global challenge for tumor treatment. Cancer stem cells (CSCs) are the main population of tumor cells for drug resistance. We have reported that high aldehyde dehydrogenase (ALDH) activity represents a functional marker for cervical CSCs. Here, we aimed at disulfiram (DSF), an ALDH inhibitor, that has the potential to be used for cervical cancer treatment.MethodsMTT assay, western blot, vector construction and transfection, cell sorting and in vivo anti-tumor assays were performed using cervical cancer cell lines SiHa and HeLa. Cell cycle distribution and cell apoptosis were carried out by flow cytometry. The cytotoxicity of DSF was detected by MTT assay and cervical cancer xenograft models.ResultsDSF was cytotoxic to cervical cancer cell lines in a copper (Cu)-dependent manner. Disulfiram/copper (DSF/Cu) complex induced deregulation of S-phase and inhibited the expression of stemness markers in cervical cancer cells. Furthermore, DSF/Cu could also reduce the cancer stem cell-like LGR5+ cells which lead to cisplatin resistance in cervical cancer cells. DSF/Cu complex had the greater antitumor efficacy on cervical cancer than cisplatin in vitro and in vivo.ConclusionOur findings indicate that the cytotoxicity of DSF/Cu complex may be superior to cisplatin because of targeting LGR5-positive cervical cancer stem-like cells in cervical cancer. Thus, the DSF/Cu complex may represent a potential therapeutic strategy for cervical cancer patients.

Cervical Cancer Stem-Like Cell Transcriptome Profiles Predict Response to Chemoradiotherapy.

Cervical cancer (CC) represents a major global health issue, particularly impacting women from resource constrained regions worldwide. Treatment refractoriness to standard chemoradiotheraphy has identified cancer stem cells as critical coordinators behind the biological mechanisms of resistance, contributing to CC recurrence. In this work, we evaluated differential gene expression in cervical cancer stem-like cells (CCSC) as biomarkers related to intrinsic chemoradioresistance in CC. A total of 31 patients with locally advanced CC and referred to Mário Penna Institute (Belo Horizonte, Brazil) from August 2017 to May 2018 were recruited for the study. Fluorescence-activated cell sorting was used to enrich CD34+/CD45- CCSC from tumor biopsies. Transcriptome was performed using ultra-low input RNA sequencing and differentially expressed genes (DEGs) using Log2 fold differences and adjusted p-value < 0.05 were determined. The analysis returned 1050 DEGs when comparing the Non-Responder (NR) (n=10) and Responder (R) (n=21) groups to chemoradiotherapy. These included a wide-ranging pattern of underexpressed coding genes in the NR vs. R patients and a panel of lncRNAs and miRNAs with implications for CC tumorigenesis. A panel of biomarkers was selected using the rank-based AUC (Area Under the ROC Curve) and pAUC (partial AUC) measurements for diagnostic sensitivity and specificity. Genes overlapping between the 21 highest AUC and pAUC loci revealed seven genes with a strong capacity for identifying NR vs. R patients (ILF2, RBM22P2, ACO16722.1, AL360175.1 and AC092354.1), of which four also returned significant survival Hazard Ratios. This study identifies DEG signatures that provide potential biomarkers in CC prognosis and treatment outcome, as well as identifies potential alternative targets for cancer therapy.

Pterostilbene Suppresses both Cancer Cells and Cancer Stem-Like Cells in Cervical Cancer with Superior Bioavailability to Resveratrol.

Increasing studies have reported that cancer stem cells (CSCs) play critical roles in therapeutic resistance, recurrence, and metastasis of tumors, including cervical cancer. Pterostilbene, a dimethylated derivative of resveratrol, is a plant polyphenol compound with potential chemopreventive activity. However, the therapeutic effect of pterostilbene against cervical CSCs remains unclear. In this study, we compared the anticancer effects of resveratrol and pterostilbene using both HeLa cervical cancer adherent and stem-like cells. Pterostilbene more effectively inhibited the growth and clonogenic survival, as well as metastatic ability of HeLa adherent cells than those of resveratrol. Moreover, the superior inhibitory effects of pterostilbene compared to resveratrol were associated with the enhanced activation of multiple mechanisms, including cell cycle arrest at S and G2/M phases, induction of ROS-mediated caspase-dependent apoptosis, and inhibition of matrix metalloproteinase (MMP)-2/-9 expression. Notably, pterostilbene exhibited a greater inhibitory effect on the tumorsphere-forming and migration abilities of HeLa cancer stem-like cells compared to resveratrol. This greater effect was achieved through more potent inhibition of the expression levels of stemness markers, such as CD133, Oct4, Sox2, and Nanog, as well as signal transducer and activator of transcription 3 signaling. These results suggest that pterostilbene might be a potential anticancer agent targeting both cancer cells and cancer stem-like cells of cervical cancer via the superior bioavailability to resveratrol.

Nanoquinacrine sensitizes 5-FU-resistant cervical cancer stem-like cells by down-regulating Nectin-4 via ADAM-17 mediated NOTCH deregulation.

Cervical cancer is a major cause of cancer-related death in women world-wide. Although the anti-metabolite 5-FU is widely used for its treatment, its clinical utility is limited due to the frequent occurrence of drug resistance during metastasis. Cancer stem-like cells (CSCs), present in the heterogeneous population of CC cells, are thought to contribute to this resistance. Nectin-4, a CSC marker, is known to play an important role in the cellular aggressiveness associated with metastatic CC. This study was designed to assess the role of Nectin-4 in the acquisition of 5-FU resistance by metastatic CC cells, including its relation to the NOTCH signalling pathway. 5FU-resistant CC cell lines were deduced from ME-180 and SiHA cells by continuous exposure to a single concentration of 5-FU. Thymidylate synthase (TS) positive cells were isolated from the 5-FU resistant cells, after which a metastatic model was developed. The role of Nectin-4 in the sensitization of 5-FU resistant metastatic CC cells upon incubation with Nano-formulated Quinacrine (NQC) was investigated using multiple bioassays including MTT, FACS, ELISA, immunoflurescence, Western blotting, comet and in vivo plasmid-based short patch and long patch base excision repair assays. We found that the expression level of Nectin-4, as well as that of other CSC markers (Oct-4, β-catenin, SOX2) and representative NOTCH signalling components (NOTCH-1, Jagged-1, γ-secretase, ADAM-17) were elevated in the 5-FU resistant metastatic cells compared to those in control cells. Increased nuclear translocation of Nectin-4 and increased proliferation and invasion rates were observed after culturing the metastatic cells under hypoxic conditions. Treatment with NQC inhibited the nuclear translocation of Nectin-4 and decreased the proliferation and invasion rates of the cells by inhibiting the induction of base excision repair (BER) pathway components and ADAM-17 expression levels. After combination treatment of Nectin-4 overexpressing metastatic CC cells with a specific ADAM-17 inhibitor (GW280264) and NQC, a decreased Nectin-4 expression, without alterations in BER and/or other NOTCH pathway components, was noted. Our data indicate that Nectin-4 may play a prominent role in 5-FU resistance of metastatic CC cells and that NQC sensitizes these cells by Nectin-4 deregulation through ADAM-17 inhibition, a major component of the NOTCH signalling pathway.

PO-296 Elucidating the potential role of CD109 as a biomarker for cancer stem-like cells in cervical cancer

IntroductionCervical cancer is a common genital tract cancer. Radiotherapy is the mainstay of management for advanced cervical cancer. Response to radiation varies widely which may be explained by the existence of cancer stem-like cells (CSCs). Since CSCs is implicated in cervical cancer and demonstrated a high degree of resistance to radiation, the identification of novel CSC markers could be critical to specifically target the cervical CSC. In our pilot study, we established attached and spheroid cells from primary cervical tumour tissue. Multiple ‘stemness’ genes were detected in spheroid cells which indicated primary cervical cancer tissue harboured CSCs population. cDNA microarray analysis was performed to compare cDNA expression profile and CD109 was significantly up-regulated in spheroid cells. Therefore, we hypothesise that CD109 may serve as a potential cervical CSCs marker.Material and methodsFlow cytometry was performed to analyse the CD109 expression and isolate the CD109 positive and negative sub-populations in cervical cancer cells. Following the sorting, cell proliferation and migration assay were performed. In order to investigate the effect on the blockade of CD109, SiHa, Caski and C4-1 cells were transfected with CD109 siRNAs. XTT assay, migration and invasion assay and spheroid formation assay were performed. Immunohistochemistry (IHC) was performed for the detection of CD109 expression in cervical cancer tissue microarray (TMA).Results and discussionsThe post-sorted CD109 (+) cells grew remarkably faster and have stronger migration capability than CD109(-) cells in Caski and C4-1. The CD109 knockdown cells with siRNA exhibited a slower cell growth, decreasing migration and self-renewal ability, as compared with the control group in SiHa, Caski and C4-1 cells. IHC of TMA indicated that CD109 was highly expressed in cancer cases than that in normal/benign cases.ConclusionCD109 increased cell proliferation rate and migration ability in post-sorted cervical cancer cell lines Caski and C4-1. On the contrary, CD109 knockdown reduced cell growth, migration and self-renewal capability in cervical cancer SiHa, Caski and C4-1 cells. Cervical carcinoma showed high expression of CD109 protein by IHC. Further in vivo and in vitro functional assays are essential to characterise the CD109-positive sub-population in cervical cancer which may provide more information of cervical CSCs-related properties and resistance of radiation therapy, with the underlying molecular mechanism involved.

Cervical cancer stem-like cells: systematic review and identification of reference genes for gene expression.

Cervical cancer is the fourth most common cancer affecting women worldwide. Among many factors, the presence of cancer stem cells, a subpopulation of cells inside the tumor, has been associated with a worse prognosis. Considering the importance of gene expression studies to understand the biology of cervical cancer stem cells (CCSC), this work identifies stable reference genes for cervical cancer cell lines SiHa, HeLa, and ME180 as well as their respective cancer stem-like cells. A literature review was performed to identify validated reference genes currently used to normalize RT-qPCR data in cervical cancer cell lines. Then, cell lines were cultured in regular monolayer or in a condition that favors tumor sphere formation. RT-qPCR was performed using five reference genes: ACTB, B2M, GAPDH, HPRT1, and TBP. Stability was assessed to validate the selected genes as suitable reference genes. The evaluation validated B2M, GAPDH, HPRT1, and TBP in these experimental conditions. Among them, GAPDH and TBP presented the lowest variability according to the analysis by Normfinder, Bestkeeper, and ΔCq methods, being therefore the most adequate genes to normalize the combination of all samples. These results suggest that B2M, GAPDH, HPRT1, and TBP are suitable reference genes to normalize RT-qPCR data of established cervical cancer cell lines SiHa, HeLa, and ME180 as well as their derived cancer stem-like cells. Indeed, GAPDH and TBP seem to be the most convenient choices for studying gene expression in these cells in monolayers or spheres.

Cervical cancer stem cells manifest radioresistance: Association with upregulated AP-1 activity

Transcription factor AP-1 plays a central role in HPV-mediated cervical carcinogenesis. AP-1 has also been implicated in chemo-radio-resistance but the mechanism(s) remained unexplored. In the present study, cervical cancer stem-like cells (CaCxSLCs) isolated and enriched from cervical cancer cell lines SiHa and C33a demonstrated an elevated AP-1 DNA-binding activity in comparison to non-stem cervical cancer cells. Upon UV-irradiation, CaCxSLCs showed a UV exposure duration-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV-irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV-irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in UV-irradiated CaCxSLCs. Thus, the study suggests a critical role of AP-1 protein in the manifestation of radioresistance but targeting with curcumin helps in radiosensitizing CaCxSLCs through upregulation of Fra-1.

Abstract 3898: Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers

Abstract Background: Activator protein-1 (AP-1) plays a central role in HPV-mediated cervical and tongue cancer (subset of HNSCC). AP-1, a homo/heterodimeric protein composed of Jun and Fos family proteins, has been implicated in chemoradioresistance but the mechanism(s) remained unexplored. In the present study, AP-1 family protein Fra-1 has been shown to play a critical role in promoting radiosensitization in enriched cervical cancer stem-like cells (CaCxSLCs) and can serve as a target for drug intervention in cervical and other HPV associated cancers. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating HPV+ve and HPV-ve human cervical cancer cell lines, SiHa, and C33a using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Both CaCxSLCs and Non CaCxSLCs were exposed to different dose of UV irradiation and a herbal compound curcumin, a potent inhibitor of AP-1 was used to examine the role of AP-1 and its family proteins in the radiosensitization of cancer stem cells. Results: Upon UV irradiation, CaCxSLCs showed a dose-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in irradiated CaCxSLCs. Conclusion: The study suggests a critical role of AP-1 protein in the manifestation of radioresistance but curcumin pretreatment helps in radiosensitizing CaCxSLCs through upregulation of Fra-1 which may serve as a potential target for elimination of radioresistant cancer stem cells in HPV-associated malignancies. Note: This abstract was not presented at the meeting. Citation Format: Abhishek Tyagi, Shilpi Gupta, Prabhat Kuamr, Alok C. Bharti, Bhudev C. Das. Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2017-3898

Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells.

Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell’s sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6.

Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is involved in cervical cancer stemness and can be a target of immunotherapy.

Cervical cancer is a major cause of cancer death in females worldwide. Cervical cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are resistant to conventional radiotherapy and chemotherapy, and CSCs/CICs are thought to be responsible for recurrence. Eradication of CSCs/CICs is thus essential to cure cervical cancer. In this study, we isolated cervical CSCs/CICs by sphere culture, and we identified a cancer testis (CT) antigen, CTCFL/BORIS, that is expressed in cervical CSCs/CICs. BORIS has 23 mRNA isoform variants classified by 6 subfamilies (sfs), and they encode 17 different BORIS peptides. BORIS sf1 and sf4 are expressed in both CSCs/CICs and non-CSCs/CICs, whereas BORIS sf6 is expressed only in CSCs/CICs. Overexpression of BORIS sf6 in cervical cancer cells increased sphere formation and tumor-initiating ability compared with those in control cells, whereas overexpression of BORIS sf1 and BORIS sf4 resulted in only slight increases. Thus, BORIS sf6 is a cervical CSC/CIC-specific subfamily and has a role in the maintenance of cervical CSCs/CICs. BORIS sf6 contains a specific c-terminal domain (C34), and we identified a human leukocyte antigen (HLA)-A2-restricted antigenic peptide, BORIS C34_24(9) encoded by BORIS sf6. A BORIS C34_24(9)-specific cytotoxic T cell (CTL) clone showed cytotoxicity for BORIS sf6-overexpressing cervical cancer cells. Furthermore, the CTL clone significantly suppressed sphere formation of CaSki cells. Taken together, the results indicate that the CT antigen BORIS sf6 is specifically expressed in cervical CSCs/CICs, that BORIS sf6 has a role in the maintenance of CSCs/CICs, and that BORIS C34_24(9) peptide is a promising candidate for cervical CSC/CIC-targeting immunotherapy.

Abstract 4079: Role of HPVE6 oncoprotein in the maintenance of cervical cancer stem cells

Abstract Background &amp; Objectives: Persistent infection of high-risk human papillomaviruses (HR-HPV) is a key etiological factor responsible for the development of cervical cancer. Deregulated expression of two viral oncoproteins E6 and E7 drive oncogenic transformation and tumour progression which may occur upon targeted infection of specific basal cervical cells with stem cell properties. However, how oncoproteins impact on putative CSCs, the stem cell signaling and their downstream functions, is not known. In this study, we have identified and characterized cervical cancer stem-like cells and unraveled the crosstalk between HPV oncoproteins and stem cell signaling that allowed maintenance of stemness in cancer cells. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa, HeLa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Moreover, differential expression level and DNA-binding activity of CSL-specific Notch1 and its downstream targets in CaCxSLCs cells along with HPVE6 specific siRNA mediated gene silencing was evaluated by gel retardation assay, immunobloting qRT-PCR followed by in vivo tumor-initiating capacity in athymic nude mice. Results: CaCxSLCs cells isolated from HPV+ve cells showed high expression of CD49f, Lrig1, Nanog, Sox2, Oct4, ABCG2 and low Notch1 DNA binding activity as compared to non- CaCxSLCs cells. Enriched CaCxSLCs were also found to be more tumorigenic and showed high expression of HPVE6, HES1, CD133 transcripts with high percentage of SP cells. CaCxSLCs differed from their parental/non-CaCxSLCs as they overexpressed E6 oncogene. Knocking down of HPVE6 by RNA interference resulted in abolition of spheroid formation through downregulation of Hes1 and induction of re-differentiation while interference of Hes-1 expression resulted in reduced cervicosphere formation through down regulation of E6 and Nanog suggesting loss of self-renewing ability. Conclusions: Our findings suggest a potential regulatory role of HPVE6 in induction and maintenance of cervical cancer stem-like cells through Hes1 expression which could be utilized as a putative target for designing drugs that can promote differentiation of cancer stem cells making the therapy most effective. Note: This abstract was not presented at the meeting. Citation Format: Bhudev C. Das, Abhishek Tyagi, Kanchan Vishnoi, Sutapa Mahata, Gaurav Verma, Alok C. Bharti. Role of HPVE6 oncoprotein in the maintenance of cervical cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2015-4079

The differential expression of OCT4 isoforms in cervical carcinoma.

OCT4 is a transcription factor involved in maintaining stem cell phenotype and pluripotential. However, it remains unclear the expression pattern and biological function of OCT4 isoforms in cervical cancer. Here, we reported that both nuclear OCT4A and cytoplasmic OCT4B were overexpressed in CC. OCT4A was responsible for self-renewal of cervical cancer stem–like cells (CCSCs). Furthermore, OCT4B overexpression in SiHa cervical cancer cell line significantly increased cell proliferation and tumorigenesis by inhibiting apoptosis. Moreover, OCT4B enhanced angiogenesis by the upregulation of CD34, VEGF, HIF-1α and IL-6, and promoted tumor cell mobility to the surrounding tissue by the upregulation of MMP2 and MMP9, and the induction of epithelial-mesenchymal transition (EMT). In conclusion, nuclear OCT4A may serve as a marker of CCSCs and the driving force for cervical cancer metastasis and recurrence, while cytoplasmic OCT4B may cooperate with OCT4A to regulate the progression of cervical cancer through inducing angiogenesis and EMT.

Apigenin inhibits HeLa sphere-forming cells through inactivation of casein kinase 2α.

The protein kinase casein kinase 2 (CK2) has been implicated in stem cell maintenance and its aberrant activation has been demonstrated in several types of cancer, including cervical cancer. In the present study, it was demonstrated that the sphere-forming cells (SFCs) of HeLa cell lines exhibited self-renewal capacity, indicating that they possessed the properties of cervical cancer stem-like cells. HeLa-derived SFCs exhibited a higher level of CK2α protein, compared with the parental cells. Apigenin, a dietary flavonoid, led to a dose-dependent inhibition of the self-renewal capacity and the protein expression of CK2α in HeLa-derived SFCs. Furthermore, forced overexpression of CK2α resulted in a decrease in the inhibition of CK2α expression and the self-renewal capacity induced by apigenin in HeLa-derived SFCs. These results suggested that apigenin inhibits the self-renewal capacity of HeLa-derived SFCs through downregulation of CK2α expression.

Silencing of E6/E7 expression in cervical cancer stem-like cells.

Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for the tumor recurrence and metastasis, the two major causes of cancer-related death. Therefore, CSC-targeted cancer therapy is important for the future development of more effective and advanced cancer therapy. One of the approaches is to specifically silence oncogene expression in CSCs and inhibit their growth. The significance of this approach is its specificity and ability to avoid multi-drug resistance of CSCs. In this chapter, we will describe a method of silencing HPV oncogenes E6/E7 in human cervical CSCs using HeLa cells as a model system.

Abstract 3028: Optical imaging of cancer stem-like cells in cervical cancer

Abstract Cancer stem cells (CSCs) defined by differentiation capacity, self-renewal capacity and tumorigenicity have been identified in many tumors, including cervical cancer. Current studies in the field of cancer stem cell research identify CSCs by several specific biomarkers, such as ALDH1, but it is difficult to monitor cervical CSCs in real-time in vitro and in vivo. Recent research reported the visualization of CSCs in breast cancer and giloma using green fluorescent protein ZsGreen fused to a degron motif ornithine decarboxylase (ODC) which is destructed by the proteasome. According to the research, CSCs are low 26S proteasome activity, while non-CSCs are high. Therefore it is possible to observe CSCs which accumulate fluorescent ZsGreen protein. In this study, we investigated optical imaging of CSCs in human cervical cancer cell lines of CaSki and Hela. We engineered CaSki and HeLa cervical cancer cells expressing ZsGeen-ODC (Zs positive cells) by a retroviral vector. The cancer stemness of their Zs positive cells was assessed by sphere formation assay, and the resistance of cancer stem cells to chemotherapy and radiation therapy was investigated by MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide)assay and clonogenic survival assay. The low 26S proteasome activity of Zs positive cells was confirmed by Proteasome-Glo™ Cell Based Assay (Promega). Zs positive cells of both CaSki and HeLa showed high ability in sphere formation assay, compared with cancer cells which did not express ZsGreen-ODC (Zs negative cells). In MTT assay, Zs positive cells of CaSki were resistant to cisplatin and paclitaxel, whereas those of HeLa were not resistant as compared with Zs negative cells of HeLa. Our data of clonogenic survival assay suggested that Zs positive cells of both CaSki and HeLa were more resistant to ionizing radiation than Zs negative cells. Thus our results indicated that Zs positive cells of CaSki and Hela had features of CSCs. In conclusion, we could visualize cervical cancer stem-like cells using a fluorescence protein system. Further researches of detecting real-time asymmetric cell division in vitro and analyzing the tumorigenicity of these cells in vivo are necessary to show evidence of cancer stemness and they are now going. In the next step, we will research the relationship between epithelial-mesenchymal transition and the radiation resistance using this real-time tracking system. Citation Format: Kazuhiko Hayashi, Keisuke Tamari, Yoshihiro Kano, Shimpei Nishikawa, Takahito Fukusumi, Masaaki Miyo, Kozo Noguchi, Hisataka Ogawa, Atsushi Hamabe, Masamitsu Konno, Yuji Seo, Hideshi Ishii, Yuichiro Doki, Masaki Mori, Kazuhiko Ogawa. Optical imaging of cancer stem-like cells in cervical cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3028. doi:10.1158/1538-7445.AM2014-3028

Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.

Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability

Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. They are also considered as an attractive target for advanced cancer therapy. Using a sphere culture method that favors the growth of self-renewal cells, we have isolated sphere-forming cells (SFCs) from cervical cancer cell lines HeLa and SiHa. HeLa-SFCs were resistant to multiple chemotherapeutic drugs and were more tumorigenic, as evidenced by the growth of tumors following injection of immunodeficient mice with 1 × 10(4) cells, compared with 1 × 10(6) parental HeLa cells required to grow tumors of similar size in the same time frame. These cells showed an expression pattern of CD44(high)/CD24(low) that resembles the CSC surface biomarker of breast cancer. We further demonstrated that HeLa-SFCs expressed a higher level (6.9-fold) of the human papillomavirus oncogene E6, compared with that of parental HeLa cells. Gene silencing of E6 with a lentiviral-short-hairpin RNA (shRNA) profoundly inhibited HeLa-SFC sphere formation and cell growth. The inhibition of cell growth was even greater than that for sphere formation after E6 silence, suggesting that the loss of self-renewing ability may be more important. We then measured the expression of self-renewal genes, transformation growth factor-beta (TGF-β) and leukemia-inhibitory factor (LIF), in shRNA-transduced HeLa-SFCs and found that expression of all three TGF-β isoforms was significantly downregulated while LIF remained unchanged. Expression of the Ras gene (a downstream component of TGF-β) was also markedly decreased, suggesting that the growth-inhibitory effect could be via the TGF-β pathway. The above data indicate RNA interference-based therapy may offer a new approach for CSC-targeted cancer therapy.

Argyrophile bodies in the human spinal cord.

<h3>ABSTRACT</h3> Cervical cancer (CC) represents a major global health issue, particularly impacting women from resource constrained regions worldwide. Treatment refractoriness to standard chemo-radiotherapy has identified cancer stem cells as critical coordinators behind the biological mechanisms of resistance, contributing to CC recurrence. In this work, we evaluated differential gene expression in cervical cancer stem-like cells (CCSC) as biomarkers related to intrinsic chemoradioresistance in CC. A total of 31 patients with locally advanced CC and referred to Mario Penna Institute (Belo Horizonte, Brazil) from August 2017 to May 2018 were recruited for the study. Fluorescence-activated cell sorting (FACS) was used to enrich CD34+/CD45-CCSC from tumor biopsies. Transcriptome was performed using ultra-low input RNA sequencing and differentially expressed genes (DEGs) using Log2 fold differences and adjusted p value &lt; 0.05 were determined. A panel of biomarkers was selected using the rank-based AUC (Area Under the ROC Curve) and pAUC (partial AUC) measurements for diagnostic sensitivity and specificity. The analysis showed 1062 DEGs comparing between the Non-Responder (n=10) and Responder (n=21) groups to chemoradiotherapy. Overlapping of the 20 highest AUC and pAUC values revealed five transcripts potentially implicated in innate chemoresistance (<i>ILF2, SNX2, COPZ1</i>, AC016722.1 and AL360175.1). This study identifies DEG signatures that serve as potential biomarkers in CC prognosis and treatment outcome, as well as identifies potential alternative targets for cancer therapy.