Showing papers by "Alok C. Bharti published in 2017"

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

Abstract: 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.

Characterization of key transcription factors as molecular signatures of HPV-positive and HPV-negative oral cancers

Abstract: Prior studies established constitutively active AP-1, NF-κB, and STAT3 signaling in oral cancer. Differential expression/activation of specific members of these transcription factors has been documented in HPV-positive oral lesions that respond better to therapy. We performed a comprehensive analysis of differentially expressed, transcriptionally active members of these pivotal signaling mediators to develop specific signatures of HPV-positive and HPV-negative oral lesions by immunohistochemical method that is applicable in low-resource settings. We examined a total of 31 prospective and 30 formalin-fixed, paraffin-embedded tissues from treatment-naive, histopathologically and clinically confirmed cases diagnosed as oral or oropharyngeal squamous cell carcinoma (OSCC/OPSCC). Following determination of their HPV status by GP5 + /GP6 + PCR, the sequential sections of the tissues were evaluated for expression of JunB, JunD, c-Fos, p50, p65, STAT3, and pSTAT3(Y705), along with two key regulatory proteins pEGFR and p16 by IHC. Independent analysis of JunB and p65 showed direct correlation with HPV positivity, whereas STAT3 and pSTAT3 were inversely correlated. A combined analysis of transcription factors revealed a more restrictive combination, characterized by the presence of AP-1 and NF-κB lacking involvement of STAT3 that strongly correlated with HPV-positive tumors. Presence of STAT3/pSTAT3 with NF-κB irrespective of the presence or absence of AP-1 members was present in HPV-negative lesions. Expression of pSTAT3 strongly correlated with all the AP-1/NF-κB members (except JunD), its upstream activator pEGFRY1092, and HPV infection-related negative regulator p16. Overall, we show a simple combination of AP-1, NF-κB, and STAT3 members’ expression that may serve as molecular signature of HPV-positive lesions or more broadly the tumors that show better prognosis.

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