Resistance to therapy and metastasis remains one of the leading causes of mortality due to cervical cancer despite advances in detection and treatment. The mechanism of epithelial to mesenchymal transition (EMT) provides conceptual explanation to the invasiveness and metastatic spread of cancer but it has not been fully understood in cervical cancer. This study aims to investigate the mechanism by which silencing of E‐cadherin gene regulates EMT leading to proliferation, invasion, and chemoresistance of cervical cancer cells through the Hedgehog (Hh) signaling pathway. We developed an in vitro EMT model by the knockdown of E‐cadherin expression in cervical cancer cell lines. To understand the role of developmental pathway like Hh in the progression of cervical cancer, we investigated the expression of Hh pathway mediators by array in E‐cadherin low cervical cancer cells and observed upregulation of Hh pathway. This was further validated on low passage patient‐derived cell lines and cervical carcinoma tissue sections from cervical cancer patients. Further, we evaluated the role of two inhibitors (cyclopamine and GANT58) of the Hh pathway on invasiveness and apoptosis in E‐cadherin low cervical cancer cells. In conclusion, we observed that inhibition of Hh pathway with GANT58 along with current therapeutic procedures could be more effective in targeting drug‐resistant EMT cells and bulk tumor cells in cervical cancer.

Cervical cancer is one of the leading causes of mortality amongst women in developing countries and therapy resistance is the main reason for its treatment failure. Recent advances suggest that cancer stem cells (CSCs) are critically involved in regulating the chemo resistant behavior of cervical cancer cells. In our study the CSC phenotype cells were isolated and the expression of stem cell marker and epithelial-mesenchymal transition (EMT) associated gene was confirmed by various assays. However, these CSC phenotype cells cannot be cultured for further cytotoxicity studies. So, we tried to establish a CSC model in cervical cancer cells. We performed the siRNA-mediated knockdown of E-cadherin (E-cad) in these cells and studied EMT associated stem cell-like properties in them. We also performed dose dependent cell viability assay using clinically relevant drugs such as cisplatin, cyclopamine and GANT58 to analyze the drug resistant behavior of these cancer cells. We found that E-cad knockdown induces EMT in cervical cancer cells imparting stem-cell like characteristics along with enhanced tumorsphere formation, migration, invasion ability and drug resistance. This is the first study to establish a CSC model in cervical cancer cells by knockdown of E-cad which can be utilized for development of anti-cancer therapies.

Cervical cancer is the second most common malignancy in women and a major cause of morbidity and mortality worldwide. Statistics show that present biologic therapy has had little impact on survival in recurrent and metastatic disease. Consequently, there is an urgent need to explore the best treatment options for advanced cervical cancer. Hedgehog (Hh) signaling pathway has been shown to play an important role in metastasis, recurrence and drug resistance of cervical cancer. Therefore, inhibition of hedgehog signaling pathway is emerging as a new strategy for the treatment of advanced cervical cancer. Reports from various solid tumors show that efficacy of targeted therapy can be compromised due to the cytoprotective effect of autophagy upregulation in cancer cells. Therefore, the present study was designed to elucidate the effects of targeting Hh pathway on autophagic flux in cervical cancer spheroids (cervicospheroids). We developed 3D cervicospheroids as an in vitro model to study the Hh signaling and role of autophagy in cervical cancer. Characterisation of the cervicospheroids showed high expression of stemness genes like oct4, sox2 , nanog and CD 133 as well as epithelial to mesenchymal trasition genes like vimentin, snail and twist indicating that spheroids were enriched in cancer stem cell population. Acridine orange staining of 3D spheroids showed that autophagy was also enhanced in the cervicospheroids compared to monolayer culture. We observed a significant positive correlation between the expression of hedgehog mediator, Gli1 and autophagy protein, LC31 in immunohistochemistry data of archival sections of 45 patients of advanced cervical cancer. A higher autophagy flux, as indicated by presence of larger number autophagic vesicle compared to normal cervix, was also observed in these sections. These results further validated our in vitro observations in the 3D model of cervicospheres. MTT data, showed that autophagy help to evade the cytotoxicity of the chemotherapeutic drug, cisplatin in the cervicospheres. We further found an aberrantly activated Hh pathway in the cervicospheroids and observed that the effect of cisplatin and Hh inhibitor, GANT61 was potentiated when the autophagy inhibitor , chloroquine, was added to the 3D spheroid culture. These results led us to conclude that that 3-D spheroid grown in nonadherent conditions can be used as surrogate model to study molecular pathways involved in metastasis. Based on our present findings, we suggest a combinatorial approach targeting both cytoprotective autophagy and Hh pathway as a new therapeutic approach for targeting cancer stem cell population in cervical cancer . Citation Format: Shalmoli Bhattacharyya, Renaissa De, Radhika Srinivasan. Combination of hedgehog targeting and autophagy inhibitor potentiate cytotoxicity in 3D spheroids of cervical cancer: A promising therapeutic strategy in metastatic disease [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A031.