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The hedgehog pathway regulates cancer stem cells in serous adenocarcinoma of the ovary

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Abstract

Purpose

Signaling by cancer stem cells (CSCs) is known to occur at least in part through conserved developmental pathways. Here, the role of one of these pathways, i.e., the hedgehog pathway, was evaluated in high-grade serous ovarian carcinoma (HGSOC).

Methods and results

We found that in HGSOC, hedgehog inhibitors (HHIs) GANT61, LDE225 and GDC0449 reduced or inhibited the formation of spheroids enriched in CSCs. Primary malignant cells (PMCs) in ascites from HGSOC patients cultured in the presence of HHIs showed significant reduction in CSCs. Sonic hedgehog (SHH) significantly increased the expression of ALDH1A1, which was inhibited by GANT61. In the presence of a SHH neutralizing antibody (5E1), a significant reduction in the number of spheroids was observed in HGSOC-derived cell lines. Further, the motility, migration and clonogenic growth of the cells were significantly reduced by HHIs. In the presence of GANT61, a reduction of cells from PMCs in the G0 phase of the cell cycle was observed. The magnitude of difference in expression of Gli1 in tumors from the same HGSOC patients at presentation and at interval debulking surgery was greater in patients who had a recurrence on follow up. GANT61 also significantly inhibited the growth of CSCs in nude mice. Finally, RNA sequencing of HGSOC cells treated with GANT61 showed a significantly reduced expression of CSC markers.

Conclusions

Our results indicate that the hedgehog pathway plays an important role in maintaining the integrity of CSCs in HGSOC and could be a potential therapeutic target.

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Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary files]. The RNA-Seq data generated and/or analyzed during the current study are available in the Sequence Read Archive (SRA): Bio Project ID PRJNA477252 and PRJNA488919 (https://submit.ncbi.nlm.nih.gov/subs/sra/SUB4484897/overview) (https://submit.ncbi.nlm.nih.gov/subs/sra/SUB4486097/overview).

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Acknowledgements

This work was supported by the Department of Biotechnology (DBT) and a research fellowship for Ms. S. Sneha was provided by the University Grants Commission (UGC). We would like to extend our gratitude to Dr. V. Sridevi and Dr. Ujwala for identifying the patients and for the tissues used in the experiments, and the staff and nurses for collecting malignant ascites samples from patients under aseptic conditions. We acknowledge Dr. Priya, Dr. Hascitha and Dr. Hemavathi, Department of Molecular Oncology, for the flow cytometry experiments and the Department of Pathology, Epidemiology and Statistics for their help and support. We also thank Dr. Srividhya and Ms. Kayalvizhi, flow cytometry core facility, IIT-Madras, for the cell sorting experiments.

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Authors and Affiliations

  1. Laboratory for Cancer Biology, Departments of Medical Oncology and Clinical Research, Cancer Institute (WIA), 38, Sardar Patel Road, Guindy, Chennai, Tamil Nadu, 600036, India

    Smarakan Sneha, Rohit P. Nagare, Chirukandath Sidhanth, Syama Krishnapriya & Trivadi S. Ganesan

  2. Amity Institute of Molecular Medicine & Stem Cell Research, Amity University Campus, Sector-125, 201303, Noida, Uttar Pradesh, India

    Manoj Garg

  3. Department of Molecular Oncology, Cancer Institute (WIA), Chennai, Tamil Nadu, India

    Balaji Ramachandran

  4. Department of Pathology, Cancer Institute (WIA), Chennai, Tamil Nadu, India

    Kanchan Murhekar & Shirley Sundersingh

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  1. Smarakan Sneha
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  2. Rohit P. Nagare
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  3. Chirukandath Sidhanth
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  4. Syama Krishnapriya
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  9. Trivadi S. Ganesan
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Contributions

All authors provided critical feedback and helped in shaping the research, results and analyses, and commented on the manuscript. SS contributed substantially to the concept and design, performed the experiments, analyzed and interpreted data and drafted the manuscript. RPN optimized flow cytometry analysis, collected clinical data of patients for immunohistochemistry experiments and helped in critical analysis of the data and interpretation of the results. CS and MG contributed in designing the biochemical studies and provided experimental suggestions. SK helped in optimizing the immunohistochemistry experiments and in SPSS analyses, while RB performed the xenograft study. KM and SS contributed in procuring tissue sections and scoring the slides of the immunohistochemistry experiments. TSG conceived the original idea, critically reviewed and edited all versions of the manuscript and approved the final version.

Corresponding author

Correspondence to Trivadi S. Ganesan.

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Sneha, S., Nagare, R.P., Sidhanth, C. et al. The hedgehog pathway regulates cancer stem cells in serous adenocarcinoma of the ovary. Cell Oncol. 43, 601–616 (2020). https://doi.org/10.1007/s13402-020-00504-w

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