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RASSF1A–Hippo pathway link in patients with urothelial carcinoma of bladder: plausible therapeutic target

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Abstract

RASSF1A is a tumor suppressor gene, and its hypermethylation has been observed in cancers. RASSF1A acts as an upstream regulator of Hippo pathway and modulates its function. The aim of this study was to analyze expression of RASSF1A, Hippo pathway molecules (YAP, MST) and downstream targets (CTGF, Cyr61 and AREG) in bladder cancer patients. Later, the link between RASSF1A and Hippo pathway and a potential therapeutic scope of this link in UBC were also studied. MSPCR was performed to study methylation of RASSF1A promoter. Expression of molecules was studied using qPCR, Western blot and IHC. The link between RASSF1A and Hippo pathway was studied using Spearman’s correlation in patients and validated by overexpressing RASSF1A in HT1376 cells and its effect on Hippo pathway was observed using qPCR and Western blot. Further therapeutic potential of this link was studied using MTT and PI assays. The expression of RASSF1A was lower, whereas the expression of YAP, CTGF and CYR61 was higher. The expression of RASSF1A protein gradually decreased, while the expression of YAP, CTGF and CYR61 increased with severity of disease. Based on Spearman’s correlation, RASSF1A showed a negative correlation with YAP, CTGF and CYR61. YAP showed a positive correlation with CTGF and CYR61. To validate this link, RASSF1A was overexpressed in HT1376 cells. Overexpressed RASSF1A activated Hippo pathway, followed by a decrease in CTGF and CYR61 at mRNA, and enhanced cytotoxicity to chemotherapeutic drugs. This study finds a previously unrecognized role of RASSF1A in the regulation of CTGF and CYR61 through mediation of Hippo pathway in UBC and supports the significance of this link as a potential therapeutic target for UBC.

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Acknowledgements

M. Khandelwal has received Fellowship from Council of Scientific and Industrial Research, India. We acknowledge Dr. P. P. Chattopadhyay (Department of Biochemistry, AIIMS) for providing HeLa cells.

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Authors and Affiliations

  1. Department of Biochemistry, AIIMS, New Delhi, India

    Madhuram Khandelwal, Vivek Anand, Sandeep Appunni & Alpana Sharma

  2. Department of Urology, AIIMS, New Delhi, India

    Amlesh Seth & Prabhjot Singh

  3. Department of Pathology, AIIMS, New Delhi, India

    Sandeep Mathur

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  1. Madhuram Khandelwal
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Khandelwal, M., Anand, V., Appunni, S. et al. RASSF1A–Hippo pathway link in patients with urothelial carcinoma of bladder: plausible therapeutic target. Mol Cell Biochem 464, 51–63 (2020). https://doi.org/10.1007/s11010-019-03648-y

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