# VHL Gene Restoration Supports RCC Reprogramming to iPSCs but Does Not Ensure Line Stability

## TL;DR

Restoring the VHL gene in kidney cancer cells allows partial reprogramming to stem-like cells but does not lead to stable stem cell lines.

## Contribution

The study shows that VHL gene restoration is necessary but insufficient for stable reprogramming of ccRCC cells into iPSCs.

## Key findings

- Restoring the VHL gene in ccRCC cells allows partial reprogramming but not stable pluripotency.
- Cancer cells reprogrammed with VHL still require continuous reprogramming factor expression.
- Genetic factors beyond VHL likely prevent full stem cell state in reprogrammed ccRCC cells.

## Abstract

The von Hippel–Lindau Syndrome, an inherited condition that predisposes to clear cell renal cell carcinoma (ccRCC), affects developmental pathways of the kidney. Studying these early stages could help develop customized, targeted treatments. One approach is to reverse-engineer cancer cells into a stem cell-like state and then grow them into organ models. We attempted this with clear cell renal cell carcinoma (ccRCC) cells and adjacent normal (AN) healthy kidney cells using cell reprogramming. While the adjacent normal cells successfully reprogrammed to induce pluripotent stem cells (iPSCs), the cancer cells did not. We then restored the VHL gene—often missing in ccRCC. This reintroduced normal cell characteristics, but the cells upon reprogramming could not maintain a stable stem cell state. Our findings suggest that while restoring VHL is necessary for reprogramming, other genetic issues block ccRCC cells from fully returning to a healthy, stem-like state, indicating the importance of other, yet to be determined factors. Similarities between cancer cells and stem cells have been recognized, as common genes can influence both outcomes. In this study, we demonstrate that the VHL gene can be added to this group, as it is involved in both cancer development and stem cell regulation.

Background: Modeling precancerous stages holds the promise to understand early transformation events, thereby offering the potential for personalized, targeted treatment. Because cancer hijacks developmental pathways, precancerous stages could potentially be modeled by reprogramming cancer cells to an induced pluripotent stem cell state and subsequently differentiating them to the target organs using organoid models. Methods: We attempted reprogramming of patient-derived clear cell renal cell carcinoma (ccRCC) cell lines and adjacent normal renal epithelial cell lines using lentivirus or episomal reprogramming vectors. Results: The cancer cells failed to reprogram while the adjacent normal cells reprogrammed with high efficiency. The von Hippel–Lindau factor (VHL) gene was re-expressed in ccRCC cells in an attempt to restore the wild-type phenotype and restore reprogramming. The VHL gene is the major tumor suppressor in ccRCC pathogenesis and a conductor of oxidative-glycolytic glucose metabolism. While its re-expression did restore the epithelial phenotype and oxidative regulation of ccRCC cells, they still failed to stably reprogram. With an optimized reprogramming workflow, VHL-corrected ccRCC cells generate NANOG+ cells; however, they remained dependent on the ectopic expression of the reprogramming factors. Conclusions: We concluded that while VHL expression is necessary for cellular reprogramming of ccRCC cells, other genetic lesions in the ccRCC cells could be preventing the stabilization of the pluripotent state.