ROCK2 regulates a stress-responsive transcriptional state and calcium signaling in renal cell carcinoma.

Renal cell carcinoma (RCC) exhibits marked intratumoral heterogeneity that contributes to disease progression and therapeutic resistance. Although ROCK2 has been implicated in RCC progression, its role in regulating tumor cell heterogeneity and cell-state plasticity remains poorly understood. Using single-cell RNA sequencing, we identified a ROCK2-associated stress-responsive transcriptional state characterized by inflammatory signaling and enhanced calcium pathway activity. ROCK2 knockdown was associated with a lower representation of this transcriptional state together with proliferative tumor cells, and integration with the TCGA-KIRC cohort supported the presence of this stress-associated transcriptional program in bulk RCC tumors. Mechanistically, ROCK2 depletion reduced intracellular calcium levels, suppressed CALM1 and CREB1 expression, promoted apoptosis, and impaired cell-cycle progression and proliferation. Pharmacological restoration of calcium signaling with Bay K8644, together with CALM1 or CREB1 overexpression, partially rescued these defects, supporting functional interactions among ROCK2, calcium signaling, CALM1, and CREB1. Collectively, our findings identify a ROCK2-associated stress-responsive transcriptional state linked to altered calcium signaling and provide new mechanistic insight into RCC cellular heterogeneity, highlighting the functional relationship between ROCK2 and calcium signaling as a potential therapeutic vulnerability in RCC.