Interactions between TGF-β type I receptor and hypoxia-inducible factor-α mediates a synergistic crosstalk leading to poor prognosis for patients with clear cell renal cell carcinoma

To investigate the significance of expression of HIF-1 alpha, HIF-2 alpha, and SNAIL1 proteins; and TGF-beta signaling pathway proteins in ccRCC, their relation with clinicopathological parameters and patient's survival were examined. We also investigated potential crosstalk between HIF-alpha and TGF-beta signaling pathway, including the TGF-beta type 1 receptor (ALK5-FL) and the intracellular domain of ALK5 (ALK5-ICD). Tissue samples from 154 ccRCC patients and comparable adjacent kidney cortex samples from 38 patients were analyzed for HIF-1 alpha/2 alpha, TGF-beta signaling components, and SNAIL1 proteins by immunoblot. Protein expression of HIF-1 alpha and HIF-2 alpha were significantly higher, while SNAIL1 had similar expression levels in ccRCC compared with the kidney cortex. HIF-2 alpha associated with poor cancer-specific survival, while HIF-1 alpha and SNAIL1 did not associate with survival. Moreover, HIF-2 alpha positively correlated with ALK5-ICD, pSMAD2/3, and PAI-1; HIF-1 alpha positively correlated with pSMAD2/3; SNAIL1 positively correlated with ALK5-FL, ALK5-ICD, pSMAD2/3, PAI-1, and HIF-2 alpha. Intriguingly, in vitro experiments performed under normoxic conditions revealed that ALK5 interacts with HIF-1 alpha and HIF-2 alpha, and promotes their expression and the expression of their target genes GLUT1 and CA9, in a VHL dependent manner. We found that ALK5 induces expression of HIF-1 alpha and HIF-2 alpha, through its kinase activity. Under hypoxic conditions, HIF-alpha proteins correlated with the activated TGF-beta signaling pathway. In conclusion, we reveal that ALK5 plays a pivotal role in synergistic crosstalk between TGF-beta signaling and hypoxia pathway, and that the interaction between ALK5 and HIF-alpha contributes to tumor progression.