P4HA3-mediated hydroxylation of SLC7A11 drives tyrosine kinase inhibitor resistance by suppressing ferroptosis in clear cell renal cell carcinoma.

Resistance to tyrosine kinase inhibitors (TKIs) severely limits their long-term efficacy in renal cell carcinoma (RCC). Here, we identify prolyl 4-hydroxylase subunit alpha 3 (P4HA3) as a critical driver of TKI resistance and elucidate its underlying mechanism. P4HA3 was markedly upregulated in TKI-resistant RCC cells, and its overexpression promoted proliferation and resistance to sunitinib and axitinib, whereas P4HA3 depletion restored drug sensitivity and suppressed tumor growth in vitro and in vivo. Mechanistically, P4HA3 inhibited ferroptosis by stabilizing the cystine transporter SLC7A11, thereby maintaining redox homeostasis and suppressing lipid peroxidation. P4HA3 directly interacted with SLC7A11 and induced hydroxyproline modification at residues P153 and P161, which attenuated NEDD4-mediated ubiquitination and proteasomal degradation of SLC7A11. Moreover, a hydroxylase-inactive P4HA3 mutant failed to stabilize SLC7A11, suppress ferroptosis, or promote TKI resistance, demonstrating the essential role of P4HA3 enzymatic activity. Importantly, pharmacological inhibition of P4HA3 using Tubuloside A or 1,4-DPCA disrupted the P4HA3-SLC7A11 axis and significantly enhanced TKI efficacy across RCC cell lines, patient-derived organoids, and xenograft models. Together, these findings establish a hydroxylation-dependent P4HA3-SLC7A11-NEDD4 axis that drives ferroptosis evasion and TKI resistance in RCC and highlight P4HA3 as a promising therapeutic target.