Loss of TIMM8A Inhibits Breast Cancer Progression Through Promoting HSPA9 Ubiquitination and Degradation via Competitive Interaction with RNF4

To investigate the role and underlying mechanisms of TIMM8A in breast cancer progression. The expression of TIMM8A in breast cancer tissues and cell lines was assessed. Functional assays were performed to evaluate the effects of TIMM8A knockdown on proliferation, metastasis, and apoptosis in BT549 and MCF-7 cells. Co-immunoprecipitation (Co-IP) and ubiquitination assays were used to examine the interaction between TIMM8A and HSPA9, as well as the involvement of the E3 ubiquitin ligase RNF4. PI3K/AKT pathway activity was assessed by Western blotting. In vivo tumor growth was evaluated using a xenograft mouse model. TIMM8A was significantly upregulated in breast cancer tissues and cell lines, and high TIMM8A expression correlated with poor prognosis. TIMM8A knockdown suppressed proliferation and metastasis while inducing apoptosis in vitro. Mechanistically, TIMM8A interacted with HSPA9 and maintained its protein stability by inhibiting RNF4-mediated ubiquitination and degradation, thereby sustaining PI3K/AKT signaling activation. Conversely, TIMM8A depletion reduced HSPA9 stability and attenuated the pathway. Rescue experiments confirmed that HSPA9 overexpression restored PI3K/AKT activation and reversed TIMM8A-knockdown-induced malignant suppression. In vivo, TIMM8A silencing efficiently inhibited tumor growth, accompanied by reduced HSPA9 and Ki-67 expression and decreased PI3K/AKT phosphorylation. TIMM8A promotes breast cancer progression through the TIMM8A/HSPA9/PI3K/AKT regulatory axis, highlighting TIMM8A as a promising prognostic biomarker and potential therapeutic target for breast cancer.