HECTD3 E3 ligase mediates ubiquitination of AKT-phosphorylated CMTM3 in HER2-overexpressed breast cancer cells

Abstract CMTM (CKLF-like MARVEL transmembrane domain-containing) proteins play pivotal roles in tumorigenesis and cancer progression across various malignancies. However, their expression profiles and regulatory mechanisms in distinct subtypes of breast cancer remain largely undefined. In this study, we systematically analyzed the expression of all nine CMTM family members across major molecular subtypes of breast cancer, including Luminal A, Luminal B, HER2-positive (HER2+), and triple-negative breast cancer (TNBC). Among these, CMTM3 was uniquely downregulated in Luminal B and HER2+ breast cancer cells and functioned as a tumor suppressor. Overexpression of HER2 in normal breast epithelial cell lines led to the phosphorylation of CMTM3. Molecular and biochemical analyses revealed that HER2 overexpression activated the downstream phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in Luminal B and HER2+ breast cancer cells. AKT1 directly phosphorylated CMTM3 at serine 181 (Ser181), a modification that facilitated its recognition and ubiquitination by the E3 ligase HECTD3 (HECT domain E3 ubiquitin protein ligase 3), ultimately targeting CMTM3 for proteasomal degradation. Functional assays demonstrated that either knockdown of HECTD3 or pharmacological inhibition of PI3K/AKT signaling stabilized CMTM3 protein levels. Moreover, reintroducing a non-phosphorylatable CMTM3 mutant (CMTM3S181A) into CMTM3 knockout breast cancer cells resulted in significantly reduced proliferation, colony formation, invasive capacity, and in vivo tumor growth compared to cells expressing wild-type CMTM3 (CMTM3WT). Collectively, these findings reveal a previously unrecognized post-translational regulatory mechanism of CMTM3 and suggest that targeting the PI3K/AKT–HECTD3–CMTM3 axis may offer a promising therapeutic approach for treating HER2+ breast cancers.