CMTM6 overexpression confers trastuzumab resistance in HER2-positive breast cancer

Abstract

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is characterized by invasive growth, rapid metastasis and chemoresistance. Trastuzumab is an effective treatment for HER2+ breast cancer; however, trastuzumab resistance leads to cancer relapse and metastasis. CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) has been considered as a new immune checkpoint for tumor-induced immunosuppression. The role of CMTM6 in trastuzumab resistance remains unknown. Here, we uncover a role of CMTM6 in trastuzumab-resistant HER2+ breast cancer. CMTM6 expression was upregulated in trastuzumab-resistant HER2+ breast cancer cell. Patients with high CMTM6 expressing HER2+ breast cancer had worse overall and progression-free survival than those with low CMTM6 expression. In vitro, CMTM6 knockdown inhibited the proliferation and migration of HER2+ breast cancer cells, and promoted their apoptosis, while CMTM6 overexpression reversed these effects. CMTM6 and HER2 proteins were co-localized on the surface of breast cancer cells, and CMTM6 silencing reduced HER2 protein levels in breast cancer cells. Co-immunoprecipitation revealed that CMTM6 directly interacted with HER2 in HER2+ breast cancer cells, and CMTM6 overexpression inhibited HER2 ubiquitination. Collectively, these findings highlight that CMTM6 stabilizes HER2 protein, contributing to trastuzumab resistance and implicate CMTM6 as a potential prognostic marker and therapeutic target for overcoming trastuzumab resistance in HER2+ breast cancer.

Keywords: CMTM6; HER2+ breast cancer; Trastuzumab resistance; Ubiquitination.

Fig. 1

CMTM6 expression is upregulated in BC tissues and cell lines. A CMTM6 expression in different types of cancers (versus non-tumor tissues) in the TCGA and GTEx datasets. B CMTM6 expression in BC tissues and matched adjacent non-tumor tissues in the TCGA-BRCA dataset. C CMTM6 expression in different subtypes of breast cancers in the TCGA-BRCA datasets. D Western blot analysis of the relative levels of CMTM6 protein expression in fresh BC tissues and matched adjacent non-tumor tissues (n = 3). E qRT-PCR analysis of the relative levels of CMTM6 mRNA transcripts in fresh BC tissues and matched adjacent non-tumor tissues (n = 30). ***P < 0.001

Fig. 2

CMTM6 is upregulated in trastuzumab-resistant BC and correlates with poor prognosis. A qRT-PCR and B Western blot analyses of CMTM6 mRNA and protein expression in different BC cell lines. C Immunohistochemical analysis of CMTM6 protein expression in HER2+ BC tissues from trastuzumab-treated patients (scale bar, 50 μM). D Immunohistochemical analysis of CMTM6 protein expression in HER2+ BC tissues from trastuzumab-treated patients with or without relapse. E, F Kaplan–Meier analysis of overall survival (OS) and relapse-free survival (RFS) in trastuzumab-treated high or low CMTM6 expressing HER2+ BC patients

Fig. 3

CMTM6 promotes the survival, migration, invasion and trastuzumab resistance of BC cells in vitro.A qRT-PCR and Western blot validated CMTM6 silencing in JIMT-1 cells and CMTM6-overexpresstion in SKBR3 cells. Negative control (NC) JIMT-1 and SKBR3 cells were transduced with lentivirus for the control shRNA or transfected with the control plasmid, respectively. B, C CCK-8 assay determined the viability of the indicated BC cells following treatment with trastuzumab (0-100 μg/ml). D, E ethynyl-2′-deoxyuridine (EdU) analysis of the proliferation of CMTM6-silenced JIMT-1 cells, CMTM6 overexpressing SKBR3 cells, control JIMT-1 cells and SKBR3 cells following treatment with trastuzumab (10 μg/ml). (scale bar, 50 μM). F, G TUNEL analysis of apoptotic CMTM6-silenced JIMT-1 cells, CMTM6 overexpressing SKBR3 cells, control JIMT-1 and SKBR3 cells following treatment with trastuzumab (10 μg/ml). H, I Cell invasion assay revealed that CMTM6 silencing inhibited JIMT-1 cell invasion while CMTM6 overexpression enhanced SKBR3 cell invasion following treatment with trastuzumab (10 μg/ml). (scale bar, 50 μM). Data are representative images or expressed as the mean ± SD of each group from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

CMTM6 directly interacts with HER2 and enhancing the HER2 signaling in BC cells. A IHC analysis of HER2 protein expression in BC tissues with low or high CMTM6 protein expression (scale bar, 50 μM). B Association between CMTM6 and HER2 protein expression in BC tissues. C Western blot analysis of CMTM6 and HER2 protein levels in BC tissues. D Correlation between CMTM6 and HER2 protein levels in BC tissues. Data are mean ± SEM. E Confocal microscopy analysis of the subcellular co-localization of CMTM6 (green) and HER2 (red) in JIMT-1 cells, with DAPI nuclear staining (blue) (scale bar, 10 μM). F, G Co-immunoprecipitation revealed the direct interaction between endogenous CMTM6 and HER2 proteins in JIMT-1 cells. H Western blot analysis of CMTM6, HER2, p-HER2, PI3K, AKT, MEK, ERK, N-cadherin and E-cadherin protein levels in CMTM6-silenced JIMT-1, CMTM6 overexpressing SKBR3, control JIMT-1 and SKBR3 cells. Data are representative images of each group of cells from three separate experiments

Fig. 5

CMTM6 stabilizes the HER2 protein by inhibiting its poly-ubiquitination in BC cells. A Western blot analysis of CMTM6 and HER2 protein levels in CMTM6-silenced JIMT-1 cells after treatment with 10 μM MG132 or vehicle control for 24 h. B Western blot analysis of CMTM6 and HER2 protein levels in CMTM6-silenced JIMT-1 cells after treatment with 10 μM CHX or vehicle control for 24 h. C CMTM6 overexpression decreased HER2 poly-ubiquitination. HeLa cells were co-transfected with the FLAG-HER2 plasmid or combined with Myc-CMTM6 plasmid, together with the HA-Ub plasmid for 24 h and treated with 10 μM MG132 for another 24 h. The cell lysates were immunoprecipitated with anti-HER2 and immunoblotted with anti-HA antibody to determine HER2 poly-ubiquitination

Fig. 6

CMTM6 promotes the growth of trastuzumab-resistant BC by preserving HER2 protein and relative signaling in BC tissues in vivo. Female node mice were injected with SKBR3, CMTM6-silenced JIMT-1 or JIMT-1 cells to establish xenograft tumors and when the tumors reached at 100 mm³, the tumor-bearing mice were randomized and treated with vehicle (Control), trastuzumab (Ttzm, 10 mg/kg body weight every 5 days for 4 times) or combination of trastuzumab and Pertuzumab (Ttzm+Ptzm, n = 4 per group). A The dynamic growth of tumors. B IHC analysis of CMTM6, HER2, Ki67, and Caspase-3 protein expression (scale bar, 50 μM). C Western blot analysis of the relative levels of CMTM6, HER2, p-HER2, PI3K, AKT, MEK, ERK protein levels in the indicated tumors. Data are presentative images of each group from at least three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

A schematic diagram illustrates the action of CMTM6 in trastuzumab resistance of BC. CMTM6 promotes trastuzumab resistance by inhibiting HER2 ubiquitination and degradation in HER2+ BC