CTTN Overexpression Confers Cancer Stem Cell-like Properties and Trastuzumab Resistance via DKK-1/WNT Signaling in HER2 Positive Breast Cancer

SIMPLE SUMMARY: Cortactin (CTTN) is an actin-binding protein that is mainly known for its ability to promote cancer progression. It is unclear, however, whether CTTN affects tumor initiation and anti-tumor drug resistance in breast cancer. Here, we investigated the potential role of CTTN as a novel poor prognostic biomarker and possible therapeutic target of trastuzumab resistance for HER2 positive breast cancer. Our findings showed that CTTN enhances tumor initiation and increases anti-HER2 drug resistance by activating the DKK-1/Wnt/β-catenin signaling pathway. CTTN-induced cancer stem cell-like properties were reversed by treatment of β-catenin/TCF inhibitor. Furthermore, combinational treatment with trastuzumab and β-catenin/TCF inhibitor overcame CTTN-induced trastuzumab resistance. These results suggest that combined treatment of trastuzumab and Wnt signaling inhibitors could be an effective therapeutic strategy to treat HER2+ breast tumors expressing high levels of CTTN. Collectively, we suggest that CTTN could be a potential target for treatment of trastuzumab resistance in HER2 positive breast cancer patients. ABSTRACT: Background: Despite the therapeutic success of trastuzumab, HER2 positive (HER2+) breast cancer patients continue to face significant difficulties due to innate or acquired drug resistance. In this study we explored the potential role of CTTN in inducing trastuzumab resistance of HER2+ breast cancers. Methods: Genetic changes of CTTN and survival of HER2+ breast cancer patients were analyzed in multiple breast cancer patient cohorts (METABRIC, TCGA, Kaplan-Meier (KM) plotter, and Hanyang University cohort). The effect of CTTN on cancer stem cell activity was assessed using the tumorsphere formation, ALDEFLUOR assay, and by in vivo xenograft experiments. CTTN-induced trastuzumab resistance was assessed by the sulforhodamine B (SRB) assay, colony formation assays, and in vivo xenograft model. RNA-seq analysis was used to clarify the mechanism of trastuzumab resistance conferred by CTTN. Results: Survival analysis indicated that CTTN overexpression is related to a poor prognosis in HER2+ breast cancers (OS, p = 0.05 in the Hanyang University cohort; OS, p = 0.0014 in KM plotter; OS, p = 0.008 and DFS, p = 0.010 in METABRIC). CTTN overexpression-induced cancer stem cell-like characteristics in experiments of tumorsphere formation, ALDEFLUOR assays, and in vivo limiting dilution assays. CTTN overexpression resulted in trastuzumab resistance in SRB, colony formation assays, and in vivo xenograft models. Mechanistically, the mRNA and protein levels of DKK-1, a Wnt antagonist, were downregulated by CTTN. Treatment of the β-catenin/TCF inhibitor reversed CTTN-induced cancer stem cell-like properties in vitro. Combination treatment with trastuzumab and β-catenin/TCF inhibitor overcame trastuzumab resistance conferred by CTTN overexpression in in vitro colony formation assays. Conclusions: CTTN activates DKK-1/Wnt/β-catenin signaling to induce trastuzumab resistance. We propose that CTTN is a novel biomarker indicating a poor prognosis and a possible therapeutic target for overcoming trastuzumab resistance.