ABSTRACT
Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Animals , Mice , Triple Negative Breast Neoplasms/genetics , Mice, Nude , Leucine Zippers , Cell Proliferation/physiology , Neoplasm Recurrence, Local , Protein Serine-Threonine Kinases/geneticsABSTRACT
Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. CD44, a putative breast cancer stem cell (CSC) marker, is overexpressed in trastuzumab-resistant breast cancer cells. While CSC-related genes may play a role in the development of trastuzumab resistance, conflicting results have been published about CSC response to trastuzumab. We hypothesized that CD44 contributes to trastuzumab resistance independently of its role as a CSC marker. We used trastuzumab-sensitive breast cancer cell lines and their trastuzumab-resistant isogenic counterparts to evaluate the role of CD44 in response to trastuzumab. miRNA and mRNA expression were analyzed using microarray chips. A gene set enrichment analysis was created and matched with response to trastuzumab in cells and patient samples. The proportions of CSC in trastuzumab-resistant cells were similar to or lower than in the trastuzumab-sensitive cells. However, CD44 expression levels were significantly higher in both trastuzumab-resistant cell lines and its knockdown led to an increased response to trastuzumab. The invasiveness and anchorage-independent growth of trastuzumab-resistant cells were higher and blocked by downregulation of CD44. Results also showed that CD44-related resistance to trastuzumab is regulated by miRNAs. We identified a CD44-related gene expression profile that correlated with response to trastuzumab in cell lines and breast cancer patients. CD44 mediates trastuzumab resistance in HER2-positive breast cancer cells independently of its role as a CSC marker and that this role of CD44 is partly regulated by miRNA.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Drug Resistance, Neoplasm/genetics , Gene Expression , Hyaluronan Receptors/genetics , Receptor, ErbB-2/metabolism , Trastuzumab/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , MicroRNAs/genetics , RNA, Small Interfering/geneticsABSTRACT
Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. Understanding the molecular pathways that contribute to the development of drug resistance is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor-specific mutations. Little is known about the clinical significance of HER family mutations in breast cancer. Because mutations within HER1/EGFR are predictive of response to tyrosine kinase inhibitors (TKI) in lung cancer, we investigated whether mutations in HER family kinase domains are predictive of response to targeted therapy in HER2-overexpressing breast cancer. We sequenced the HER family kinase domains from 76 HER2-overexpressing invasive carcinomas and identified 12 missense variants. Patients whose tumors carried any of these mutations did not respond to HER2 directed therapy in the metastatic setting. We developed mutant cell lines and used structural analyses to determine whether changes in protein conformation could explain the lack of response to therapy. We also functionally studied all HER2 mutants and showed that they conferred an aggressive phenotype and altered effects of the TKI lapatinib. Our data demonstrate that mutations in the finely tuned HER kinase domains play a critical function in breast cancer progression and may serve as prognostic and predictive markers.
