Cx43 Expression Correlates with Breast Cancer Metastasis in MDA-MB-231 Cells In Vitro, In a Mouse Xenograft Model and in Human Breast Cancer Tissues

Abstract

Connexins regulate multiple cellular functions and are considered tumor suppressors. Connexin43 (Cx43) is frequently down-regulated in breast tumors. However, Cx43 regulation during cancer onset and metastasis is complex and context-dependent. We investigated the effect of Cx43 over-expression or knock-down on the metastatic potential of MDA-MB-231 breast cancer cells in vitro and in vivo and in human breast cancer tissues. MDA-MB-231 cells over-expressing (Cx43D) or down-regulating Cx43 (shCx43) were generated and used in proliferation, migration, and invasion assays. The regulation of genes/proteins implicated in progression, invasion and metastasis was assessed in vitro and in immune-compromized mice injected with MDA-MB-231, Cx43D or shCx43 cells. Primary tumor onset/growth, metastasis and overall survival of these animals was monitored and evaluated. In addition, Cx43 expression in human breast carcinoma samples was assessed by qPCR. Cx43 over-expression increased protein levels of epithelial markers E-cadherin and zonula occludens 1 expression and resulted in the sequestration of β-catenin at the cell membrane, while Cx43 knock-down induced protein expression of the mesenchymal marker N-cadherin and an increased invasive potential of shCx43 cells. In vivo, in mice xenografted with breast cancer cells, Cx43 over-expression decreased tumor volume, attenuated cell metastasis to lungs and liver and increased overall mice survival. Importantly, the expression of Cx43 in triple negative human breast cancer tissues is also down-regulated. Collectively, Cx43 over-expression induced an epithelial-like phenotype in MDA-MB-231 cells and suppressed tumor growth and metastasis to secondary organs in vivo. In contrast, Cx43 knock-down in MDA-MB-231 cells induced a mesenchymal phenotype with increased cell invasion leading to an enhanced metastatic phenotype. These data provide evidence for a pivotal role of Cx43 in breast cancer metastasis and support the potential targeting of connexins in breast cancer therapy.

MYC as a Multifaceted Regulator of Tumor Microenvironment Leading to Metastasis

“…It has been reported that in brain metastases derived from breast cancer the up-regulation of MYC participates in the recruitment of macrophages and plays an important role in the formation of gap junctions between metastatic cells and astrocytes, which seems crucial for the metastatic growth within the brain [ 198 ]. MYC takes part in the generation of gap junctions through the up-regulation of Connexin 43 (Cx43), a crucial protein in favoring close interactions between cancer cells and endothelial cells, hence, facilitating extravasation and local survival of metastatic cells [ 198 , 199 ]. The correlation between the up-regulation of Cx43 and metastasis is reported not only in breast cancer, but also in prostate cancer, where the increased expression of Cx43 is involved in bone-specific metastasis, the most common target site for advanced prostate cancers [ 200 ].…”

Pannexin1 Is Associated with Enhanced Epithelial-To-Mesenchymal Transition in Human Patient Breast Cancer Tissues and in Breast Cancer Cell Lines

“…Real-Time Cell Analysis (RTCA) was used to study invasion and proliferation of MDA-MB-231 and MDA-PANX1cells. Cells were seeded on a cellular invasion plate (CIM-plate 16) with micro-electronic sensors on the underside of an 8 µm microporous polyethylene terephthalate membrane [18,[96][97][98] of a Boyden-like upper chamber, and real-time evaluation of cell impedance was performed using xCELLigence RTCA [A2] DP instrument (Roche). For invasion assays, 30 µL of growth factor-reduced Matrigel TM (BD Biosciences, San Jose, CA, USA) diluted in serum-free medium at a ratio of 1:20 were used to coat the upper surface of the membrane, followed by incubation at 37 • C in 5% CO 2 for 4 h in a humidified incubator and then washed with PBS.…”

Lipoprotein receptor‐related protein 6 is required to maintain intercalated disk integrity

“…Cardiac‐specific loss of N‐cadherin leads to lethal arrhythmogenesis and dissolution of the ID structure, with a significance decrease in the expression of Cx43 and Cx40 (Kostetskii et al, ; Li et al, ). However, Cx43 knockdown promotes N‐cadherin expression in MDA‐MB‐231 breast cancer cells in vitro (Kazan et al, ). The present study demonstrated that LRP6‐CKO induced a sharp decrease in Cx43 expression, which may contribute to the increased N‐cadherin expression in LRP6‐deficient hearts.…”