Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis

Abstract

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of EMT by cell dilution, TGFbeta, or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral shRNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18 year period among breast cancer patients compared to lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue

“…22 Thus, hCLCA2 and its murine orthologue have been proposed as tumour suppressor genes in breast cancer. 23, 24 However, other investigators have demonstrated an upregulation of hCLCA2 in triple-negative breast cancer patients with a poor prognosis, 25 perhaps suggesting breast cancer subtype specific roles, and exemplifying the need for further characterization of this poorly understood protein family.…”

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

“…The chloride channel calcium-activated (CLCA) 3 proteins are a complex family targeted for a role in cancer (1,2) and inflammatory diseases (3) but are poorly understood in terms of molecular structure and function. The original annotation of this family as calcium-activated chloride channels (CaCCs) was based on the observation that overexpression of several different CLCA paralogues from various species all induced chloride current in response to cytosolic calcium flux (4,5).…”

Plasma membrane ion channels and epithelial to mesenchymal transition in cancer cells

“…Similarly, reduced levels of the related isoform CLCA4 is a feature of subpopulations of cells from the HMLE cell line that are enriched in mesenchymal markers and a consequence of TGFβ-induced EMT (Yu et al 2013). Consistent with the loss of CLCA2 and CLCA4 in the mesenchymal phenotype, low levels of CLCA2 and CLCA4 appear likely to be associated with an increased incidence of metastasis (as assessed through metastasis or relapse free survival) using specific cohorts of breast cancer patients (Walia et al 2012, Yu et al 2013. In addition to their remodelling as a consequence of EMT, CLCA2 and CLCA4 have also been implicated in the regulation of the transition of breast cancer cells towards a more mesenchymal state.…”

“…MCF-7). Indeed, expression of the EMT Shiwarski et al (2014) transcription factor Snail supresses CLCA2 protein in the human breast cell line MCF10A, and CLCA2 levels are reduced in subpopulations of cells from the human mammary epithelial (HMLE) cell line that are enriched in mesenchymal markers (Walia et al 2012). Moreover, CLCA2 levels are reduced during EMT induced by TGFβ (Yu et al 2013).…”

“…In addition to their remodelling as a consequence of EMT, CLCA2 and CLCA4 have also been implicated in the regulation of the transition of breast cancer cells towards a more mesenchymal state. Knockdown of CLCA2 or CLCA4 is sufficient in HMLE cells to induce the expression of the mesenchymal marker vimentin and supress the epithelial marker E-Cadherin (Walia et al 2012, Yu et al 2013). In the case of CLCA2, the regulation of EMT may at least in part be through interactions with the cell junctional protein EVA1 (Ramena et al 2016).…”