CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines

From Orai to E-Cadherin: Subversion of Calcium Trafficking in Cancer to Drive Proliferation, Anoikis-Resistance, and Metastasis

The common currency of epithelial differentiation and homeostasis is calcium, stored primarily in the endoplasmic reticulum, rationed according to need, and replenished from the extracellular milieu via store-operated calcium entry (SOCE). This currency is disbursed by the IP3 receptor in response to diverse extracellular signals. The rate of release is governed by regulators of proliferation, autophagy, survival, and programmed cell death, the strength of the signal leading to different outcomes. Intracellular calcium acts chiefly through intermediates such as calmodulin that regulates growth factor receptors such as epidermal growth factor receptor (EGFR), actin polymerization, and adherens junction assembly and maintenance. Here we review this machinery and its role in differentiation, then consider how cancer cells subvert it to license proliferation, resist anoikis, and enable metastasis, either by modulating the level of intracellular calcium or its downstream targets or effectors such as EGFR, E-cadherin, IQGAP1, TMEM16A, CLCA2, and TRPA1. Implications are considered for the roles of E-cadherin and growth factor receptors in circulating tumor cells and metastasis. The discovery of novel, cell type-specific modulators and effectors of calcium signaling offers new possibilities for cancer chemotherapy

Homeostatic Signaling by Cell–Cell Junctions and Its Dysregulation during Cancer Progression

The transition of sessile epithelial cells to a migratory, mesenchymal phenotype is essential for metazoan development and tissue repair, but this program is exploited by tumor cells in order to escape the confines of the primary organ site, evade immunosurveillance, and resist chemo-radiation. In addition, epithelial-to-mesenchymal transition (EMT) confers stem-like properties that increase efficiency of colonization of distant organs. This review evaluates the role of cell–cell junctions in suppressing EMT and maintaining a quiescent epithelium. We discuss the conflicting data on junctional signaling in cancer and recent developments that resolve some of these conflicts. We focus on evidence from breast cancer, but include other organ sites where appropriate. Current and potential strategies for inhibition of EMT are discussed

Loss of CLCA4 promotes epithelial-to-mesenchymal transition in breast cancer cells.

The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor CLCA2 is expressed in differentiated, growth-arrested mammary epithelial cells but is downregulated during tumor progression and EMT. We further demonstrated that CLCA2 is a p53-inducible proliferation-inhibitor whose loss indicates an increased risk of metastasis. We show here that another member of the CLCA gene family, CLCA4, is expressed in mammary epithelial cells and is similarly downregulated in breast tumors and in breast cancer cell lines. Like CLCA2, the gene is stress-inducible, and ectopic expression inhibits colony formation. Transcriptional profiling studies revealed that CLCA4 and CLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of CLCA4 in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin and CLCA2, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated. Double knockdown of CLCA2 and CLCA4 enhanced the mesenchymal profile. These findings suggest that CLCA4 and CLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of CLCA4 signaled lower relapse-free survival in basal and luminal B breast cancers

Tet-On lentiviral transductants lose inducibility when silenced for extended intervals in mammary epithelial cells

Silencing of virally transduced genes by promoter methylation and histone deacetylation has been a chronic problem both experimentally and therapeutically. We observed frequent silencing of the tetracycline-inducible Tet-On promoter borne by the Tripz lentivirus in mammary epithelial cell lines. We found that silencing could be prevented by continuous induction, but uninduced Tet-On gradually became uninducible, suggesting promoter modification. Accordingly, silencing was reversible by a common inhibitor of histone deacetylases, sodium butyrate. The effect was cell-line dependent, as HEK293 cells exhibited only moderate silencing that could be partly reversed by extended induction. These results indicate the need to test individual cell lines prior to using this system for studies that require induction after long periods of repression such as in animal models or RNA interference screens. Keywords: pTripz, Tet-On, Inducibility, Promoter silencing, Sodium butyrate, Histone deacetylase, Breast cance

CLCA4 expression inhibits breast cancer cell proliferation.

<p><b>A</b> western blot showing expression of Flag-tagged CLCA4 and CLCA2 transfected into 293 T cells. <b>B</b>, clonogenicity assays. CLCA4, CLCA2, and pLex vector were packaged and transduced into MCF7, and colonies were selected with puromycin for two weeks then stained with crystal violet in methanol. <b>C</b>, microimages of the surviving colonies. Bar, 200 microns. Data in B and C are representative of three repeats. The well marked “No DNA” was a non-transduced control for puromycin selection.</p

CLCA4 and relapse-free survival in breast cancer patients.

<p>Kaplan-Meier plots of relapse-free survival of patients with (A) basal-like, ER-, PR-, LN+ or (B) luminal B breast cancer relative to CLCA4 expression. Upper curve, red, indicates higher than median expression, and lower curve, black, lower than median expression.</p

Knockdown of CLCA4 promotes cell migration and invasion.

<p><b>A</b>, microimages showing loss of cobblestone and acquisition of hummingbird morphology with knockdown. Bar, 200 microns. <b>B</b>, Boyden chemotaxis chamber migration assay (left) and invasion assay (right).</p

Knockdown of CLCA4 induces EMT.

<p>HMLE cells were lentivirally transduced with shRNAs GipzH4A, GipzH4B and GipzH4C. GipzNC contained a non-silencing control. <b>A</b>, transcriptional profile of CLCA4 and EMT markers. Values were normalized to HMLE parent. P<0.01 for each pairwise comparison between GipzNC and GipzH4A or GipzH4C. <b>B</b>, immunoblot of whole cell lysates probed for EMT marker proteins. Band intensities in each lane were normalized to the beta actin value. <b>C</b>, CLCA2 downregulation in CLCA4-knockdown cells. Values normalized to HMLE parent. P<0.001.</p

CLCA4 is moderately induced by doxorubicin.

<p><b>A</b>, induction of CLCA4 mRNA in HMLE treated with 30 nM doxorubicin measured by RT-qPCR. <b>B</b>, induction of CLCA4 in MCF7. Cells were treated with 30 nM doxorubicin continuously for 6 days or 3 µM for 2 h then released into fresh medium for 2 days.</p