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

Abstract

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.

Figure 1. CLCA4 downregulation in colon and breast cancers.

A and B, CLCA4 mRNA expression in normal tissue compared to cancer in colon/rectum and breast. The Cancer Genome Atlas (TCGA) datasets were searched using Oncomine. The log2 median-centered ratios for CLCA4 expression level are depicted in box-and-whisker plots. Dots represent maximum and minimum outliers from the main dataset. For each plot, the following pathological subtypes were evaluated separately. A, colorectal: 0, normal tissue (22); 1, cecum adenocarcinoma (22); 2, colon adenocarcinoma (101); 3, colon mucinous adenocarcinoma (22); 4, rectal adenocarcinoma (60); 5, rectal mucinous adenocarcinoma (6); 6, rectosigmoid adenocarcinoma (3); 7, rectosigmoid mucinous adenocarcinoma (1). B, breast: 0, normal tissue (61); 1, apocrine carcinoma (1); 2, large cell neuroendocrine (1); 3, ductal carcinoma (1); 4, intraductal cribriform adenocarcinoma (3); 5, invasive carcinoma (76); 6, invasion cribriform carcinoma (1); 7, invasive ductal carcinoma (395); 8, invasive ductal and lobular carcinoma (3); 9, invasive lobular breast carcinoma (36); 10, invasive papillary breast carcinoma (1); 11, metaplastic breast carcinoma (1); 12, mixed lobular and ductal breast carcinoma (7); 13, mucinous breast carcinoma (4); 14, papillary carcinoma (1); 15, pleomorphic carcinoma (1). Number in parentheses indicates sample size for each category. C, CLCA4 mRNA downregulation in breast cancer cell lines. RT-qPCR data from cultured cells.

Figure 2. CLCA4 expression inhibits breast cancer cell proliferation.

A western blot showing expression of Flag-tagged CLCA4 and CLCA2 transfected into 293 T cells. 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. C, 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.

Figure 3. CLCA4 is moderately induced by doxorubicin.

A, induction of CLCA4 mRNA in HMLE treated with 30 nM doxorubicin measured by RT-qPCR. 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.

Figure 4. Expression of CLCA4 correlates with epithelial differentiation.

A, HMLE cells were separated into epithelioid and mesenchymal subpopulations by differential trypsinization and subjected to RT-qPCR. The transcriptional profile reveals that CLCA4 is highest in the most trypsin-resistant fraction, correlating with E-cadherin and CLCA2. The 5 min fraction was normalized as 1. P<0.001 for all comparisons between 5 min and 20 min samples. B, EMT induced by TGF-beta treatment (2.5 ng/ml) downregulates CLCA4 and CLCA2. Values are normalized to no-drug control. P<0.01 for each comparison to no-drug control.

Figure 5. Knockdown of CLCA4 induces EMT.

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

Figure 6. Knockdown of CLCA4 promotes cell migration and invasion.

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

Figure 7. Double knockdown of CLCA4 and CLCA2 enhances EMT.

HMLE cell lines bearing CLCA4 knockdown constructs were transduced with the CLCA2 knockdown construct Tripz1. Cells were selected in the presence of puromycin and doxycycline for two weeks. At least 90% of Tripz1-infected cells expressed RFP. Values were normalized to the non-silencing control, GipzNC.

Figure 8. CLCA4 and relapse-free survival in breast cancer patients.

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.