hCLCA2 Is a p53-Inducible Inhibitor of Breast Cancer Cell Proliferation

Abstract

hCLCA2 is frequently down-regulated in breast cancer and is a candidate tumor suppressor gene. We show here that the hCLCA2 gene is strongly induced by p53 in response to DNA damage. Adenoviral expression of p53 induces hCLCA2 in a variety of breast cell lines. Further, we find that p53 binds to consensus elements in the hCLCA2 promoter and mutation of these sites abolishes p53-responsiveness and induction by DNA damage. Adenoviral transduction of hCLCA2 into immortalized cells induces p53, CDK inhibitors p21 and p27, and cell cycle arrest by 24 hours, and caspase induction and apoptosis by 40 hours postinfection. Transduction of the malignant tumor cell line BT549 on the other hand does not induce p53, p21, or p27 but instead induces apoptosis directly and more rapidly. Knockout and knockdown studies indicate that growth inhibition and apoptosis are signaled via multiple pathways. Conversely, suppression of hCLCA2 by RNA interference enhances proliferation of MCF10A and reduces sensitivity to doxorubicin. Gene expression profiles indicate that hCLCA2 levels are strongly predictive of tumor cell sensitivity to doxorubicin and other chemotherapeutics. Because certain Cl(-) channels are proposed to promote apoptosis by reducing intracellular pH, we tested whether, and established that, hCLCA2 enhances Cl(-) current in breast cancer cells and reduces pH to approximately 6.7. These results reveal hCLCA2 as a novel p53-inducible growth inhibitor, explain how its down-regulation confers a survival advantage to tumor cells, and suggest both prognostic and therapeutic applications.

Figure 1

Induction of hCLCA2 by chemotherapeutic agents in breast cancer cell lines. A, diverse forms of DNA damage induce hCLCA2. Cells were treated with doxorubicin or 1μg/ml aphidicolin and induction of hCLCA2 was measured by RT-qPCR. For IR, CA1d cells were treated with 8 Gy of ionizing radiation and allowed to recover for the indicated times, followed by RT-qPCR. Expression is relative to untreated cells (0h). B, inhibition of induction by p53 knockdown. MCF7 cells transduced with p53shRNA or non-silencing control were treated with 0.25μM doxorubicin for 24h, followed by RT-qPCR. C-D, kinetics and scale of induction vary with dosage. MCF7 cells were treated with different doses of drug and kinetics of induction were compared for hCLCA2, p21, and Bax by RT-qPCR. For drug treatments, expression is relative to that of the vehicle control, DMSO.

Figure 2

p53 binds to a consensus element in the hCLCA2 promoter. A, top, p53 binding consensus, reprinted from Wei et al. (9) with permission from Elsevier. Lower, matches for the consensus in the hCLCA2 promoter are conserved in orthologs from mouse, rat, and dog. Positions within the human promoter relative to the translation initiation codon are indicated. B, ChIP analysis with anti-p53 antibody 1C12 (Cell Signaling) showing p53 binding to the consensus elements in the hCLCA2 promoter (h2 p53) and to an established binding site from the p21 promoter (p21) but not to an adjacent region in the hCLCA2 promoter (h2 nc). An anti-CD34 mAb (Pharmingen) served as an additional negative control. Input, 10% of the lysate was subjected to PCR directly. Relative positions of the PCR products from the hCLCA2 promoter are depicted in the schematic. C, demonstration that the putative p53 binding sites from the hCLCA2 promoter are responsible for transcriptional activation by p53. Right, PCR products containing these sites or not were fused to a firefly luciferase reporter gene in pGL4. The constructs were transfected into 293T cells alone or along with a plasmid encoding p53 or no insert (C1). A plasmid expressing Renilla luciferase was co-transfected and readings were normalized to this control. Activities are expressed relative to the pGL4 vector control. Promoter segments include the interval from the indicated upstream nucleotide to the A of the translation initiation codon (+1). Inset, mutations introduced into the p53 binding site in the -184M construct are shown. Luc, luciferase. D, p53 role in induction by DNA damage. MCF7 cells were transfected for 8h with constructs bearing or lacking the p53 binding site, treated with aphidicolin for two hours, and lysed 24h later. Bars indicate standard error.

Figure 3

Induction of hCLCA2 by adenoviral transduction of p53. A, RT-qPCR shows 200-fold induction of hCLCA2 by 24h post-infection. Subconfluent MCF10A cells were infected with adenoviral supernatants at an m.o.i. of 30, and RNA was extracted and analysed. B, induction of hCLCA2 protein in parallel with p53. CA1d cells were infected with adenoviral supernatants and lysed at indicated intervals. Lysates were immunoblotted directly for p53 and tubulin or subjected to immunoprecipitation for hCLCA2 followed by immunoblotting. C, timing of hCLCA2 induction relative to p21. BT549 cells were infected and immunoblotted 20 or 40h later. Ad-GFP indicates Ad-Easy vector sans insert; p53, p53-Ad-Easy; h2, hCLCA2-Ad-Easy.

Figure 4

Restoration of hCLCA2 expression in breast cancer cells is growth-inhibitory. A-C, BT549 breast cancer cells were infected with adenoviruses bearing a GFP marker, p53, or hCLCA2 (h2) at an m.o.i. of 10. A, growth curves measured on a Vi-Cell instrument. B, flow cytometry of propidium iodide-stained cells 48h after infection showed a steep increase in the sub-G1 DNA peak. C, lysates were analyzed by immunoblotting for apoptotic markers. Signals were quantified by fluorometry (Licor) and normalized to actin. Units are relative to Ad-GFP control. Lower right, mitochondrial fractions were analyzed for translocation of Bax, Bad, Bim, and Bmf.

Figure 5

Growth inhibition and apoptosis of MCF10A cells infected with hCLCA2 adenovirus. A, cells were infected as in Figure 4. Adherent (left) and detached (right) cells were counted daily. B, immunoblots of lysates processed at 20h intervals post-infection. Stars indicate proteolytic products diagnostic of apoptosis. C, immunoblots of subconfluent MCF10A cells 24h post-infection show induction of p53, p21, and p27 but not caspase 3. Star indicates absence of caspase 3 proteolytic product. This experiment was repeated four times with similar results. In B and C, equal amounts of total protein were loaded per lane (50 μg) based on the BCA assay. D, top, scheme for cell synchronization by sequential double block and release. Bottom, quantification of cells in G0/G1 versus G2/M after release from the double block as measured by flow cytometry. E, measurement of pHi in cells transduced with hCLCA2-Ad or control by the SNARF-AM method.

Figure 6

Effect of hCLCA2 attenuation on cell proliferation and survival. Stable knockdown cell lines were generated by infection with lentiviruses expressing shRNAs (H2sh1; H2sh2; or NC, negative control), followed by puromycin selection. The extent of knockdown was measured by immunoblot (A, inset). To measure proliferation rate, identical numbers of cells were seeded in multiwell plates and counted at one day intervals on a Vi-cell instrument (A). B-C, surviving cell number after treatment with doxorubicin (B, 3 μM for 12h; C, 0.5 μM for 3 days) followed by medium change and recovery for 3 or 20 days.