Tumor-specific silencing of COPZ2 gene encoding coatomer protein complex subunit ζ 2 renders tumor cells dependent on its paralogous gene COPZ1

Abstract

Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of coatomer protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform coatomer protein complex ζ2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.

Fig. 1.

Effects of COPZ1, COPZ2, and COPA knockdown on tumor and normal cells. (A) siRNAs targeting COPA, COPZ1, COPZ2, or no human genes (siCont), obtained from Dharmacon (DH) or Qiagen (Q), were transfected into PC3 cells at the indicated concentrations. Cell numbers were determined 4 d posttransfection by flow cytometry (in three independent transfections) and are expressed as mean ± SD. (B) Experiment similar to A carried out with lower siRNA concentrations, demonstrating dose-dependent inhibition, and including a combination of COPZ1 and COPZ2 siRNAs. Cell numbers were measured 8 d posttransfection (in three independent transfections) and are expressed as mean ± SD. (C) Effects of the siRNAs in A on the proliferation of BJ-HTERT cells. Cell numbers were measured 7 d posttransfection (in triplicate) and are expressed as mean ± SD. (D) Effects of COPA, COPZ1, and COPZ2 siRNAs and a combination of COPZ1 and COPZ2 siRNAs (all at 5-nM concentrations) on the proliferation of BJ-HTERT cells. Cell numbers were measured 7 d posttransfection (six replicates) and are expressed as mean ± SD. (E) Effects of COPA, COPZ1, and COPZ2 siRNAs and a combination of COPZ1 and COPZ2 siRNAs (at the same concentrations as in B on the proliferation of normal human prostate epithelial cells (HPEC). Cell numbers were measured 8 d posttransfection (in triplicate) and are expressed as mean ± SD.

Fig. 2.

Effects of COPZ1 knockdown on Golgi apparatus, autophagosomes, and cell death. (A) PC3 cells expressing the autophagosome marker GFP-LC3 were transfected with control siRNA or siRNAs targeting COPA, COPZ1, or COPZ2 and were analyzed by fluorescence microscopy for GFP fluorescence (green), indirect immunofluorescence staining for Golgi marker GM130 (red), and nuclear DNA staining with DAPI 72 h posttransfection with the indicated siRNAs. (Scale bars: 10 μM.) (B) GFP-LC3 electrophoretic mobility of the cells in A analyzed by immunoblotting with anti-GFP antibody. (C) Changes in the number of membrane-permeable (PI⁺) PC-3 cells upon transfection with control siRNA or siRNAs targeting COPA or COPZ1, as determined by flow cytometry (mean ± SD, triplicate measurements). (D) Changes in the number of apoptotic (TUNEL⁺) PC-3 cells upon transfection with control siRNA or siRNAs targeting COPA or COPZ1, as determined by flow cytometry (mean ± SD, triplicate measurements). (E) Effects of COPZ1, COPA, and CDC2 siRNAs on cell number of proliferating and growth-arrested HT1080 cells. HT1080 p21-9 cells with IPTG-inducible expression of the cell-cycle inhibitor p21 were transfected with the indicated siRNAs. Transfected cells were pIated in the absence (proliferating) or in the presence (arrested) of 50 μM IPTG, in triplicate; cell numbers were determined 5 d later. Data are shown as mean ± SD.

Fig. 3.

Down-regulation of the COPZ2 gene in transformed cell lines. (A) qRT-PCR analysis of expression of the indicated COPI subunit genes in tumor cell lines and BJ-hTERT fibroblasts. Expression is presented relative to BJ-hTERT; data are shown as mean ± SD. (B) qRT-PCR analysis of expression of the indicated COPI subunit genes in immortalized normal BJ-EN fibroblasts and their partially transformed (BJ-ELB) and fully transformed (BJ-ELR) derivatives. Expression is presented relative to BJ-EN. (C) qRT-PCR analysis of expression of the indicated COPI subunit genes in two normal melanocyte preparations (NMP) and four melanoma cell lines. Expression is presented relative to NMP 241.

Fig. 4.

COPZ2 expression in normal, benign, and malignant tissues of different tumor types (microarray data from GEO database). P values (student's t test) are indicated for significant differences between the groups. (A) Bladder cancer study (43): normal urothelium (NU) and superficial tumors (ST). (B) Two prostate cancer studies (44, 45): normal prostate (NP), primary tumors (PT), and metastatic tumors (MT). (C) Colon cancer study (46): normal mucosa (NM) and adenocarcinomas (A). (D) Melanoma study (47): normal melanocytes (NMel), benign nevi (BN), and metastatic melanoma (MM).

Fig. 5.

Relationship between COPZ1 and COPZ2 expression and siRNA sensitivity. (A) Immunoblotting of COPZ1 and COPZ2 proteins in PC3 cells transduced with control lentivirus or with lentiviral vectors expressing FLAG-tagged COPZ1 or COPZ2 and probed with FLAG, COPZ1, and COPZ2 antibodies. (B) In vivo growth of PC3 xenograft tumors transduced with a control lentiviral vector or with vectors expressing COPZ1 or COPZ2. Data shown are tumor weights (mean ± SD) at the end of the experiment (41 d postinoculation). (C) Effects of the indicated siRNAs on the proliferation of PC3 cells transduced with control siRNA (siCont) or vectors expressing COPA, COPZ1, or COPZ2 (six replicates). Cell numbers were measured 4 d posttransfection and are expressed as mean ± SD.

Fig. 6.

Effects of miR-152 in tumor cells. (A) Effects of transfection with miR-152 precursors from Dharmacon (DH) or Ambion (AM) and miRNA mimic negative control (Dharmacon) on the proliferation of MDA-MB-231, HeLa, and PC3 cells (in triplicate). Cell numbers were measured 7 d posttransfection and are expressed relative to cells transfected with negative control. Data are shown as mean ± SD. (B) In vivo growth of PC3 xenograft tumors transduced with a control lentiviral vector or with a vector expressing miR-152 precursor. (Upper) Tumor weights (mean ± SD) at the end of the experiment (42 d postinoculation). (Lower) Photographs of tumors.