The mitochondrial protein C1qbp promotes cell proliferation, migration and resistance to cell death

Abstract

Complement 1q-Binding Protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration, and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation, and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP, and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels, and decreased cell migration compared to control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than anti-tumor, role for C1qbp, and indicate that C1qbp could serve as a molecular target for cancer therapeutics.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by National Institutes of Health grants HL092327 and HL094404 (to Christopher P. Baines).

Figures and Tables

Figure 1 C1qbp suppresses staurosporine-induce apoptosis in fibroblasts. (A) Mouse embryonic fibroblasts (MEFs) were infected with an adenovirus encoding Myc-tagged C1qbp, or β-galactosidase (β-gal, control) and then protein gel blotted for Myc, cleaved caspase-3 and cleaved PARP following exposure to 300 nM staurosporine for 18 h. GAPDH was used to demonstrate equivalent loading. (B) TUNEL staining in the infected MEFs exposed to increasing concentrations of staurosporine for 18 h. (C) MEFs were transfected with 100 nM of control (Con) or C1qbp-specific siRNA and protein gel blotted for C1qbp, cleaved caspase-3, cleaved PARP and GAPDH following exposure to 300 nM staurosporine for 18 h. (D) TUNEL staining in the siRNA-transfected MEFs exposed to increasing concentrations of staurosporine for 18 hrs. The results shown are representative of 3 and 4 independent experiments performed in duplicate. Error bars indicate SEM and *p < 0.05 vs. β-gal or Con siRNA.

Figure 2 C1qbp promotes proliferation and migration in fibroblasts. (A) Phospho-histone H3 (pH3) staining, a marker of cell proliferation, in MEFs either infected with β-galactosidase (β-gal) or C1qbp adenoviruses or transfected with control (Con) or C1qbp siRNAs. (B) ATP levels in the virus-infected and siRNA-transfected MEFs. (C) Wound-healing assay in MEFs infected with either β-gal or C1qbp adenoviruses. (D) Wound-healing assay in MEFs transfected with either Con or C1qbp siRNAs. The results shown are representative of 3 and 4 independent experiments performed in duplicate. Error bars indicate SEM and *p < 0.05 vs. β-gal or Con siRNA.

Figure 3 Mitochondrial C1qbp is upregulated in human breast cancer cells and tumors. (A) Protein gel blotting for C1qbp in the normal breast epithelial line MCF12A and in the MCF7, BT474, T47D and MDA-MB-231 breast cancer cell lines. GAPDH was used as a loading control. The results shown are representative of 3 and 4 independent experiments performed in duplicate. (B–G) Fluorescent immunohistochemical staining for C1qbp in sections from: (B and C) normal human breast tissue; (D and E) breast invasive ductal carcinoma and (F and G) breast mucous adenocarcinoma. Results are representative of staining from 4 different patient samples in each group.

Figure 4 Knockdown of C1qbp reduces proliferation in MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were stably transfected with either a control shRNA (Con) or one of 3 different C1qbp-specific shRNAs and then protein gel blotted for C1qbp. GAPDH was used as a loading control. (B) Quantification of the efficiency of C1qbp knockdown in the MDA-MB-231 shRNA cell lines. (C) Each of the MDA-MB-231 shRNA cell lines was plated at an equal density, and the increase in cell numbers after 48 hrs measured by bis-benzamide staining of the nuclei. (D) ATP levels in the control and C1qbp shRNA MDA-MB-231 cell lines. The results shown are representative of 3 and 4 independent experiments performed in duplicate. Error bars indicate SEM and *p < 0.05 vs. Con sh.

Figure 5 Knockdown of C1qbp reduces migration and resistance to doxorubicin in MDA-MB-231 breast cancer cells. (A) Wound-healing assay in MDA-MB-231 cells stably transfected with either a control shRNA (Con) or one of 3 different C1qbp-specific shRNAs. (B) TUNEL staining in the different MDA-MB-231 shRNA cell lines at baseline and following exposure to 2 µM doxorubicin for 24 h. The results shown are representative of 3 and 4 independent experiments performed in duplicate. Error bars indicate SEM and *p < 0.05 vs. Con shRNA.

Figure 6 C1qbp is upregulated in human lung and colon cancer cell lines and tumors. (A) Protein gel blotting for C1qbp in the normal lung Wi38 cell line and in the H441, Calu-6 and A549 lung cancer cell lines. GAPDH was used as a loading control. (B) Protein gel blotting for C1qbp and GAPDH in the normal colon FHC cell line and in the HT-29 and HCT-116 colon cancer cell lines. (C–F) Fluorescent immunostaining for C1qbp in sections from: (C) normal human lung tissue; (D) small cell lung carcinoma; (E) normal human colon tissue and (F) colon invasive adenocarcinoma (well differentiated, mucinous). The results shown are representative of 3 and 4 independent experiments performed in duplicate (cell lines) or 4 different patient samples (tumors).