A novel crosstalk between CCAR2 and AKT pathway in the regulation of cancer cell proliferation

Abstract

Human CCAR2 has recently emerged as having a pivotal role in the DNA damage response, promoting apoptosis and repair of heterochromatic DNA breaks. However, less is known about the function of CCAR2 in tumor formation and cancer progression. Here, we demonstrate, for the first time, that CCAR2 loss inhibits the proliferation of cancer cells, but preserves the growth of normal cells. Investigating the mechanisms responsible for this differential effect, we found that CCAR2 depletion specifically impairs the activation of AKT pathway in cancer cells, but not in normal cells, by reducing AKT phosphorylation on Ser473. This effect is achieved through the transcriptional upregulation of TRB3 gene and accumulation of TRB3 protein, which then binds to and inhibits the phosphorylation and activation of AKT. The defective activation of AKT finally results in reduced GSK3β phosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. These results establish an important role for CCAR2 in cancer cells proliferation and could shed new light on novel therapeutic strategies against cancer, devoid of detrimental side effects.

Figure 1

CCAR2 depletion strongly inhibits U2OS cell proliferation. (a) Western blot (WB) analysis demonstrating CCAR2 depletion for up to 10 days after siRNA transfection. (b) Cell proliferation rate in control and CCAR2 silenced U2OS cells. **P<0.01. (c) Colony forming assays in U2OS cells silenced for CCAR2 (top). Colonies were counted and expressed as a percentage of siLUC control. Results are mean +/− S.D. from three inedependent experiments. **P<0.01 (d) Cell proliferation rate (top) and WB analysis (bottom) of BJ-hTERT control cells and CCAR2-KO mass culture

Figure 2

CCAR2 depletion reduces AKT phosphorylation. (a) Pie chart indicating the percentage of up- and downregulated genes identified by the gene expression analysis. (b) WB analysis of U2OS cells silenced for CCAR2 with the indicated antibodies. (c) WB analysis of control and CCAR2-KO mass culture BJ-hTERT cells with the indicated antibodies

Figure 3

CCAR2 depletion decreases cancer cell proliferation but preserves the growth of normal cells. Proliferation rate analyses in the indicated cancer (a) and normal immortalized (b) cell lines silenced for CCAR2 and siLUC as negative control. *P<0.05, **P<0.01, ***P<0.001

Figure 4

CCAR2 depletion reduces AKT activation in cancer cells, but increases phosphorylated AKT in normal cells. WB analyses of AKT phosphorylation in the indicated cancer (a) and normal immortalized (b) cell lines silenced for CCAR2. (c) Cell proliferation rate in U2OS cells silenced for CCAR2, PTEN or both CCAR2 and PTEN. ***P<0.001 (d) WB analysis with the indicated antibodies of U2OS cells transfected with siRNA against CCAR2, PTEN and both siCCAR2 and siPTEN

Figure 5

CCAR2 protein levels do not affect malignant transformation. Representative images of colonies formed in soft agar by HME (a) and A549 (b) cells silenced for CCAR2. (c) Representative images of HME colonies formed in soft agar by a clone stably expressing FLAG-CCAR2

Figure 6

CCAR2 depletion prevents G1/S-phase transition in cancer cell lines. Normal and cancer cells silenced for CCAR2 were analyzed by western blot for the levels of cleaved PARP protein (a) and by trypan blue exclusion test for the percentage of dead cells (b). (c) A549 and IOSE80 cells transfected with siCCAR2 and siLUC were incubated in EdU containing medium for 1 h 30 min. EdU-positive cells were stained, enumerated and data reported in the chart (top). **P<0.01 WB analysis of A459 and IOSE80 cells transfected with siCCAR2 and siLUC, with the indicated antibodies (bottom). (d) WB analysis of cancer (U2OS and A549) and normal immortalized cells (HME and IOSE80) silenced for CCAR2 with the indicated antibodies

Figure 7

CCAR2 depletion induces the expression of TRB3 in cancer cell lines. (a) Expression of TRB3 was analyzed by RT-qPCR in cancer (U2OS and A549) and normal cells (HME and IOSE80) transfected with siCCAR2 and siLUC. *P<0.05, **P<0.01. (b) WB analyses of cancer and normal immortalized cells transfected with siCCAR2 and siLUC with the indicated antibodies. AKT immunoprecipitates from A549 (c) and U2OS (d) cells silenced for CCAR2 and LUC were analyzed by WB with the indicated antibodies. PC= negative control. (e) Graphical representation of our working model