XB130 mediates cancer cell proliferation and survival through multiple signaling events downstream of Akt

Abstract

XB130, a novel adaptor protein, mediates RET/PTC chromosome rearrangement-related thyroid cancer cell proliferation and survival through phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway. Recently, XB130 was found in different cancer cells in the absence of RET/PTC. To determine whether RET/PTC is required of XB130-related cancer cell proliferation and survival, WRO thyroid cancer cells (with RET/PTC mutation) and A549 lung cancer cells (without RET/PTC) were treated with XB130 siRNA, and multiple Akt down-stream signals were examined. Knocking-down of XB130 inhibited G(1)-S phase progression, and induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Knocking-down of XB130 reduced phosphorylation of p21Cip1/WAF1, p27Kip1, FOXO3a and GSK3β, increased p21Cip1/WAF1protein levels and cleavages of caspase-8 and-9. However, the phosphorylation of FOXO1 and the protein levels of p53 were not affected by XB130 siRNA. We also found XB130 can be phosphorylated by multiple protein tyrosine kinases. These results indicate that XB130 is a substrate of multiple protein tyrosine kinases, and it can regulate cell proliferation and survival through modulating selected down-stream signals of PI3K/Akt pathway. XB130 could be involved in growth and survival of different cancer cells.

Figure 1. XB130 controls cell cycle progression of cancer cells.

(A and B) Down-regulation of XB130 inhibited G₁-S phase progression in WRO and A549 cells. Cells transfected with control or XB130 siRNA were stained with PI and analyzed by flow cytometry. Mean ± SEM. n = 6. *p<0.05 (compared with control siRNA). (C) Reduced Ki67 and PCNA levels in XB130 siRNA treated WRO and A549 cells, as determined by western blotting. n = 3. *p<0.05 (compared with control siRNA treated group). (D) Presence of RET/PTC rearrangement was confirmed in both TPC-1 and WRO cells using RT-PCR. A549 cells do not contain RET/PTC rearrangement.

Figure 2. XB130 controls cell survival of cancer cells.

(A) Down-regulation of XB130 enhanced spontaneous and induced cell death of WRO cells cultured with 10% FBS. Cells transfected with control or XB130 siRNA were treated with staurosporine (STS, 200 nM), or Fas antibody (FasAb, clone CH11, 100 ng/ml) for 24 h. Apoptosis was determined by flow cytometry using PI/Annexin V double staining. (B) Down-regulation of XB130 enhanced staurosporine-induced apoptosis in serum free condition in A549 cells. A549 cells transfected with control or XB130 siRNA were incubated in serum free medium with or without 200 nM STS for 24 h. n = 6. Mean ± SEM. *p<0.05 (compared with control siRNA). (C) XB130 can be phosphorylated by protein tyrosine kinases other than RET/PTC. Immunoprecipitation with anti-myc antibody in HEK293 cells transfected with myc-tagged XB130, along with RET/PTC3, EGFR, Src, Abl, or ERBB2, showed an increase in XB130 tyrosine phosphorylation using anti-phosphotyrosine antibody.

Figure 3. XB130 binds to p85α subunit of PI3K and controls Akt activity in cancer cells.

(A) Schematic representation of protein structures of XB130 and p85α subunit of PI3K. Interactions of XB130 with p85α were detected by co-immunoprecipitation in WRO and A549 cells. (B) XB130 siRNA effectively reduced XB130 protein levels and Akt phosphorylation in WRO and A549 cells. siRNA transfected cells were harvested at 48 h after transfection. n = 5. *p<0.05 (compared with control siRNA).

Figure 4. XB130 controls cell cycle progression and survival via PI3K/Akt in cancer cells.

(A and B) Down-regulation of XB130 decreased phosphorylation of p21 and p27, and increased expression of p21 in WRO and A549 cells. Expressions of p27 and p53 were not affected by down-regulation of XB130. (C and D) Down-regulation of XB130 decreased phosphorylations of FOXO3a and GSK3β in WRO and A549 cells. n = 4. *p<0.05 (compared with control siRNA).

Figure 5. XB130 regulates cell survival through caspase-8 and caspase-9 signaling.

(A and B) Cleaved fragments of both caspase-8 and-9 were distinctly increased by knocking-down of XB130 in WRO cells. In A549 cells, down-regulation of XB130 decreased procaspase-8 and increased cleaved caspase-9. n = 4. Mean ± SEM. *p<0.05 (compared with control siRNA).

Figure 6. Roles of XB130 in cell cycle progression and survival of cancer.

XB130 specifically binds p85α subunit of PI3K, which subsequently activate Akt. Akt plays an essential role in cell proliferation and survival. Down-regulation of XB130 with siRNA affected multiple molecules down-stream of Akt. This suggests that XB130 is an important regulator in PI3K/Akt related cancer cell proliferation and survival.