We have investigated some roles of splicing factor polypyrimidine tract-binding protein (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in progressively transformed human mammary epithelial cells (HMECs), as well as in breast tumor cell lines compared with HMECs with finite growth potential and found that the level of PTBP1 correlated with the transformation state of HMECs. Knockdown of PTBP1 expression substantially inhibited tumor cell growth, colony formation in soft agar and in vitro invasiveness of breast cancer cell lines, a result similar to what we have reported in ovarian cancer. However, ectopic expression of PTBP1 (as a PTBP1–EGFP fusion protein) did not enhance the proliferation of immortalized HMEC. Rather, PTBP1 expression promoted anchorage-independent growth of an immortalized HMEC as assessed by increased colony formation in soft agar. In addition, we found that knockdown of PTBP1 expression led to upregulation of the expression of the M1 isoform of pyruvate kinase (PKM1) and increase of the ratio of PKM1 vs PKM2. PKM1 has been reported to promote oxidative phosphorylation and reduce tumorigenesis. Correspondingly, we observed increased oxygen consumption in PTBP1-knockdown breast cancer cells. Together, these results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of transformed properties. PTBP1 exerts these effects, in part, by regulating the splicing of pyruvate kinase, and consequently alters glucose metabolism and contributes to the Warburg effect.

Background: Cancer-specific alternative splicing as well as aberrant splicing factor expression in tumors have been reported (Korner M et al, Am. J. Pathol. 175:461-472, 2009). Splicing factor SRp20 belongs to a family of highly conserved serine-arginine-rich proteins with multiple functions in RNA processing such as spliceosome assembly and alternative splicing (Graveley BR, RNA. 6(9):1197-211, 2000). Materials and Methods: We established stable MDA-MB-231, MCF-7, and T47D cell lines carrying doxycycline (Doxy)-inducible SRp20shRNA and assessed Doxy-induced knockdown (KD) of SRp20 by western blot. We performed cell growth curves in triplicate using the MTT assay to determine the effect of SRp20 KD in these cell lines after Doxy induction of SRp20shRNA, and compared these to controls. For apoptosis assays, we stained SRp20shRNA sublines with Hoechst 33342 and counted apoptotic cells under the microscope. We also carried out western blots to examine the expressions of Bcl-2 and cleaved caspases-3, -6, -7, and -9. Results and Discussion: We found that the expression of SRp20 was upregulated in immortalized human mammary epithelial cells (HMECs), compared to controls, and that the level of SRp20 correlated with the transformation state. Knockdown of SRp20 was seen to be 88%, 58%, and 52% in MDA-MB-231, MCF-7, and T47D cells, respectively. However, SRp20 KD suppressed breast cancer cell growth in a cell line-dependent manner. Cell growth after 7 days was decreased in estrogen receptor-negative (ER-) MDA-MB-231 cells (91.4%), and estrogen receptor-positive (ER+) MCF-7 (73.2%), while SRp20 KD had no effect on growth of ER+ T47D cells. The differences between MCF-7 and T47D were notable, since SRp20 was suppressed by -50% in both lines. We found that the number of apoptotic cells in MDA-MB-231 line remarkably increased after Doxy treatment, indicating that the inhibition of cell growth was due to apoptosis. Further, immunoblot analysis of Bcl-2 and cleaved caspases in MDA-MB-231 cells indicated that the SRp20 KD activates the intrinsic apoptotic pathway in these breast cancer cells. We are presently assessing the expressions of these proteins in MCF-7 and T47D cells and investigating the mechanistic basis for this cell line-dependent effect of SRp20 KD. In summary, we report here a novel finding that inhibition of splicing factor SRp20 inhibits cell growth in some breast cancer cell lines. The intrinsic apoptotic pathway appears to be the main mechanism of cell growth inhibition in these cells. Our results indicate that SRp20 plays a major role in the survival of some breast cancer cell lines. This splicing factor may represent a novel therapeutic target for the treatment of some breast cancers. (Supported in part by CA40570 and CA138762 from NCI to WTB, in part by IDPH to XH, and in part by UIC) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-11.