The genes of the piwi family are defined by conserved PAZ and Piwi domains and play important roles in stem-cell self-renewal, RNA silencing and translational regulation in various organisms. Both, mouse and human Piwil2 genes, members of the piwi gene family, are specifically expressed in testis. We report here enhanced expression of the human Piwil2 gene in testicular seminomas, but not in testicular non-seminomatous tumors. Expression of the Piwil2 gene was also found in different tumors examined, including prostate, breast, gastrointestinal, ovarian and endometrial cancer of human and in breast tumors, rhabdomyosarcoma and medulloblastoma of mouse. Therefore, Piwil2 can be categorized as a novel member of cancer/testis antigens. To identify genes activated by Piwil2, RNA isolated from NIH-3T3 cells expressing constitutively Piwil2 were compared with RNA samples from control NIH-3T3 cells using a cancer gene array. Induction of high-level expression of the antiapoptotic gene Bcl-X(L) was observed in cells expressing Piwil2. Furthermore, increased Bcl-X(L) expression correlated with increase of signal transducer and activator of transcription 3 (Stat3) expression. Gene silencing of Piwil2 with its small interference RNA suppressed Stat3 and Bcl-X(L) expression and induced apoptosis. A causal link between Piwil2 expression and inhibition of apoptosis and enhanced proliferation was demonstrated in cells expressing Piwil2. Furthermore, results of soft agar assay indicated that Piwil2 overexpression induced transformation of fibroblast cells. In summary, our results demonstrate that Piwil2 is widely expressed in tumors and acts as an oncogene by inhibition of apoptosis and promotion of proliferation via Stat3/Bcl-X(L) signaling pathway. Expression of Piwil2 in a wide variety of tumors could be a useful prognostic factor that could have also diagnostic and therapeutic implications.

Cancer stem cell studies may improve understanding of tumor pathophysiology and identify more effective strategies for cancer treatment. In a variety of organisms, Piwil2 has been implicated in multiple roles including stem cell self-renewal, RNA silencing, and translational control. In this study, we documented specific expression of the stem cell protein Piwil2 in breast cancer with predominant expression in breast cancer stem cells. In patients who were evaluated, we determined that 90% of invasive carcinomas and 81% of carcinomas in situ exhibited highest expression of Piwil2. In breast cancer cells, Piwil2 silencing suppressed the expression of signal transducer and activator of transcription 3, a pivotal regulator of Bcl-X L and cyclin D1, whose downregulation paralleled a reduction in cell proliferation and survival. Our findings define Piwil2 and its effector signaling pathways as key factors in the proliferation and survival of breast cancer stem cells. Cancer Res; 70(11); 4569-79. ©2010 AACR.

Supplementary Table 1 from Pathways of Proliferation and Antiapoptosis Driven in Breast Cancer Stem Cells by Stem Cell Protein Piwil2

Supplementary Table 2 from Pathways of Proliferation and Antiapoptosis Driven in Breast Cancer Stem Cells by Stem Cell Protein Piwil2

Supplementary Figure Legends 1-7 from Pathways of Proliferation and Antiapoptosis Driven in Breast Cancer Stem Cells by Stem Cell Protein Piwil2

Supplementary Table 1 from Pathways of Proliferation and Antiapoptosis Driven in Breast Cancer Stem Cells by Stem Cell Protein Piwil2

Supplementary Figures 1-7 from Pathways of Proliferation and Antiapoptosis Driven in Breast Cancer Stem Cells by Stem Cell Protein Piwil2

Supplementary Table 2 from Pathways of Proliferation and Antiapoptosis Driven in Breast Cancer Stem Cells by Stem Cell Protein Piwil2