3s Ser69/74Ala protein on the Smad3-dependent transcription, its overexpression significantly inhibits the putative CSC population even without disturbing the Smad3-dependent transcription. This observation can potentially suggest that abrogation of TGFb1 dependent phosphorylation of 14-3-3s at two sites can trigger the Smad3 independent 16494499 mechanisms of CSC regulation by TGFb1 axis. Tumorigenic properties of MCF7 cells stably expressing wild type and the mutant 14-3-3s proteins were analyzed in vivo using NOD/SCID mouse NVP-BHG712 xenograft models. Our data indicates that the MCF7 xenograft tumors expressing 14-3-3s Ser74Ala and 14-33s Ser69/74Ala mutant proteins showed statistically significant decrease in the growth rate in vivo compared with a control group. Analysis of cell apoptosis in the xenograft tumors using TUNEL staining confirmed a higher number of apoptotic cells in the MCF7 xenograft tumors expressing 14-3-3s Ser74Ala mutant protein. This result is in agreement with in vitro data for TGFb dependent inhibition of sphere formation and suggests that phosphorylation of 14-3-3s at Ser69 plays an important role in cell tumorigenicity and survival. In breast cancers, a subset of the cells with high aldehyde dehydrogenase activity and CD44+/CD242/low pheno- 15 Phosphoproteomics of TGFb1 Signaling type has been characterized as CSCs population with tumorigenic and radioresistant phenotype,. Histological analysis of MCF7 xenograft tumors expressing 14-3-3s wild type or Ser74Ala mutant proteins revealed a decrease in CD44+/CD242/low cell phenotype as compared with a control group or with the MCF7 xenograft tumors expressing 14-3-3s Ser69Ala or double mutant proteins. As suggested by preclinical studies and clinical observation, resistance to conventional therapy, including irradiation, has been reported to be a defining characteristic of CSCs from various tumor types, including breast cancer. One of the bestcharacterized chromatin modification events in responses to cell irradiation is histone H2A.X phosphorylation by ATM or ATR serine/threonine protein kinase. The phosphorylated form of H2A.X forms nuclear foci on the sites of DNA double strand breaks 17984313 and the number of c-H2AX foci approximates the number of DSBs induced. Recent studies demonstrated that in murine mammary gland CSCs, c-H2A.X foci resolved faster than in non-CSC population that perhaps is reflective of more efficient DSB repair in CSCs. Immunofluorescent detection of phosphorylated c-H2A.X revealed more residual 
DNA damage at 4 hours after irradiation in the cell overexpressing 14-3-3s Ser74Ala mutant protein and treated with TGFb as compared to the control cells. Because successful DNA reparation in the irradiated cells is associated with their survival, we further assessed the surviving fraction of the cells treated or non-treated with TGFb and irradiated with 4 Gy X-ray dose. Our data suggest that activation of TGFb signaling pathway decreases cell survival after irradiation and, besides of that, surviving fraction of the cells overexpressing 14-3-3s Ser74Ala mutant protein was significantly reduced as compared to the control cells. These results showed an impaired repair capability in the cells overexpressing 14-3-3s Ser74Ala mutant after TGFb treatment, which is consistent with the decreased cell survival. Preclinical assays and clinical findings suggest that a higher proportion of tumor initiating cells correlates with higher tumor radioresistance. ALDH activity selectively
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