A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBFb, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment.

Abbreviations: AIP4 (atrophin 1-interacting protein 4); BMP (bone morphogenetic protein); CHIP (carboxyl terminus of Hsc70-interacting protein); GSK3b (glycogen synthase kinase 3-b); HECT (homologous to E6-AP carboxyl terminus); MAPK (mitogen-activated protein kinase); NEDD4-2 (neural precursor cell expressed, developmentally downregulated 4-2); PIAS (protein inhibitor of activated Stat); Smurf (Smad ubiquitylation regulatory factor); STRAP (serine-threonine kinase receptorassociated protein); SUMO (small ubiquitin-like modifier); TbRI/TbRII (TGFb receptor type I and II); TGFb (transforming growth factor b); WWP1 (WW domain-containing protein 1); Ub (ubiquitin) npg Transforming growth factor β (TGFβ) controls cellular behavior in embryonic and adult tissues. TGFβ binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that together regulate gene expression. In this review, mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of TGFβ are presented. We discuss how the activity and duration of TGFβ receptor/Smad signaling can be regulated by post-translational modifications that affect the stability of key proteins in the pathway. We highlight links between these mechanisms and human diseases, such as tissue fibrosis and cancer. Regulating the stability of TGFβ receptors and Smads

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Bone morphogenetic protein (BMP) signaling exerts antitumor activities in glioblastoma; however, its precise mechanisms remain to be elucidated. Here, we demonstrated that the BMP type I receptor ALK-2 (encoded by the ACVR1 gene) has crucial roles in apoptosis induction of patient-derived glioma-initiating cells (GICs), TGS-01 and TGS-04. We also characterized a BMP target gene, Distal-less homeobox 2 (DLX2), and found that DLX2 promoted apoptosis and neural differentiation of GICs. The tumor-suppressive effects of ALK-2 and DLX2 were further confirmed in a mouse orthotopic transplantation model. Interestingly, valproic acid (VPA), an anti-epileptic compound, induced BMP2, BMP4, ACVR1 and DLX2 mRNA expression with a concomitant increase in phosphorylation of Smad1/5. Consistently, we showed that treatment with VPA induced apoptosis of GICs, whereas silencing of ALK-2 or DLX2 expression partially suppressed it. Our study thus reveals BMP-mediated inhibitory mechanisms for glioblastoma, which explains, at least in part, the therapeutic effects of VPA.