Title Page Title: Genistein induces phenotypic reversion of endoglin deficiency in human prostate cancer cells Running Title Page Running Title: Endoglin deficiency phenotype reversed by genistein Correspondence to

Abstract

Abstract Genistein has been shown to inhibit human prostate cancer (PCa) cell motility. Endoglin has been identified as an important suppressor of PCa cell motility, and its expression is lost during PCa progression. It is therefore important to determine whether endoglin loss affects genistein's efficacy, and if so, by what mechanism. In the current study, genistein was shown to induce reversion of endoglin deficient cells to a low motility, endoglin replete, phenotype. As endoglin suppresses PCa cell motility in an ALK2 and Smad1 dependent manner, we sought to determine whether genistein was activating the ALK2-Smad1 pathway. While treatment with genistein, or over expression of Smad1 or ALK2, all increased Smad1 responsive promoter activity and decreased cell motility, genistein's efficacy was abrogated by either Smad1 or ALK2 knock down. Further, transfection of cells with a kinase dead mutant of ALK2 abrogated genistein's efficacy. Together, these findings demonstrate that genistein therapeutically induces reversion to a low motility phenotype in aggressive endoglin deplete PCa cells. It does so by activating ALK2-Smad1 endoglin-associated signaling. These findings support the notion that individuals with low endoglin expressing PCa will benefit from genistein treatment. MOL #38935 4 Prostate cancer (PCa) is a leading cause of cancer associated death in the United States and world wide We have previously demonstrated that genistein (4',5,7-trihydroxyflavone) inhibits PCa cell invasion Dysregulated cell motility is a basic characteristic of cancer, including PCa, and is seen during PCa progression. Molecular changes which relate to the regulation of cell motility underlie this abnormal cellular phenotype. To be effective, anti-cancer therapeutics must retain efficacy in the face of molecular aberrations associated with cancer progression. Alternatively, their use must be tailored to specific molecular profiles. In either situation, optimal therapeutic implementation requires an MOL #38935 5 understanding of the relationship between therapeutic intervention, and the underlying molecular profile. A series of prior studies by us have identified endoglin as a key regulator of PCa cell motility, and have shown that its expression is lost during PCa progression Endoglin is a 180 kDa homodimeric type I transmembrane auxiliary receptor in the TGFβ superfamily A consideration of a series of studies by us and others supports the notion that genistein may exert effects upon the endoglin signaling pathway. Both endoglin and genistein act to suppress PCa cell invasion. Further, we have recently demonstrated that endoglin suppresses PCa cell motility by activating Smad1, in an ALK2 dependent manner Smad1 promoter luciferase reporter assays. Cells were co-transfected with BRE2-Luc and β -gal, and luciferase and β -gal activity were measured as described Quantitative reverse transcription/polymerase chain reaction (qRT/PCR). RNA isolation and real time qRT/PCR were performed as described Given the small magnitude of the increase, and the lack of statistical significance, this increase was felt not to be responsible for genistein's effects. This notion is supported by other findings. First, genistein retained efficacy in the face of endoglin knock down. Second, though endoglin expression did not increase with increases in genistein concentration, we have previously shown enhanced anti-invasion efficacy by genistein across this concentration range As endoglin has been shown to suppress PCa cell invasion by activating Smad1, we hypothesized that genistein was activating Smad1 promoter activity was not altered by endoglin knock down. Genistein cooperates with Smad1 to inhibit ALK2 is necessary for genistein-mediated inhibition of cell invasion. Type I (RI) TGFβ superfamily receptors have kinase domains which function as activators of Smad proteins Further, these findings suggest that individuals with endoglin deficient PCa may in fact experience a greater therapeutic benefit from therapy, than those with normal endoglin expression. We also show for the first time that genistein compensates for endoglin deficiency by activating endoglin-associated signaling pathways. In particular, endoglin activated Smad1 transcriptional activity. This in turn was shown to require ALK2, and in particular, a kinase competent ALK2. We have recently shown that endoglin inhibits PCa cell motility through a mechanism involving the type I TGFβ superfamily receptor ALK2 and Smad1 18 treatment conditions, genistein decreases PCa cell invasion but not cell viability Finally, in the current study, cell viability was closely monitored, and was not adversely altered under experimental conditions, compared to relevant controls. We thus propose the schema depicted in MOL #38935 19 The current study identifies ALK2, and in particular, kinase competent ALK2 as necessary for genistein-mediated reversion to an endoglin replete phenotype. However, additional studies will be required to further elucidate the underlying mechanism. One possibility is that genistein may alter the molecular make up of heteromeric cell surface receptor complexes. Canonical signaling through TGFβ superfamily receptors requires the formation of a multi-protein cell surface complex which contains two or more RI subtypes, two or more RII subtypes, and with and without one or more endoglin subunits In summary, genistein was shown to induce reversion of low endoglin PCa cells to a low motility, high endoglin, phenotype. This was due to genistein-mediated activation of Smad1, which in turn was dependent upon kinase competent ALK2. As endoglin is lost during PCa progression, and contributes to its metastatic phenotype, the current study supports the notion that individuals with low endoglin expressing PCa may derive relatively high therapeutic benefit from genistein. These findings may help interpret ongoing phase II molecular efficacy studies of genistein in prostate and other cancers. MOL #38935 2