Genistein suppresses FLT4 and inhibits human colorectal cancer metastasis

Dietary consumption of genistein, found in soy, has been associated with a potentially protective role in colorectal cancer (CRC) development and progression. Herein we demonstrate that genistein will inhibit human CRC cell invasion and migration, that it does so at non-cytotoxic concentrations and we demonstrate this in multiple human CRC cell lines. After orthotopic implantation of human CRC tumors into mice, oral genistein did not inhibit tumor growth, but did inhibit distant metastasis formation, and was non-toxic to mice. Using a qPCR array, we screened for genistein-induced changes in gene expression, followed by Western blot confirmation, demonstrating that genistein downregulated matrix metalloproteinase 2 and Fms-Related Tyrosine Kinase 4 (FLT4; vascular endothelial growth factor receptor 3). After demonstrating that genistein suppressed neo-angiogenesis in mouse tumors, we examined FLT4 expression in primary CRC and adjacent normal colonic tissue from 60 human subjects, demonstrating that increased FLT4 significantly correlates with increased stage and decreased survival. In summary, we demonstrate for the first time that genistein inhibits human CRC metastasis at dietary, non-toxic, doses. FLT4 is identified as a marker of metastatic disease, and as a response marker for small molecule therapeutics that inhibit CRC metastasis

A Phase I Trial of Aminolevulinic Acid-Photodynamic Therapy for Treatment of Oral Leukoplakia

Background Photodynamic therapy with aminolevulinic acid (ALA PDT) for oral leukoplakia has shown promising effects in regression of oral leukoplakia. Although ALA has been extensively studied and is an ideal photosensitizer, the optimal light dose for treatment of oral leukoplakia has not been determined. We conducted a phase I study to determine MTD and DLT of PDT in patients treated with ALA for leukoplakia. Methods Patients with histologically confirmed oral leukoplakia received a single treatment of ALA PDT in cohorts with escalating doses of light (585 nm). Clinical, histologic, and biologic markers were assessed. Results Analysis of 11 participants is reported. No significant toxicity from ALA PDT was observed in patients who received ALA with a light dose of up to 4 J/cm2. One participant experienced transient grade 3 transaminase elevation due to ALA. One participant had a partial clinical response 3 months after treatment. Biologic mucosal risk markers showed no significant associations. Determination of MTD could not be accomplished within a feasible timeframe for completion of the study. Conclusions ALA PDT could be safely administered with a light dose up to 4 J/cm2 and demonstrated activity. Larger studies are needed to fully elucidate the MTD and efficacy of ALA-PDT

A phase II single-arm study of pembrolizumab with enzalutamide in men with metastatic castration-resistant prostate cancer progressing on enzalutamide alone.

BACKGROUND: Checkpoint inhibitors can induce profound anticancer responses, but programmed cell death protein-1 (PD-1) inhibition monotherapy has shown minimal activity in prostate cancer. A published report showed that men with prostate cancer who were resistant to the second-generation androgen receptor inhibitor enzalutamide had increased programmed death-ligand 1 (PD-L1) expression on circulating antigen-presenting cells. We hypothesized that the addition of PD-1 inhibition in these patients could induce a meaningful cancer response. METHODS: We evaluated enzalutamide plus the PD-1 inhibitor pembrolizumab in a single-arm phase II study of 28 men with metastatic castration-resistant prostate cancer (mprogressing on enzalutamide alone. Pembrolizumab 200 mg intravenous was given every 3 weeks for four doses with enzalutamide. The primary endpoint was prostate-specific antigen (PSA) decline of ≥50%. Secondary endpoints were objective response, PSA progression-free survival (PFS), time to subsequent treatment, and time to death. Baseline tumor biopsies were obtained when feasible, and samples were sequenced and evaluated for the expression of PD-L1, microsatellite instability (MSI), mutational and neoepitope burdens. RESULTS: Five (18%) of 28 patients had a PSA decline of ≥50%. Three (25%) of 12 patients with measurable disease at baseline achieved an objective response. Of the five responders, two continue with PSA and radiographic response after 39.3 and 37.8 months. For the entire cohort, median follow-up was 37 months, and median PSA PFS time was 3.8 months (95% CI: 2.8 to 9.9 months). Time to subsequent treatment was 7.21 months (95% CI: 5.1 to 11.1 months). Median overall survival for all patients was 21.9 months (95% CI: 14.7 to 28 .4 months), versus 41.7 months (95% CI: 22.16 to not reached (NR)) in the responders. Of the three responders with baseline biopsies, one had MSI high disease with mutations consistent with DNA-repair defects. None had detectable PD-L1 expression. CONCLUSIONS: Pembrolizumab has activity in mCRPC when added to enzalutamide. Responses were deep and durable and did not require tumor PD-L1 expression or DNA-repair defects. TRIAL REGISTRATION NUMBER: clinicaltrials.gov (NCT02312557)

Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

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 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

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 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 The current study was undertaken to determine whether genistein retained its antiinvasion efficacy in human PCa in the face of endoglin loss, and to determine whether there was any mechanistic overlap between the endoglin pathway and genistein. Here we demonstrate for the first time that genistein can cooperate with endoglin associated signaling molecules, ALK2 and Smad1, to inhibit cell invasion in endoglin deficient PCa cells. MOL #38935 7 Materials and Methods Materials. Genistein (4',5,7-trihydroxyflavone) (Sigma Chemical Co., St. Louis, MO), was prepared and stored as previously described 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 Reactions were run in duplicate on a single Applied Biosystems 7500 Real Time PCR workstation, using a TaqMan universal PCR kit and validated gene-specific exon spanning primers and probe sets (all from Applied Biosystems). Gene expression was normalized to GAPDH. MOL #38935 11 Genistein increases Smad1 promoter activity. The above findings raised the possibility that genistein could be activating the endoglin pathway. Others have reported that genistein could increase endoglin expression in human PCa cells 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 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 However, siA2 abrogated genistein's anti-invasion activity. As the above findings implicated ALK2 in mediating genistein function, additional studies were performed. Human prostate contains high levels of acid phosphatase, complicating the accurate measurement protein phosphorylation. Experience has taught us that measurement of in vivo function represents the optimal approach . Cells were therefore transfected with either wild type (WT) or K233R kinase dead (KD) ALK2, Figs 4C-E. The K233R mutation results in an non-phosphorylated and kinase inactive receptor 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 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 We have previously shown that genistein inhibits TGFβ-mediated increases in cell invasion by blocking TGFβ-mediated activation of p38 MAP kinase, and it's downstream effector, HSP27 Further, we have shown that p38 MAP kinase can activate Smad3 through signaling pathway cross-talk, and that Smad3 is pro-invasive . The effect of TGFβ ligand was not evaluated in the current study for a number of reasons. Recently, we demonstrated that endoglin inhibits cell motility and activates Smad1 regardless of the activation state of the TGFβ-Smad3 pathway 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