Xiaoke Huang

Genistein Inhibits p38 Map Kinase Activation, Matrix Metalloproteinase Type 2, and Cell Invasion in Human Prostate Epithelial Cells

Epidemiologic studies associate consumption of genistein, in the form of dietary soy, with lower rates of metastatic prostate cancer. We have previously shown that genistein inhibits prostate cancer cell detachment in vitro, that it is well tolerated in an older cohort of men with prostate cancer, and that it alters cell signaling in that same cohort. We have also shown that p38 mitogen-activated protein kinase (MAPK) is necessary for transforming growth factor beta (TGF-beta)-mediated increases in prostate cancer adhesion. Although cell invasion is closely linked to metastatic behavior, little is known about how this process is regulated in prostate cancer or what effect, if any, genistein has on associated processes. We now show that genistein inhibits matrix metalloproteinase type 2 (MMP-2) activity in six of seven prostate cell lines tested, blocks MMP-2 induction by TGF-beta, and inhibits cell invasion. Efficacy was seen at low nanomolar concentrations, corresponding to blood concentrations of free genistein attained after dietary consumption. Inhibition of p38 MAPK by either SB203580 or dominant-negative construct blocked induction of MMP-2 and cell invasion by TGF-beta. Genistein exerted similar effects and was found to block activation of p38 MAPK by TGF-beta. This study shows that p38 MAPK is necessary for TGF-beta-mediated induction of MMP-2 and cell invasion in prostate cancer and that genistein blocks activation of p38 MAPK, thereby inhibiting processes closely linked to metastasis, and does so at concentrations associated with dietary consumption. Any potential causal link to epidemiologic findings will require further investigation.

MEK4 Function, Genistein Treatment, and Invasion of Human Prostate Cancer Cells

BACKGROUND Dietary intake of genistein by patients with prostate cancer has been associated with decreased metastasis and mortality. Genistein blocks activation of p38 mitogen-activated protein kinase and thus inhibits matrix metalloproteinase-2 (MMP-2) expression and cell invasion in cultured cells and inhibits metastasis of human prostate cancer cells in mice. We investigated the target for genistein in prostate cancer cells. METHODS Prostate cell lines PC3-M, PC3, 1532NPTX, 1542NPTX, 1532CPTX, and 1542CPTX were used. All cell lines were transiently transfected with a constitutively active mitogen-activated protein kinase kinase 4 (MEK4) expression vector (to increase MEK4 expression), small interfering RNA against MEK4 (to decrease MEK4 expression), or corresponding control constructs. Cell invasion was assessed by a Boyden chamber assay. Gene expression was assessed by a quantitative reverse transcription-polymerase chain reaction. Protein expression was assessed by Western blot analysis. Modeller and AutoDock programs were used for modeling of the structure of MEK4 protein and ligand docking, respectively. MMP-2 transcript levels were assessed in normal prostate epithelial cells from 24 patients with prostate cancer from a phase II randomized trial comparing genistein treatment with no treatment. Statistical significance required a P value of .050 or less. All statistical tests were two-sided. RESULTS Overexpression of MEK4 increased MMP-2 expression and cell invasion in all six cell lines. Decreased MEK4 expression had the opposite effects. Modeling showed that genistein bound to the active site of MEK4. Genistein inhibited MEK4 kinase activity with a half maximal inhibitory concentration of 0.40 microM (95% confidence interval [CI] = 0.36 to 0.45 muM). The MMP-2 transcript level in normal prostate epithelial cells was statistically significantly higher in the untreated group (100%) than in the genistein-treated group (24%; difference = 76%, 95% CI = 38% to 115%; P = .045). CONCLUSIONS We identified MEK4 as a proinvasion protein in six human prostate cancer cell lines and the target for genistein. We showed, to our knowledge for the first time, that genistein treatment, compared with no treatment, was associated with decreased levels of MMP-2 transcripts in normal prostate cells from prostate cancer-containing tissue.

p38 MAP kinase modulates Smad-dependent changes in human prostate cell adhesion

Transforming growth factor beta (TGFβ) regulates cell adhesion, proliferation, and differentiation in a variety of cells. Smad proteins are receptor-activated transcription factors that translocate to the nucleus in response to TGFβ. We demonstrate here that TGFβ increases cell adhesion in metastatic PC3-M prostate cancer cells. TGFβ treatment of PC3-M cells leads to nuclear translocation of R-Smad proteins. We show that Smad proteins are necessary, but not sufficient, for TGFβ-mediated cell adhesion. After showing that TGFβ upregulated p38 MAP kinase activity in PC3-M cells, we show that inhibition of p38 MAP kinase partially blocked TGFβ-mediated increase in cell adhesion, as well as nuclear translocation of Smad3. Finally, we show that Smad3 is phosphorylated by p38 MAP kinase in vitro. These findings implicate crosstalk between the MAP kinase and Smad signaling pathways in TGFβs regulation of cell adhesion in human prostate cells. This represents a mechanism by which the pleiotropic effects of TGFβ may be channeled to modulate cell adhesion.

Endoglin suppresses human prostate cancer metastasis

Endoglin is a transmembrane receptor that suppresses human prostate cancer (PCa) cell invasion. Small molecule therapeutics now being tested in humans can activate endoglin signaling. It is not known whether endoglin can regulate metastatic behavior, PCa tumor growth, nor what signaling pathways are linked to these processes. This study sought to investigate the effect of endoglin on these parameters. We used a murine orthotopic model of human PCa metastasis, designed by us to measure effects at early steps in the metastatic cascade, and implanted PCa cells stably engineered to express differing levels of endoglin. We now extend this model to measure cancer cells circulating in the blood. Progressive endoglin loss led to progressive increases in the number of circulating PCa cells as well as to the formation of soft tissue metastases. Endoglin was known to suppress invasion by activating the Smad1 transcription factor. We now show that it selectively activates specific Smad1-responsive genes, including JUNB, STAT1, and SOX4. Increased tumor growth and increased Ki67 expression in tissue was seen only with complete endoglin loss. By showing that endoglin increased TGFβ-mediated suppression of cell growth in vitro and TGFβ-mediated signaling in tumor tissue, loss of this growth-suppressive pathway appears to be implicated at least in part for the increased size of endoglin-deficient tumors. Endoglin is shown for the first time to suppress cell movement out of primary tumor as well as the formation of distant metastasis. It is also shown to co-regulate tumor growth and metastatic behavior in human PCa.

Mitogen-activated protein kinase kinase 4 (MAP2K4) promotes human prostate cancer metastasis

Prostate cancer (PCa) is the second leading cause of cancer death in the US. Death from PCa primarily results from metastasis. Mitogen-activated protein kinase kinase 4 (MAP2K4) is overexpressed in invasive PCa lesions in humans, and can be inhibited by small molecule therapeutics that demonstrate favorable activity in phase II studies. However, MAP2K4s role in regulating metastatic behavior is controversial and unknown. To investigate, we engineered human PCa cell lines which overexpress either wild type or constitutive active MAP2K4. Orthotopic implantation into mice demonstrated MAP2K4 increases formation of distant metastasis. Constitutive active MAP2K4, though not wild type, increases tumor size and circulating tumor cells in the blood and bone marrow. Complementary in vitro studies establish stable MAP2K4 overexpression promotes cell invasion, but does not affect cell growth or migration. MAP2K4 overexpression increases the expression of heat shock protein 27 (HSP27) protein and protease production, with the largest effect upon matrix metalloproteinase 2 (MMP-2), both in vitro and in mouse tumor samples. Further, MAP2K4-mediated increases in cell invasion are dependent upon heat shock protein 27 (HSP27) and MMP-2, but not upon MAP2K4s immediate downstream targets, p38 MAPK or JNK. We demonstrate that MAP2K4 increases human PCa metastasis, and prolonged over expression induces long term changes in cell signaling pathways leading to independence from p38 MAPK and JNK. These findings provide a mechanistic explanation for human studies linking increases in HSP27 and MMP-2 to progression to metastatic disease. MAP2K4 is validated as an important therapeutic target for inhibiting human PCa metastasis.

Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII’s Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

Spectral biomarkers for chemoprevention of colonic neoplasia: a placebo-controlled double-blinded trial with aspirin

Objective A major impediment to translating chemoprevention to clinical practice has been lack of intermediate biomarkers. We previously reported that rectal interrogation with low-coherence enhanced backscattering spectroscopy (LEBS) detected microarchitectural manifestations of field carcinogenesis. We now wanted to ascertain if reversion of two LEBS markers spectral slope (SPEC) and fractal dimension (FRAC) could serve as a marker for chemopreventive efficacy. Design We conducted a multicentre, prospective, randomised, double-blind placebo-controlled, clinical trial in subjects with a history of colonic neoplasia who manifested altered SPEC/FRAC in histologically normal colonic mucosa. Subjects (n=79) were randomised to 325 mg aspirin or placebo. The primary endpoint changed in FRAC and SPEC spectral markers after 3 months. Mucosal levels of prostaglandin E2 (PGE2) and UDP-glucuronosyltransferase (UGT)1A6 genotypes were planned secondary endpoints. Results At 3 months, the aspirin group manifested alterations in SPEC (48.9%, p=0.055) and FRAC (55.4%, p=0.200) with the direction towards non-neoplastic status. As a measure of aspirins pharmacological efficacy, we assessed changes in rectal PGE2 levels and noted that it correlated with SPEC and FRAC alterations (R=−0.55, p=0.01 and R=0.57, p=0.009, respectively) whereas there was no significant correlation in placebo specimens. While UGT1A6 subgroup analysis did not achieve statistical significance, the changes in SPEC and FRAC to a less neoplastic direction occurred only in the variant consonant with epidemiological evidence of chemoprevention. Conclusions We provide the first proof of concept, albeit somewhat underpowered, that spectral markers reversion mirrors antineoplastic efficacy providing a potential modality for titration of agent type/dose to optimise chemopreventive strategies in clinical practice. Trial Number NCT00468910

Abstract 5331: The effect of heat shock protein 27 (HSP27) on prostate cancer cell adhesion, invasion and metastasis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Prostate cancer (PCa) is the most commonly diagnosed form of cancer among American men, and the second leading cause of all cancer-related deaths. PCa death is caused by the process of metastasis. In order to metastasize, tumor cells must acquire a series of phenotypic alterations, collectively known as the metastatic cascade. We have demonstrated that the small heat-shock protein, HSP27, affects key early steps of the metastatic cascade, and therefore may be a regulator of human PCa metastasis. HSP27 protein expression increases during PCa progression. It is phosphorylated in response to signaling from transforming growth factor-β (TGF-β), which in turn increases matrix metalloproteinase 2 (MMP-2) and cell invasion in vitro. We now demonstrate that HSP27 does not affect human PCa cell adhesion, cell detachment or cell migration, all of which contribute to the composite function of cell invasion. We went on to show that proteolysis by MMPs is necessary for HSP27-driven cell invasion by demonstrating that it was abrogated Marimastat, a broad-spectrum MMP inhibitor. By orthotopically implanting HSP27 variant human PCa cell lines, we demonstrated that HSP27 increases metastasis in a murine model. We also found that HSP27 increased tumor size. HSP27 increases human PCa metastasis and tumor growth in vivo. These findings provide a mechanistic explanation for the poor prognosis associated with increased HSP27 expression in human prostate tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5331. doi:1538-7445.AM2012-5331

Abstract 4752: Selective inhibition of cancer metastasis with a novel small therapeutic molecule

Prostate cancer (PCa) is the second most common cause of cancer death in US males. Death is typically caused by metastasis. Naturally occurring isoflavanones have been reported to be a class of compounds that effectively inhibit PCa motility and metastasis. This led us to use these compounds as a synthetic scaffold starting point. By integrating fragment-based diversification synthesis with chemi-driven biological selection, we discovered novel small molecule therapeutics with increased selectivity and potent efficacy. We thereby efficiently synthesized a new class of bioactive compounds that inhibit cell motility in vitro and inhibit human PCa metastasis in a murine model at low nanomolar concentrations. Extensive investigations indicate high specificity at the molecular and cellular levels, and failed to identify toxicity, even at high doses administered over extended periods. Importantly, efficacy against several cancer types was also demonstrated. Target validation studies used our lead as a chemical probe, and point to inhibition of ATM/ATR interaction with specific substrate proteins as important. Together, these studies indicate that we have successfully discovered a novel compound, acting upon a novel pharmacologic target, which selectively inhibits human PCa metastasis. Taken with our favorable preclinical toxicological data, these findings support movement of our lead compound into early phase human trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4752. doi:1538-7445.AM2012-4752

Abstract 3277: Increased mitogen-activated protein kinase kinase 4 (MEK4) induces metastasis in an orthotopic murine model of prostate cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mitogen-activated protein kinase kinase 4 (MEK4) is a dual-specificity kinase that has been implicated in prostate cancer (PCa) progression. Increased MEK4 expression is observed in invasive cancer lesions in human prostate tissue. We have demonstrated the clinical importance of this in vitro, as MEK4 increases human PCa invasion. This phenotype is driven by increased protease production, and not via changes in cell migration. However, it is not known whether MEK4 regulates human PCa metastasis. This is of high importance as MEK4 has shown differential effects in various cancer models in a cell type specific fashion, and may therefore represent an important human PCa specific target. To emulate the situation of sustained altered MEK4 expression in humans, we engineered MEK4 clonal variant cell lines, generating multiple clones each for vector control (VC), increased wild type (WT), and increased constitutively-active mutant MEK4 (CA). Using a murine orthotopic implantation model designed to characterize in vivo behavior, we implanted 75 mice with these clones and demonstrated that both WT and CA MEK4 increase human PCa metastasis to the lung, a clinically relevant site in humans. In the primary tumor, interestingly, WT tumors were not increased compared to VC, while CA tumors were. Additionally, increased MMP-2 and MMP-10 were observed in both WT and CA tumors. However, MMP-9 was increased in WT tumors only, and MMP-13 in CA tumors only. Currently, we are expanding our efforts at characterizing additional molecular changes in primary tumor between these groups. Outside of the primary tumor, we isolated circulating tumor cells in the blood and bone marrow. We are currently characterizing their relevant cellular and molecular parameters. Using a clinically relevant murine model we have shown that increased MEK4 increases human PCa metastasis to the lung and protease production within the primary tumor. Further, a constitutively active phenotype drives tumor growth and circulating tumor cell formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3277. doi:1538-7445.AM2012-3277