Janet M. Pavese

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.

Genistein inhibits human prostate cancer cell detachment, invasion, and metastasis

Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the United States and the second leading cause of cancer death. Death is not caused by the primary tumor but rather by the formation of distinct metastatic tumors. Therefore, prevention of metastasis is of utmost importance. The natural product genistein, found in high amounts in soy products, has been implicated in preventing PCa formation and metastasis in men who consume high amounts of soy. In vitro studies and in vivo rodent models that used human PCa cells, as well as prospective human clinical trials, provide a mechanistic explanation directly supporting genistein as an antimetastatic agent. Specifically, our group showed that genistein inhibits cell detachment, protease production, cell invasion, and human PCa metastasis at concentrations achieved in humans with dietary intake. Finally, phase I and phase II clinical trials conducted by us and others showed that concentrations of genistein associated with antimetastatic efficacy in preclinical models are achievable in humans, and treatment with genistein inhibits pathways that regulate metastatic transformation in human prostate tissue.

Circulating tumor cells exhibit a biologically aggressive cancer phenotype accompanied by selective resistance to chemotherapy

With prostate cancer (PCa), circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) portend a poor clinical prognosis. Their unknown biology precludes rational therapeutic design. We demonstrate that CTC and DTC cell lines, established from mice bearing human PCa orthotopic implants, exhibit increased cellular invasion in vitro, increased metastasis in mice, and express increased epithelial to mesenchymal transition biomarkers. Further, they are selectively resistant to growth inhibition by mitoxantrone-like agents. These findings demonstrate that CTC formation is accompanied by phenotypic progression without obligate reversion. Their increased metastatic potential, selective therapeutic resistance, and differential expression of potential therapeutic targets provide a rational basis to test further interventions.

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.

An orthotopic murine model of human prostate cancer metastasis.

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

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

Abstract C177: Chemical-driven biological probing: Discovery of a novel antimetastatic drug.

Chemicals constitute richly informative probes, have high resolving potential and are able to uncover complex biological processes. Harnessing this potential provides an avenue for the discovery of new therapeutics that act via novel mechanisms. We focused these principles upon an intractable problem: cancer metastasis. We started with a chemical scaffold with broad bioactivity, with desirable drug-like properties and that would support synthetic diversification. Initially focusing upon human prostate cancer (PCa) we then coupled fragment-diversification and novel synthetic routes to upfront positive selection screens (inhibition of cell motility) and negative selection screens (cell toxicity) in an iterative fashion. We thereby efficiently synthesized a new class of bioactive compounds that inhibits systemic PCa metastasis at low nanomolar concentrations. Efficacy against other cancer types was demonstrated. Extensive investigations indicate high specificity and no toxicity. Target validation studies point to inhibition of protein-protein interaction motifs. Together, these studies support the notion that this approach is powerful, can be broadly applied across biological systems, and constitutes a paradigm. Specifically, they have led to the discovery of a novel acting drug that inhibits human cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C177.

Abstract 1407: Mitogen-activated protein kinase kinase 4 (MEK4) is a key regulator of prostate cancer progression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Mitogen-activated protein kinase kinase 4 (MEK4) is a dual-specificity kinase that has been implicated in cancer progression in multiple cancer types, including prostate cancer (PCa). MEK4 is upregulated in invasive PCa lesions in human tissue. We hypothesize that increased MEK4 promotes PCa invasion and metastasis. Our group has created PC3-M cells stably transfected with either constitutively-active or increased levels of wild-type MEK4. In a Matrigel Boyden chamber assay, constitutively-active or wild-type MEK4 was shown to increase PCa invasion. Cancer cell invasion requires the coupling of cell migration with increased production of proteases. We hypothesized that MEK4 promoted invasion via production of proteases, but not via cell migration. Using an uncoated boyden chamber, MEK4 was shown not to affect migration. Using quantitative real time polymerase chain reaction (qRT/PCR), we went on to demonstrate that MEK4 increased MMP-2 and MMP-9 transcript expression, but did not affect MMP-10 expression. In addition to these studies, we have also looked at the effect of increased or constitutively-active MEK4 on an orthotopic mouse model of PCa. In this model, PCa cells are injected into the prostates of nude mice and tumor size, circulating tumor cells, and lung metastasis are quantified. Preliminary data shows that constitutively-active MEK4 causes an increase in primary tumor size as well as an increase in circulating tumor cells in both the blood and the bone marrow. In summary, we demonstrated that chronic high expression of MEK4 increases human PCa invasion, that this is not due to increased migration, but is associated with increases in MMP-2 and MMP-9. Further, constitutively-active MEK4 appears to have different biological effects than high levels of wild type MEK4 in vivo. Ongoing studies are evaluating the effect of wild type MEK4 and of constitutively-active MEK4 on the formation of distant soft tissue metastasis in the above murine model. In related studies we are seeking to identify downstream proteins critical for MEK4s effects on cell invasion and metastasis both in vitro and in vivo. We are also seeking to examine the effects of sustained MEK4 knockdown on PCa invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1407. doi:10.1158/1538-7445.AM2011-1407

Abstract B58: Discovery of a novel drug KBU2046 that inhibits conversion of human prostate cancer to a metastatic phenotype

Epidemiological evidence, preclinical studies and prospective phase II studies in humans indicate that the isoflavone, genistein, will inhibit the conversion of human prostate cells to an invasive, and ultimately, a metastatic phenotype. Though promising, genistein exerts many additional effects that have the potential for future toxicity in humans. We therefore sought to discover a new drug with improved efficacy and most importantly, with high specificity. Starting from an isoflavone chemical scaffold, we employed a fragment-based chemical synthesis diversification approach, and coupled it to three in vitro screens: 1) cell invasion (efficacy), 2) cell growth inhibition (an indicator of general toxicity), and 3) several measures of estrogenic activity. From multiple synthesis/biological assay iterations we developed a refined structure-activity relationship map, thereby leading us to discover KBU2046. KBU2046 represents a new and chemically distinct class of bioactive compounds. It has greater anti-invasion efficacy than genistein, and more importantly, no cell toxicity or estrogenic activity. Extensive toxicity studies in mice were negative. At low nanomolar blood concentrations, KBU2046 will prevent orthotopically implanted human prostate cancer cells from forming metastasis in a dose-responsive fashion. In summary, we have successfully discovered and developed a compound that prevents progression to a metastatic phenotype for human prostate cancer. We are in the process of bringing KBU2046 into the clinic, with the goal of preventing death from the second most common cause of cancer related death in men. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B58.