Manonmani Ganapathy

Akt/cAMP-Responsive Element Binding Protein/Cyclin D1 Network: A Novel Target for Prostate Cancer Inhibition in Transgenic Adenocarcinoma of Mouse Prostate Model Mediated by Nexrutine, a Phellodendron Amurense Bark Extract

Purpose: Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)–mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protect TRAMP mice from developing prostate cancer. Experimental Design: Eight-week-old TRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules. Results: We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylation-defective CREB inhibited cyclin D1 transcriptional activity. Conclusions: The current study shows that Nexrutine-mediated targeting of Akt/CREB–induced activation of cyclin D1 prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.

Involvement of FLIP in 2-Methoxyestradiol–Induced Tumor Regression in Transgenic Adenocarcinoma of Mouse Prostate Model

Purpose: The purpose of this study is to investigate whether Fas-associated death domain interleukin-1 converting enzyme like inhibitory protein (FLIP) inhibition is a therapeutic target associated with 2-methoxyestradiol (2-ME2)–mediated tumor regression. Experimental Design: Expression and levels of FLIP were analyzed using (a) real-time PCR and immunoblot analysis in androgen-independent PC-3 cells treated with the newly formulated 2-ME2 and (b) immunohistochemistry in different Gleason pattern human prostate tumors. Transient transfections and chromatin immunoprecipitation (ChIP) assays were used to identify the transcription factors that regulate FLIP. Involvement of FLIP in 2-ME2–induced tumor regression was evaluated in transgenic adenocarcinoma mouse prostate (TRAMP) mice. Results: High Gleason pattern (5+5) human prostate tumors exhibit significant increase in FLIP compared with low Gleason pattern 3+3 (P = <0.04). 2-ME2 reduced the levels and promoter activity of FLIP (P = 0.001) in PC-3 cells. Transient expression assays show sequences between −503/+242 being sufficient for 2-ME2–induced inhibition of FLIP promoter activity. Cotransfection experiments show that overexpression of Sp1 activated, whereas Sp3 inhibited, Sp1 transactivation of FLIP promoter activity (P = 0.0001). 2-ME2 treatment reduced binding of Sp1 to the FLIP promoter as evidenced by ChIP. Further, levels of FLIP associated with Fas or FADD decreased, whereas cleavage of caspase-8, levels of Bid, and apoptosis increased in response to 2-ME2 treatment in PC-3 cells. Administration of 2-ME2 regressed established prostate tumors in TRAMP mice that were associated with reduced expression of FLIP and Sp1. Conclusion: Targeting Sp1-mediated FLIP signaling pathway may provide a novel approach for prostate cancer management.

Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells

Lack of effective treatment options for the management of hormone refractory prostate cancer (PCA) reinforce the great need to develop novel compounds that act singly or in combination. 2-Methoxyestradiol (2-ME(2)) is an endogenous estrogenic metabolite that has been reported to work as an antiproliferative agent in various tumor models including prostate. Recently conducted clinical trial in hormone refractory prostate cancer (HRPC) patients concluded that 2-ME(2) was safe and well tolerated. However this study identified bioavailability of 2-ME(2) as a limiting factor. Here we report the ability of a combination of 2-ME(2) and eugenol (4-allyl-2-methoxyphenol) as an approach for enhancing anticancerous activities in prostate cancer cells. Combining 2-ME(2) with eugenol (i) inhibited growth of prostate cancer cells and induced apoptosis at lower concentrations than either single agent alone; (ii) analysis of the data using combination index (CI) showed CI values of 0.4 indicating strong synergistic interaction; (iii) increased population of cells G(2)/M phase by 4.5-fold (p=0.01); (iv) significantly reduced expression of antiapoptotic protein Bcl-2 and enhanced expression of proapoptotic protein Bax. Combination induced apoptosis was not affected in PC-3 cells that over-express or lack Bcl-2 but was associated with loss of mitochondrial membrane potential. Since 2-ME(2) was well tolerated in phase II trail in patients with HRPC; and eugenol is consumed by humans in the form of spices, the combination of 2-ME(2) with eugenol may offer a new clinically relevant treatment regimen. Combining these agents may allow ameliorating any adverse effects of either 2-ME(2) or eugenol alone by reducing their individual concentrations should these two agents be developed for human use.

Estrogen receptor-β activation in combination with letrozole blocks the growth of breast cancer tumors resistant to letrozole therapy

Treatment with anti-estrogens or aromatase inhibitors (AI) is the main therapeutic strategy used against estrogen receptor ERα-positive breast cancer. Resistance to these therapies presents a major challenge in the management of breast cancer. Little is known about ERβ in breast carcinogenesis. Our aim in this study is to examine potential novel strategies utilizing ERβ activity to overcome AI resistance. We provide evidence that ERβ agonist can reduce the growth of AI-resistant breast cancer cells. Our data further confirm that therapeutic activation of ERβ by DPN, an ERβ agonist, blocks letrozole-resistant tumor growth in a xenograft model. Interestingly, DPN exerted tumor growth inhibition only in the presence of the AI letrozole, suggesting that combination therapy including ERβ activators and AI may be used in the clinical setting treating AI resistant breast cancer. An increase in ERβ levels, with diminished ERα/ERβ ratio, was observed in the tumors from mice treated with DPN/letrozole combination compared to single agents and control. Decreased Cyclin D1 and increased CyclinD1/CDK inhibitors p21 and p27 levels in DPN/letrozole treated tumors were observed, suggesting that the combination treatment may inhibit tumor growth by blocking G1/S phase cell cycle progression. Our data show a decrease in MAPK phosphorylation levels without affecting total levels. In addition to providing evidence suggesting the potential use of ERβ agonists in combination with letrozole in treating AI resistant breast cancer and prolonging sensitivity to AI, we also provide mechanistic evidence supporting the role of ERβ in altering the expression profile associated with resistance.

Estrogen Receptor-beta Mediates the Protective Effects of Aromatase Induction in the MMTV-Her-2/neu x Aromatase Double Transgenic Mice

Breast cancers amplified for the tyrosine kinase receptor Her-2/neu constitute ~30% of advanced breast cancer cases, and are characterized by hormone independence and aggressive growth, implicating this pathway in breast oncogenesis. The induction of Her-2/neu leads to tumor development in 60% of transgenic mice. We have previously examined the effects of estrogen in the MMTV-Her-2/neu background by generating the MMTV-Her-2/neu x aromatase double transgenic mouse strain. MMTV-Her-2/neu x aromatase mice developed fewer mammary tumors than the Her-2/neu parental strain. Our present data show the induction of several estrogen-related genes, including the tumor suppressors BRCA1 and p53, and a decrease in several angiogenic factors. The phosphorylated forms of MAPK p42/44 and AKT were lower in the MMTV-Her-2/neu x aromatase double transgenic mice compared to the MMTV-Her-2/neu parental strain; conversely, phospho-p38 levels were higher in the double transgenic strain. The ERβ-selective antagonist THC reversed these changes. The regulation of these factors by ERβ was confirmed in clones of MCF7 breast cancer cells overexpressing Her-2/neu in combination with ERβ, suggesting that ERβ may play a direct role in regulating MAPK and AKT pathways. In summary, the data suggest that ERβ may play a major role in decreasing tumorigenesis and that it may affect breast cancer cell proliferation and survival by altering MAPK and AKT activation as well as modulation of tumor suppressor and angiogenesis factors. Treatment with selective ERβ agonist may provide therapeutic advantages for the treatment and prevention of breast cancer.

Raf-1, a potential therapeutic target, mediates early steps in endometriosis lesion development by endometrial epithelial and stromal cells.

Endometriosis is a hormone-sensitive gynecological disorder characterized by the benign growth of endometrial-like tissue in the pelvic cavity. Endometriotic lesions composed of endometrial stromal cells (ESC) and glandular epithelial cells (EEC) are thought to arise from menstrual endometrial tissue reaching the pelvic cavity via retrograde menstruation. The cause of endometriotic lesion formation is still not clear. Recent evidence suggest that cytokines may play a role in the early development of endometriosis lesions. Because cytokines and growth factors signal via the v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase pathway, we have examined the role of Raf-1 in early steps of endometriosis lesion formation, specifically attachment of endometrial cells to peritoneal mesothelial cells (PMC) and invasion of endometrial cells through PMC (trans-mesothelial invasion). Raf-1 antagonist GW5074 decreased attachment to PMC and trans-mesothelial invasion by primary EEC and ESC. Raf-1 also mediated TGFβ-induced trans-mesothelial invasion by the established, low-invasive EEC line EM42. TGFβ treatment of EEC resulted in Raf-1 phosphorylation at S338 and phosphorylation of ERK, suggesting that TGFβ activates Raf-1 signaling in these cells. GW5074 had little effect on ESC proliferation but inhibited EEC growth significantly under reduced serum conditions. Antagonizing Raf-1 activity and expression via GW5074 and specific Raf-1 small interfering RNA, respectively, did not alter EEC resistance to growth inhibition by TGFβ. Raf-1 inhibition blocked induction of EEC growth by epidermal growth factor. Our data suggest that Raf-1 may mediate pathologic steps involved in early endometriosis lesion formation and may be a mediator of TGFβ and epidermal growth factor actions in endometriosis.

Abstract 5753: Potential therapeutic use of ER beta modulators in treating endocrine therapy resistant breast cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Although systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) such as letrozole or block actions of estrogen/ER actions by anti-estrogens (AEs) like tamoxifen are well tolerated, unfortunately, essentially all breast cancers in women with advanced breast cancer develop resistance to these agents, and the benefit of adjuvant therapy is also limited by the development of resistance. In addition, side effects associated with the systemic inhibition of aromatase with current compounds limits their long-term utility of them as chemopreventative agents. Using in vitro and preclinical postmenopausal breast cancer models, we have investigated whether treatment of endocrine therapy resistant (AI or AE) breast cancer cells with selective plant-derived ER beta agonists (Liquiritigenin; LIQ or Nyasol; NYA) alone or in combination with AE or AI affects the growth of resistant breast cancer cells, thereby restores sensitivity to AE or AI. To test whether combination therapy restores sensitivity to letrozole or tamoxifen, we have treated resistant breast cancer cells with either letrozole or tamoxifen alone or with a combination of AE or AI and LIQ/NYA, active ingredient of MF101 used for treatment hot flashes in postmenopausal women. In addition, an effect of these compounds on cell growth was also tested using different breast cancer cells with and without ERα including triple negative breast cancer cells. Efficacy of these drugs was also tested in these model cells with or without ERβ expression. Compared to single agents, combination treatment not only restored sensitivity to letrozole but also resulted in decreased cell proliferation and increased apoptosis as well as increased ERβ and decreased ERα levels in resistant cells. Combination of LIQ or NYA not only diminished cell growth various breast cancer cells (that express endogenous aromatase) but also affected the expression of aromatase. Detailed investigations indicated that LIQ/NYA affects the induction of aromatase by specifically inhibiting breast cancer specific aromatase promoter 1.3 as well as its activity through inflammatory cytokine-mediated actions. ERβ-mediated inhibition of tumor cell growth appears to be regulated by different molecular mechanisms. Some of these molecular pathways are different than that are known to be responsible for resistance to hormonal therapeutic agents. These studies suggest the therapeutic benefit of LIQ or NYA ERβ agonist to resensitize breast cancer cells that are resistant to AEs or AI and their ability to selectively inhibit tumor induction of aromatase. Our study suggests a novel role of natural ER beta agonists as valuable therapeutic modulators to treat endocrine sensitive as well as resistant tumors. 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 5753. doi:1538-7445.AM2012-5753

Abstract 2297: ERβ agonists reduce letrozole resistant breast cancer cell growth and diminish aromatase expression and activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Aromatase inhibitors (AI) have shown efficacy against estrogen receptor (ER) positive breast cancer; however, development of resistance to AI is a major concern. The need to develop therapeutic agents that overcome AI resistance is urgent. We investigated whether synthetic and natural selective ERβ agonists restored sensitivity to AI and anti-estrogens in hormone resistant breast cancer cells. Our data suggest that ERβ agonists such as DPN (a synthetic compound) and plant derived Nyasol, Liquiritigenin, and MF101 (Menerba) ERβ agonists may restore sensitivity against resistance by the current drugs. Natural ERβ agonists in combination with AI reduced cell proliferation in MCF7/ARO (overexpressing aromatase), MCF7/HER2 (overexpressing Her2/neu) LTLT-Ca (letrozole resistant), MCF7/TAM (tamoxifen resistant), SKBR3, and T47D breast cancer cells by 70 % to 90% when compared with DPN/AI combination. Our Western blot and RT-PCR analyses showed that combination treatment with natural ERβ agonist in combination with AI resulted in decreased levels of ERα, Cyclin D1, TNF-α, IL-6 and increased ERβ levels in LTLT-Ca and MCF7/ARO cells. In addition, aromatase expression and activity were decreased due to ERβ agonists/AI combination treatment. The activity of aromatase promoter 1.3, which is activated in breast cancer, was found to be down regulated with Nyasol, Liquiritigenin and MF101 combinations with AI when compared to single compounds in various breast cancer cells. Decreased promoter 1.3 activity is consistent with the combination-treatment decrease in the expression of inflammatory cytokines known to induce this promoter (e.g., IL-6). Our data suggest that inhibition of aromatase expression/activity by natural ERβ agonists (in combination with AI) may be a factor in overcoming AI-resistant breast cancer cell growth by these drugs. Our data also suggest that modulation of inflammatory cytokines may be factors by which ERβ agonists may influence aromatase expression and sensitizing AI-resistant breast cancer to AI therapy. Further work is required to understand the mechanistic aspects of this regulation and to advance natural ERβ agonists to the clinical treatment of AI-resistant breast cancer. 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 2297. doi:10.1158/1538-7445.AM2011-2297

Abstract 1131: Differential induction of c-fms promoters by TGFβ in cervical cancer and macrophage cell lines

Cervical cancer is the second most common gynecological malignancy in the world. An expected 12,200 total new diagnosed cases were estimated by the American Cancer Society for 2010 (Cancer Facts and Figures, 2010). Human papillomavirus infection, a major cause for cervical cancer, results in a persistent and pathological inflammatory response, contributing to establishment of disease and malignant progression. Our previous studies have shown that inflammatory factors, like Cox2, tumor associated macrophages, and cytokines such as macrophage colony-stimulating factor (CSF-1) and transforming growth factor beta (TGFβ) are elevated in cervical carcinomas. The receptors for CSF-1 (encoded by the c-fms oncogene) and TGFβ were also elevated in cervical carcinomas compared to normal cervical tissue. Both CSF-1 and c-fms have been implicated in aggressive gynecological malignancies, including cervical cancer. CSF-1/c-fms signaling is involved in proliferation, differentiation and recruitment of macrophages as well as cancer cell growth. We have previously demonstrated that TGFβ induced the expression of c-fms mRNA in cervical cancer cell lines and macrophage cell line THP-1. Using flow cytometry in this study, we have confirmed induction of c-fms protein at the cell surface due to TGFβ treatment, suggesting that TGFβ may enhance CSF-1 signaling by inducing cell surface expression of its receptor. Our goal was to examine the mechanisms underlying the regulation of c-fms in macrophages, relevant leukocytes associated with inflammation and pro-malignant growth within the tumor microenvironment. Two promoters for the c-fms gene exist, promoter one driving expression in trophoblasts (trophoblast specific promoter) and promoter two driving expression in macrophages (macrophage specific promoter). Consistent with our previous findings, TGFβ induced c-fms expression via MAPK p38 mainly at the trophoblast specific promoter in cervical cancer cells, as shown by the use of the p38 inhibitor SB203580. Our data also suggest that Casein kinase 1 mediates c-fms expression via this promoter in cervical cancer cells. In the macrophage cell line THP-1, p38 mediated the expression of c-fms via both promoters, as demonstrated by using exon specific RT-PCR primer sets. The c-fms mRNA induced at the macrophage specific promoter is the major c-fms transcript in THP-1 cells and is likely to mainly contribute to c-fms expression in these cells. Understanding the regulation of c-fms in macrophages and cervical cancer cells may pave the way to targeting c-fms expression to interrupt aberrant CSF-1 signaling in cervical cancer. 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 1131. doi:10.1158/1538-7445.AM2011-1131

Abstract 4607: Novel role of PARP4 in hormone-dependent breast cancer progression and metastasis

Estrogen/estrogen receptor-mediated actions play an important role in regulating a variety of biological process including development, homeostasis and breast cancer progression. Even though systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) or block actions of estrogen/estrogen receptor (ER) by antiestrogens (AE) is well tolerated, the development of resistance to these agents is still a major concern. Since multiple signaling pathways involved in ER activation, combination of endocrine and non endocrine agents that block these signaling pathways may delay the onset of resistance to hormonal therapy. Recent studies from our laboratory have identified Poly(ADP-ribose) polymerase 4(PARP4) as a novel ER co-regulator. We have investigated with hormone resistant breast cancer cells to demonstrate whether blocking of PARP4 results in resensitization to hormone therapies. PARP comprise of a family of enzymes which catalyses poly(ADPribosyl) action of DNA-binding proteins. PARP-1, the best characterized member, however PARP4 is an understudied protein with numerous capabilities. PARP4 has both N-terminal PXXP and PPXXP motifs which are known to interact with SH3 domain of c-Src as well as LXXLL motif which interacts with estrogen receptor (ER). We show that PARP4 is widely expressed in breast cancer cells (MCF-7, MDA-MB-231, ZR-75, and SKBR3) and its expression is significantly higher in these cells compared to non malignant MCF-10A cells. To understand the important role of PARP4-ER axis in hormonal resistance, metastasis and to test whether blocking of PARP4 mediated actions provide a therapeutic advantage to treat endocrine resistant breast tumors, we have used hormone resistant breast cancer cells that over-express PARP4 or and treated with PARP4 siRNA nanoparticles. The PLGA-siRNA nanoparticles were designed multifunctionally using chemical conjugation. The nanoparticles were characterized physiochemically. Our studies show that PARP4 functionally interacts with ER. As demonstrated by immunoprecipitation, PARP4 interacts with ER upon ligand (E2)-mediated stimulation. PARP4 induces cell cycle progression, local aromatase activity and promotes in vitro tumorigenic potential of breast cancer cells. siRNA nanoparticles of PARP4 inhibit the metastatic incidence in vivo. Our study also shows that PARP4-ER axis enhances multiple signaling cross-talk leading to hormonal independence/resistance and metastasis. Our results suggests that targeting PARP4 may provide a therapeutic advantage to treat endocrine resistant tumors and blocking the development of resistance to hormonal therapeutic agents and as well as decrease the metastatic potential in therapy resistant breast tumors. (Supported by NIH/NCI P30 CA 54174, RRT) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4607.