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Supplementary Movie S2 from Critical Role of Endogenous Heme Oxygenase 1 as a Tuner of the Invasive Potential of Prostate Cancer Cells

Supplementary Movie S1 from Critical Role of Endogenous Heme Oxygenase 1 as a Tuner of the Invasive Potential of Prostate Cancer Cells

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Inflammation has been recognized as a risk factor for this disease. Heme oxygenase 1 (HO-1), the inducible isoform of the rate-limiting enzyme in heme degradation, counteracts oxidative and inflammatory damage. Here, we investigated the regulated expression of HO-1 and its functional consequences in PCa. We studied the effect of genetic and pharmacologic disruption of HO-1 in the growth, invasion, and migration in androgen-sensitive (MDA PCa2b and LNCaP) and androgen-insensitive (PC3) PCa cell lines. Our results show that HO-1 levels are markedly decreased in PC3 compared with MDA PCa2b and LNCaP. Hemin treatment increased HO-1 at both protein and mRNA levels in all cell lines and decreased cell proliferation and invasion. Furthermore, overexpression of HO-1 in PC3 resulted in markedly reduced cell proliferation and migration. Accordingly, small interfering RNA–mediated silencing of HO-1 expression in MDA PCa2b cells resulted in increased proliferation and invasion. Using reverse transcription-quantitative PCR–generated gene array, a set of inflammatory and angiogenic genes were upregulated or downregulated in response to HO-1 overexpression identifying matrix metalloprotease 9 (MMP9) as a novel downstream target of HO-1. MMP9 production and activity was downregulated by HO-1 overexpression. Furthermore, PC3 cells stably transfected with HO-1 (PC3HO-1) and controls were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth and MMP9 expression was significantly reduced in PC3HO-1 tumors compared with control xenografts. Taken together, these results implicate HO-1 in PCa cell migration and proliferation suggesting its potential role as a therapeutic target in clinical settings. (Mol Cancer Res 2009;7(11):1745–55)

Supplementary Data from Critical Role of Endogenous Heme Oxygenase 1 as a Tuner of the Invasive Potential of Prostate Cancer Cells

PDF file - 556K, Table S1: Genesets enriched in PC3.pGIPZ xenografts. Table S2: Genesets enriched in PC3.shCtBP1 xenografts. Supplemental Figure 1: (A) Crystal violet stained NIH3T3 cells foci transfected with pcDNA3.CtBP1 or beta-gal expression vectors. (B) CtBP1 RT-qPCR from PC3.CtBP1, PC3.shCtBP1 and PC3.pGIPZ stable cells using specific CtBP1 and Actin beta primers. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation of one representative experiment.***p < 0.001. Supplemental Figure 2: Box plots for (A) triglycerides, (B) glycemia and (C) estradiol serum levels for CD or HFD fed mice were shown. Boxes represent the interquartile range, the horizontal line within each box represents the median, and the upper and lower whiskers represent the standard deviation of one independent experiment. (D-E) Representative photograph of H&E staining from kidney and liver of mice fed with CD or HFD (Magnification 400x & 250x respectively). Supplemental Figure 3: (A) Testosterone and (B) estradiol levels from CD or HFD xenografts mice. Plotted boxes represent the interquartile range, the horizontal line within each of the boxes represents the median, and the upper and lower whiskers represent the standard deviation. * p < 0.05 (C) Specific CtBP1 RT-qPCR from tumors of CD or HFD fed mice. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 independent mice. * p < 0.05. Supplemental Figure 4: (A) GSEA from microarray data from HFD tumor xenografts. enrichment plots from genesets enriched in PC3.shCtBP1 xenografts. We showed the most significant over-representive cell adhesion categories selected from GSEA analysis from microarray data. (B) CDH1 RT-qPCR from the HFD fed mice tumors was performed. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 mice. * p < 0.05 (C) GSEA from microarray data from HFD tumor xenografts. The most significant enrichment plots for hormone pathways genesets in PC3.shCtBP1 xenografts were shown. Supplemental Figure 5: (A) Enrichment plots from genesets enriched in PC3.pGIPZ xenografts. Enrichment plots for the most significant over-representive categories selected from GSEA analysis from microarray data were shown. Listed above we found enrichment plots associated to cell cycle regulation processes and proliferation. Supplemental Figure 6: (A-B) BRCA1 RT-qPCR from (A) 22Rv1 or (B) LNCaP cells grew in charcoal FBS-medium and exposed to testosterone or vehicle for 24h. Fold induction was calculated normalizing data to Actin beta and control. (C-D) BRCA1 RT-qPCR from (C) 22Rv1 or (D) LNCaP cells, pre-treated for an hour with letrozole, were then grown as indicated before. Fold induction was calculated normalizing data to Actin beta and control. Data represents the average and standard deviation of one representative experiment. * p < 0.05.

Purpose: Clinical and epidemiologic data suggest that obesity is associated with more aggressive forms of prostate cancer, poor prognosis, and increased mortality. C-terminal–binding protein 1 (CtBP1) is a transcription repressor of tumor suppressor genes and is activated by NADH binding. High calorie intake decreases intracellular NAD+/NADH ratio. The aim of this work was to assess the effect of high-fat diet (HFD) and CtBP1 expression modulation over prostate xenograft growth.Experimental Design: We developed a metabolic syndrome-like disease in vivo model by feeding male nude mice with HFD during 16 weeks. Control diet (CD)–fed animals were maintained at the same conditions. Mice were inoculated with PC3 cells stable transfected with shCtBP1 or control plasmids. Genome-wide expression profiles and Gene Set Enrichment Analysis (GSEA) were performed from PC3.shCtBP1 versus PC3.pGIPZ HFD-fed mice tumors.Results: No significant differences were observed in tumor growth on CD-fed mice; however, we found that only 60% of HFD-fed mice inoculated with CtBP1-depleted cells developed a tumor. Moreover these tumors were significantly smaller than those generated by PC3.pGIPZ control xenografts. We found 823 genes differentially expressed in shCtBP1 tumors from HFD-fed mice. GSEA from expression dataset showed that most of these genes correspond to cell adhesion, metabolic process, and cell cycle.Conclusions: Metabolic syndrome–like diseases and CtBP1 expression cooperate to induce prostate tumor growth. Hence, targeting of CtBP1 expression might be considered for prostate cancer management and therapy in the subset of patients with metabolic syndromes. Clin Cancer Res; 20(15); 4086–95. ©2014 AACR.

Table S1: Genesets enriched in PC3.pGIPZ xenografts. Table S2: Genesets enriched in PC3.shCtBP1 xenografts. Supplemental Figure 1: (A) Crystal violet stained NIH3T3 cells foci transfected with pcDNA3.CtBP1 or beta-gal expression vectors. (B) CtBP1 RT-qPCR from PC3.CtBP1, PC3.shCtBP1 and PC3.pGIPZ stable cells using specific CtBP1 and Actin beta primers. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation of one representative experiment.***p < 0.001. Supplemental Figure 2: Box plots for (A) triglycerides, (B) glycemia and (C) estradiol serum levels for CD or HFD fed mice were shown. Boxes represent the interquartile range, the horizontal line within each box represents the median, and the upper and lower whiskers represent the standard deviation of one independent experiment. (D-E) Representative photograph of H&E staining from kidney and liver of mice fed with CD or HFD (Magnification 400x & 250x respectively). Supplemental Figure 3: (A) Testosterone and (B) estradiol levels from CD or HFD xenografts mice. Plotted boxes represent the interquartile range, the horizontal line within each of the boxes represents the median, and the upper and lower whiskers represent the standard deviation. * p < 0.05 (C) Specific CtBP1 RT-qPCR from tumors of CD or HFD fed mice. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 independent mice. * p < 0.05. Supplemental Figure 4: (A) GSEA from microarray data from HFD tumor xenografts. enrichment plots from genesets enriched in PC3.shCtBP1 xenografts. We showed the most significant over-representive cell adhesion categories selected from GSEA analysis from microarray data. (B) CDH1 RT-qPCR from the HFD fed mice tumors was performed. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 mice. * p < 0.05 (C) GSEA from microarray data from HFD tumor xenografts. The most significant enrichment plots for hormone pathways genesets in PC3.shCtBP1 xenografts were shown. Supplemental Figure 5: (A) Enrichment plots from genesets enriched in PC3.pGIPZ xenografts. Enrichment plots for the most significant over-representive categories selected from GSEA analysis from microarray data were shown. Listed above we found enrichment plots associated to cell cycle regulation processes and proliferation. Supplemental Figure 6: (A-B) BRCA1 RT-qPCR from (A) 22Rv1 or (B) LNCaP cells grew in charcoal FBS-medium and exposed to testosterone or vehicle for 24h. Fold induction was calculated normalizing data to Actin beta and control. (C-D) BRCA1 RT-qPCR from (C) 22Rv1 or (D) LNCaP cells, pre-treated for an hour with letrozole, were then grown as indicated before. Fold induction was calculated normalizing data to Actin beta and control. Data represents the average and standard deviation of one representative experiment. * p < 0.05.

Loss of normal development is a hallmark of cancer. Thus, understanding the mechanisms of tissue-specific developmental regulation and the changes that occur during tumorigenesis may provide insights of both diagnostic and therapeutic importance. In breast cancer, several members of the receptor tyrosine kinases (RTK) family that are well known to promote aggressive breast cancers also have roles in normal breast. We found that Ret, a RTK member, is normally expressed in the mouse glands in lactation. We determined that inhibition of Ret activity in vivo does not alter lactation, however impacts in the transition to involution. Involution is the period with high inflammation which returns the lactating mammary gland to a quiescent state after weaning. Involution has been well described as a post-lactation stage that drives cancer progression. Ret is overexpressed in about 40% of human breast tumors. Previously, using a doxycycline-inducible transgenic mouse model (Ret/MTB) we determined that chronic expression of Ret is oncogenic in the mammary epithelium. However, the stage of development at which Ret expression results in increase mammary tumor incidence has not been identified. To address this, we used the Ret/MTB system, to conditionally overexpress Ret during discrete periods of mammary gland development. We found that Ret is required for efficient transition to involution. We determined that the induction of Ret in Ret/MTB females promotes the expression of factors that drives involution, including premature Stat3 activation. RNA-seq data in Ret-overexpressing glands is supporting these findings, which were confirmed by several techniques. In addition, sustained expression of Ret during post-lactation enhances cancer potential showing a significant increase in pre-neoplastic lesions, defective milk recycling and disrupting Stat3 signaling. These results demonstrate that Ret deregulation increases cancer potential in post-lactation and might be considered as a prognostic marker for post-partum breast cancer. Citation Format: Sabrina A. Vallone, Martín García Solá, Robert D. Cardiff, Roberto P. Meiss, Lewis A. Chodosh, Carolina Shere-Levy, Edith C C. Kordon, Nancy E. Hynes, Albana Gattelli. Sustained Ret expression during mammary gland post-lactation induces premature involution and enhances cancer potential [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3685.

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although previous studies indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In a recently published study, we identified this factor as meta-tyrosine and ortho-tyrosine, 2 isomers of tyrosine that would not be present in normal proteins. In 3 different murine models of cancer that generate CR, both meta- and orthotyrosine inhibited tumor growth. Additionally, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isomers were mediated in part by early inhibition of the MAP/ERK pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy in G 0-phase. Other mechanisms, putatively involving the activation of an intra-S-phase checkpoint, would also inhibit tumor proliferation by accumulating cells in S-phase. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors or after other stressors that may promote the escape of metastases from dormancy, an issue that is of pivotal importance to oncologists and their patients. ©2012 AACR. Fil: Ruggiero, Raul Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bruzzo Iraola, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Chiarella, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bustuoabad, Oscar David. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Meiss, Roberto P.. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Pasqualini, Christiane D.. Academia Nacional de Medicina de Buenos Aires; Argentina

Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients. ©2011 AACR. Fil: Ruggiero, Raul Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Bruzzo Iraola, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Chiarella, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Di Gianni, Pedro. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Isturiz, Martín Amadeo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Linskens, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Speziale, Norma. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Meiss, Roberto P.. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Bustuoabad, Oscar David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Pasqualini, Christiane D.. Academia Nacional de Medicina de Buenos Aires; Argentina

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy. Citation Format: Nicolás Anselmino, Paula Chiarella, Emiliano G. Ortiz, Daiana B. Leonardi, Felipe M. Jaworski, Sofia Lage Vikers, Jimena Giudice, Anna Woloszynska-Read, Roberto P. Meiss, Raul Rugiero, Geraldine Gueron, Elba S. Vazquez. Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4180.

Mouse mammary tumor virus (LA) induces pregnancy-dependent mammary tumors that progress toward autonomy. Here we show that in virgin females, pregnancy-dependent tumor transplants are able to remain dormant for up to 300 days. During that period, these tumors synthesize DNA, express high levels of estrogen and progesterone receptors (ER+PR+) and are able to resume growth after hormone stimulation. Surprisingly, in a subsequent transplant generation, all these tumors are fully able to grow in virgin females, they express low levels of ER and PR (ER−PR−) and have a monoclonal origin; i.e., show all of the features we have described previously in pregnancy-independent tumors. Histologically, mouse mammary tumor virus (LA)-induced tumors are morphologically similar to genetically engineered mouse (GEM) mammary tumors that overexpress genes belonging to the Wnt pathway. Interestingly, in the virus-induced neoplasias, pregnancy-independent passages arising after a dormant phase usually display a lower level of glandular differentiation together with epithelial cell trans-differentiation, a specific feature associated to Wnt pathway activation. In addition, dormancy can lead to the specific selection of Int2/Fgf3 mutated and overexpressing cells. Therefore, our results indicate that during hormone-dependent tumor dormancy, relevant changes in cell population occur, allowing rapid progression after changes in the animal internal milieu.

Clinical and experimental evidence suggest that primary tumors may exert a controlling action on its metastases. The phenomenon, by which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis, is known as concomitant tumor resistance (CR). We have previously showed in murine T-lymphoma (LB) tumors, that meta-tyrosine (m-Tyr) an isomer of tyrosine not present in normal proteins, is the main serum anti-tumoral factor responsible for CR. In this work, we assess for the first time the CR phenomenon in human prostate cancer (PCa). Athymic nude mice were inoculated with PC3 cells (primary implant) and after 14 days the animals received a second inoculation (secondary implant). Strikingly, the growth of the secondary implant was significantly reduced after 27 days, in animals carrying the primary xenograft. When phenylalanine (Phe), a protective amino acid highly present in primary tumors, and precursor of p-tyrosine, was periodically inoculated at the site of a secondary tumor implant (otherwise inhibited by CR), this secondary implant grew similarly to controls. On the contrary, when m-Tyr was inoculated at the site of a primary tumor implant or systemically, this implant did not grow. Tumor inhibition was associated with low expression of Ki-67 and STAT3. In vitro analyses demonstrate the higher inhibitory activity of the serum from tumor-bearing mice on PC3 cell proliferation, compared to serum from control animals. m-Tyr could account for most of the growth-inhibitory activity present in the serum. Furthermore, we observed an increase in the frequency of Gr1+ CD11b+ MDSCs in bone marrow, spleen and lymph nodes from tumor-bearing mice compared to control mice. This expansion correlated with a significantly higher production of reactive oxygen species and enhanced suppressor function upon CD8+ T cell proliferation. Further, in vitro studies also showed that exposure of PC3 cells to m-Tyr inhibited cell growth, induced G0/G1 cell cycle arrest, altered the expression levels of survivin, Ki67 and Hes1; impaired the NFκB/STAT3 pathway and induced autophagy; effects reversed by Phe treatment. Strikingly, m-Tyr periodic intravenous administration provoked a dramatic reduction of experimental lung metastases generated in mice bearing PC3 human tumors. Altogether, we demonstrate for the first time that RC occurs in experimental human solid tumors, that this effect is mediated by m-Tyr, a non-cytotoxic metabolite with high potential clinical implications for metastatic PCa. Citation Format: Geraldine Gueron, Nicolás Anselmino, Paula Chiarella, Emiliano Ortiz, Alejandra Paez, Jimena Giudice, Federico Schuster, Daiana Leonardi, Felipe Jaworski, Estefania Labanca, Verónica Manzano, Javier Cotignola, Roberto Meiss, Norma D′Accorso, Nora Navone, Raul Ruggiero, Elba Vazquez. Clinical implications for m-tyrosine, an isomer of p-tyrosine, for the treatment of aggressive prostate tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4717.

Concomitant tumor resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of secondary tumor implants. Ehrlich first described it in 1906, but this phenomenon remained forgotten for about 60 years. After its renascence, some groups have demonstrated that both immunogenic and non-immunogenic tumors can induce CR in different animal models. Metastases could be considered as secondary tumor implants developed spontaneously during the primary tumor growth, thus CR could be relevant for cancer progression. Clinical and experimental evidence suggest that the removal of human and murine tumors might be followed by an abrupt increase in metastatic growth, hence the primary tumor could exert a controlling action on its metastases. In previous papers we demonstrated that, in mice, two temporally separate peaks of CR can be detected during murine T-lymphoma (LB) primary tumor growth. The second peak of CR is mediated by most large-sized immunogenic and non-immunogenic tumors and is associated with the anti-tumor and anti-metastatic serum factor meta-tyrosine (m-tyr), an isomer of tyrosine not present in normal proteins. Based on this background, in this work we assessed whether CR was also occurring in human tumor experimental models. Athymic nude mice were inoculated s.c. in the right flank, with the human prostate cancer cell line PC3 (1 × 106, primary implant). After 14 days the animals received a second inoculation of PC3 cells in the left flank (1 × 106, secondary implant). The control group only received the secondary implant. The growth of the secondary implant was significantly reduced (92%; P Citation Format: Geraldine Gueron, Nicolás Anselmino, Damian Manchuca, Emiliano G. Ortiz, Maria Noelia Carabelos, Federico Schuster, Paula Chiarella, Alejandra Paez, Felipe M. Jaworski, Javier Cotignola, Roberto Meiss, Raul Ruggiero, Elba S. Vazquez. A second round for concomitant resistance in human cancer: A restraint upon metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2015-5199

Breast cancer is still one of the most important public health problems in the entire world. Obesity and metabolic syndrome (MS) increases the incidence and aggressiveness of breast cancer. C-Terminal Binding Protein (CtBP1) is a transcriptional corepressor of tumor suppressor genes and is considered a molecular sensor of cell metabolic state due to is activated in high energy conditions (high NADH). In this work we studied the effects of the activation of CtBP1 pathway by metabolic syndrome on breast tumor development and progression. We generated a murine model of MS by chronic high fat diet (HFD) administration. By histological and whole mount methods, we found that breast tissue of animals receiving HFD presented higher levels of immature adipose tissue and an increased glandular area with more generation of lateral branches and terminal end buds of mammary ducts. Breast tissue of HFD animals also showed higher expression of the proliferation markers (cyclin D1) and epithelial markers (E-cadherin). Interestingly, HFD induced CtBP1 expression in the mammary ducts. Furthermore, the number and size of mamospheres generated with LM38-LP breast cancer cells were significantly increased when cells were incubated with serum from HFD fed mice compared to the serum of animals under control diet (CD). In addition, to investigate CtBP1 role in tumor progression we performed xenografts in nude mice fed with CD or HFD by subcutaneous injecton of breast tumor cells MDA MB 231 with depleted CtBP1 expression (shRNA CtBP1) or control cells (shRNA scramble). We found that CtBP1 depletion dramatically decreased tumor growth and KI67 expression relative to control tumors. Furthermore, xenografts developed in HFD fed mice were less differentiated compared to CD. Finally, CtBP1 diminished expression tumors showed lower mesenchymal markers expression, progenitor cells markers and markers involved in mammary development. Our studies demonstrated for the first time that gene transcription regulation by CtBP1 provides an important molecular link among MS, CtBP1 function and tumor growth. Hence, these results suggest an association to understand metabolism and breast cancer. Citation Format: Paola De Luca, Nicolás Dalton, Cristian Pablo Moiola, Carolina Flumian, Georgina Scalise, Juliana Porretti, Cintia Massillo, Edith Kordon, Laura Todaro, Elba Vazquez, Roberto Meiss, Adriana De Siervi. CtBP1 is the molecular link that associates breast cancer and metabolic syndrome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1160. doi:10.1158/1538-7445.AM2015-1160

Clinical and epidemiological data suggest that obesity is associated with more aggressive forms of prostate cancer (PCa), poor prognosis and increased mortality. In addition, high calorie intake decreases intracellular NAD+/NADH ratio. C-terminal Binding Protein 1 (CtBP1) is a transcription repressor of several important tumor suppressor genes and is activated by NADH binding. The aim of this work was to assess the effect of a high fat diet (HFD) and CtBP1 expression modulation over PCa tumor development in a murine xenograft model. We developed a metabolic syndrome-like disease in vivo model. Male nude mice fed with HFD or control diet (CD) for 16 weeks showed hypercholesterolemia, low testosterone serum levels, liver steatosis and glomeruli enlargement with edema at the kidney ephitelium collecting duct. In addition, at week 12 of diet, prostate tumor PC3 cells stable transfected with shCtBP1 or control (pGIPZ) plasmids were s.c. inoculated into randomly divided mice groups. No significant differences were observed in tumor growth on CD fed mice; however, we found that only 60 % of HFD fed mice inoculated with CtBP1 depleted cells developed a tumor; even more these tumors were significantly smaller than those generated by PC3.pGIPZ control xenografts. Furthermore, CtBP1 depletion in xenograft tumors was validated by IHQ and RT-qPCR. Genome-wide expression profiles (HUGENE ST1.0 Affymetrix) from PC3.shCtBP1 versus PC3.pGIPZ HFD fed mice tumors showed 823 genes differentially expressed (1.5 fold change and p < 0.05). By biological process GO classification, we found that most of these genes correspond to cell adhesion, metabolic process, apoptosis and cell cycle among others GO terms. In addition, we performed Gene Set Enrichment Analyses (GSEA) from our expression datasets and contrasted our results using MSigDB (C2 and C5 gene sets collection). We identified gene sets associated with metabolic and cellular processes. Interestingly, E-cadherin (CDH1) and aromatase (CYP19A1) expression were up-regulated in CtBP1 depleted tumors, and we validated this evidence by RT-qPCR. Our results suggested that metabolic syndrome-like diseases and CtBP1 expression might cooperate to PCa tumor development. Hence, CtBP1 expression might be considered for PCa management and therapy in the subset of patient with metabolic syndromes. Citation Format: Cristian P. Moiola, Paola De Luca, Florencia Zalazar, Santiago Rodriguez Segui, Javier Cotignola, Roberto Meiss, Elba Vazquez, Nicolas Dalton, Estefania Labanca, Kevin Gardner, Adriana De Siervi. CtBP1 is implicated in prostate tumor development in a metabolic syndrome-like disease in vivo model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 247. doi:10.1158/1538-7445.AM2014-247

Prostate Cancer (PCa) is one of the most common invasive tumors in men. Epidemiological studies indicate that diet and overweight are important factors implicated in prostate carcinogenesis. Obesity is associated with PCa aggressiveness, poorer prognosis and increased mortality. Breast cancer susceptibility gene 1 (BRCA1) interacts with several transcriptional regulators to modulate the androgen receptor (AR) signaling in PCa cell lines. Germline mutations in this gene increase breast cancer risk and are associated with high grade PCa. Previously, it had been reported that C-terminal Binding Protein 1 (CtBP1) acts as a switch to control BRCA1 transcription in response to the metabolic status of the cells. The release of CtBP1 from BRCA1 promoter through estrogen induction and high NAD+/NADH ratio (similar to high caloric intake) increases BRCA1 transcription in breast cancer cells. The aim of this work was to assess the effect of androgens and/or high fat diet over the BRCA1/CtBP1 axis and PCa tumor growth. We found that BRCA1 and CtBP1 proteins associate to BRCA1 proximal promoter region in PC3 cells and suppress BRCA1 transcription. Testosterone stimulation released these factors from BRCA1 promoter increasing its transcription. To assess whether this activation is mediated by testosterone or the estrogen, synthesized from testosterone by the aromatase (CYP19A1), we investigated this mechanism in the presence of letrozol (LTZ), an aromatase inhibitor. We found that LTZ abolished BRCA1 induction by testosterone, suggesting that BRCA1 activation is mediated by estrogen in these cells. Furthermore, we generated PC3 cell lines transfected with pcDNA3-CtBP1 (PC3-CtBP1) or shRNA-CtBP1 (PC3-shCtBP1) plasmids, to overexpress or knock down CtBP1 expression, respectively. CtBP1 induction decreased BRCA1 expression in these cells and this effect was reverted by CtBP1 depletion. In addition, PC3-CtBP1 cells showed increased clonogenic capacity and proliferation compared to PC3-shCtBP1 cells. Moreover, we developed an in vivo model to investigate the effect of high caloric diet on PCa growth after CtBP1 modulated-expression. High fat or control diet fed male nude mice were inoculated with PC3-CtBP1 and PC3-shCtBP1 stable cells. We found that CtBP1 depleted cells growing as xenografts in high fat diet fed mice dramatically decreased prostate tumor growth. Molecular analysis of tumors by RT-qPCR showed that CtBP1 depletion correlated with high BRCA1 expression. In addition, serum from high fat fed mice significantly induced PC3-CtBP1 cell proliferation in vitro. These results strongly suggest that the potential oncogenic role of CtBP1 is dependent on the caloric diet intake. Hence, BRCA1 regulation by CtBP1 provides an important molecular link between caloric intake and tumor suppressor expression. Citation Format: Cristian P. Moiola, Paola De Luca, Florencia Zalazar, Javier Cotignola, Estefania Labanca, Roberto Meiss, Elba S. Vazquez, Kevin Gardner, Adriana De Siervi. Molecular link that associates high fat diet and prostate tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3697. doi:10.1158/1538-7445.AM2013-3697

Prostate cancer (PCa) is a leading cause of death among males. Angiogenesis is critical during tumor initiation and progression. The arrival of anti-angiogenic agents as cancer treatments joining the ranks of surgery, chemotherapy and radiotherapy has been a source of renewed hope. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage. It has become increasingly clear that in addition to its effect on blocking proliferation, invasion and migration, other mechanisms including a direct effect on angiogenic factors may account for its anti-tumoral role in PCa. To further assess its properties, we investigated its potentiality to modulate PCa associated-angiogenesis. In the present study, we identified in PCa cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α, KDR and α5β1 integrin. Our results indicated also that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Furthermore, using immunofluorescence and structured illumination microscopy we visualized NFκB retention in cytoplasm and demonstrated a higher rate of co-localization with HO-1 under HO-1 over-expression. These observations correlated with repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, induced accumulation of IκB and decreased IKK mRNA levels under HO-1 modulation. These evidences strongly suggest that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports an unprecedented role of HO-1, challenging the angiogenic-switch in prostate carcinogenesis outlining a rationale for its development as an anticancer target in PCa. 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 5290. doi:1538-7445.AM2012-5290

Prostate cancer (PCa) still ranks as the second most commonly diagnosed cancer and metastatic castration-resistant prostate cancer (CRPC) is a leading cause of cancer death in men around the world. The common treatment for patients with CRPC is chemotherapy based on docetaxel. While there are currently seven agents approved for CRPC and four regimens have shown some survival benefit, those survival prolongations have been modest and unfortunately all patients will eventually progress. Thus, there is a need for new agents and regimens for this disease. 2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin dependent kinases and also provokes lethality against leukemic cells. We previously found that CPS49 and flavopiridol combination induced selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. The goal of this study was to investigate the selectivity and efficacy of CPS49-flavopiridol combination in prostate cancer preclinical models. Our results showed that flavopiridol enhanced CPS49 cytotoxicity in all human prostate tumor cell lines analyzed (PC3, C4-2, LNCaP and 22RV1); however non tumor cell lines (293HEK and MCF10) were resistant to the tested doses. Furthermore, it was previously reported, high doses of flavopiridol (10 μM) or CPS49 (12 μM) were needed to inhibit tumor growth in PC3 xenograft mice compared with vehicle treated mice. Here, we demonstrated that combining these two agents, antitumor activity was synergistically enhanced with low doses. Injecting subcutaneously PC3 cells in nu/nu mice, we found that CPS49-flavopiridol administration reduced tumor volume approximately 83% after 2 weeks of co-treatment and 54% after 1 week of low dose flavopiridol pretreatment and 2 weeks of drug combination. In addition, we performed RT-qPCR array containing 26 genes from PC3 cells exposed to CPS49 and flavopiridol combination. We determined that this treatment shut down the expression of several genes involved in cell cycle, DNA damage and tumor progression. Histological analysis of xenograft PC3 tumor samples showed extensive areas of necrosis induced by the treatment. Furthermore, we assessed the efficacy of CPS49 in combination with paclitaxel (docetaxel analog). All the prostate tumor cell lines tested were highly sensitive to this combination. However, this combination did not reduce the tumor volume in PC3 xenografts. These results indicate that the CPS49 and flavopiridol is a promising new alternative for the treatment of CRPC. 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 2340. doi:1538-7445.AM2012-2340

Prostate cancer metastasis is well characterized by its tropism to the bone with metastatic lesions being typically osteoblastic. Osteoblasts produce factors that promote growth and survival of prostate cancer cells, but the molecular nature of this interaction is still unknown. Previous reports from our laboratory documented that heme oxygenase-1 (HO-1) is expressed in primary prostate carcinomas and is localized in the nucleus. Recently, the involvement of HO-1 in bone metabolism has been reported. The aim of this study is to investigate the role of HO-1 in the interaction between prostate cancer cells and bone. We analyzed the expression of HO-1 by immunohistochemistry using a tissue microarray generated from 12 castrate-resistant prostate cancer specimens (MDA Anderson Cancer Center), growing as xenografts in male CB17 SCID wild or castrated mice. Each of these tumors led to clinically relevant biological models to study human prostate cancer progression. All samples showed cytoplasmic HO-1 staining of varying intensity. Intense positive nuclear staining was also detected in 3/5 tumors with neuroendocrine morphology. HO-1 expression was correlated with other markers such as PSA, AR and Ki-67 expression. Co-cultures of the osteolytic PC3 cancer cell line with PMO (primary mice osteoblasts) demonstrated that HO-1 induction in PC3 cells abolished the PC3-induced loss of osteoblasts and significantly increased Dickkopf-1 expression in tumor cells. Furthermore, using PC3-PMO co-cultures, we found that HO-1 induction in prostate cancer cells increased mRNA levels of Runx-2 and Cyclin D1 and diminished β-catenin protein expression in PMO. These results clearly indicate the involvement of HO-1 in the interaction between prostate cancer cells and bone. The study of the molecular mechanisms of this interaction will provide effective therapeutic interventions. 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 5260. doi:10.1158/1538-7445.AM2011-5260

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. The metastatic spread of PCa cells and the ability to survive when reaching the metastatic nitch is affected by growth factors, chemokines, angiogenic factors and hormones. Heme oxygenase-1 (HO-1), the inducible isoform of the rate-limiting enzyme in heme degradation, counteracts oxidative and inflammatory damage. Here we investigated its nuclear translocation in androgen sensitive (MDAPCa2b and LNCaP) and insensitive (PC3) PCa cell lines. Our results show that endogenous levels of HO-1 are markedly lower in PC3 compared to MDAPCa2b and LNCaP. Hemin treatment, a potent and specific inducer of HO-1, increased protein levels with a resulting nuclear translocation in PCa cell lines. Stable transfection of HO-1 in PC3 (PC3HO-1) also showed a significant nuclear translocation. Furthermore, human prostate cancer PC3HO-1 cells growing subcutaneously in athymic nude mice showed significant HO-1 nuclear staining (detected by immunohistochemsitry and immunofluorescence) compared to PC3pcDNA3 xenografts. Using RT-qPCR-generated gene array we observed a set of inflammatory and angiogenic genes up- or down-regulated in response to HO-1 overexpression, identifying VEGFA as a novel downstream target of HO-1. HO-1 modulation limited the metastatic potential of neoplastic cells by down-regulating VEGFA production. An in vivo angiogenic assay also demonstrated that PC3HO-1 tumors presented less neovascularization than tumors derived from control cells, giving further evidence to support HO-1 anti-angiogenic function. These results implicate for the first time HO-1 in the regulation of key proinflammatory factors, involved in PCa progression. Taken together our results suggest that HO-1 may be a potential target for therapeutic interventions. 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 LB-245.