Felipe Jaworski

Galectins as New Prognostic Markers and Potential Therapeutic Targets for Advanced Prostate Cancers

A better understanding of multimolecular interactions involved in tumor dissemination is required to identify new effective therapies for advanced prostate cancer (PCa). Several groups investigated protein-glycan interactions as critical factors for crosstalk between prostate tumors and their microenvironment. This review both discusses whether the “galectin-signature” might serve as a reliable biomarker for the identification of patients with high risk of metastasis and assesses the galectin-glycan lattices as potential novel targets for anticancer therapies. The ultimate goal of this review is to convey how basic findings related to galectins could be in turn translated into clinical settings for patients with advanced PCa.

Stable and high expression of Galectin-8 tightly controls metastatic progression of prostate cancer

Two decades ago, Galectin-8 was described as a prostate carcinoma biomarker since it is only expressed in the neoplastic prostate, but not in the healthy tissue. To date, no biological function has been attributed to Galectin-8 that could explain this differential expression. In this study we silenced Galectin-8 in two human prostate cancer cell lines, PC3 and IGR-CaP1, and designed a pre-clinical experimental model that allows monitoring the pathology from its early steps to the long-term metastatic stages. We show for the first time that the natural and conserved expression of Gal-8 in tumour cells is responsible for the metastatic evolution of prostate cancer. In fact, Gal-8 controls the rearrangement of the cytoskeleton and E-Cadherin expression, with a major impact on anoikis and homotypic aggregation of tumour cells, both being essential processes for the survival of circulating tumour cells during metastasis. While localized prostate cancer can be cured, metastatic and advanced disease remains a significant therapeutic challenge, urging for the identification of prognostic markers of the metastatic process. Collectively, our results highlight Galectin-8 as a potential target for anti-metastatic therapy against prostate cancer.

In vivo hemin conditioning targets the vascular and immunologic compartments and restrains prostate tumor development

Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model. Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes. Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1–conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo. A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels. Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response. Clin Cancer Res; 23(17); 5135–48. ©2017 AACR.

Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis article

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.

High RAC3 expression levels are required for induction and maintaining of cancer cell stemness

RAC3 is a transcription coactivator, usually overexpressed in several tumors and required to maintain the pluripotency in normal stem cells. In this work we studied the association between RAC3 overexpression on cancer cell stemness and the capacity of this protein to induce cancer stem properties in non tumoral cells. We performed in vitro and in vivo experiments using two strategies: by overexpressing RAC3 in the non tumoral cell line HEK293 and by silencing RAC3 in the human colorectal epithelial cell line HCT116 by transfection. Furthermore, we analysed public repository microarrays data from human colorectal tumors in different developmental stages. We found that RAC3 overexpression was mainly associated to CD133+ side-population of colon cancer cells and also to early and advanced stages of colon cancer, involving increased expression of mesenchymal and stem markers. In turn, RAC3 silencing induced diminished tumoral properties and cancer stem cells as determined by Hoechst efflux, tumorspheres and clonogenic growth, which correlated with decreased Nanog and OCT4 expression. In non tumoral cells, RAC3 overexpression induced tumoral transformation; mesenchymal phenotype and stem markers expression. Moreover, these transformed cells generated tumors in vivo. Our results demonstrate that RAC3 is required for maintaining and induction of cancer cell stemness.

Abstract 4717: Clinical implications for m-tyrosine, an isomer of p-tyrosine, for the treatment of aggressive prostate tumors

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, Nicolas Anselmino, Paula Chiarella, Emiliano Ortiz, Alejandra Paez, Jimena Giudice, Federico Schuster, Daiana Leonardi, Felipe Jaworski, Estefania Labanca, Veronica 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.

Abstract 5199: A second round for concomitant resistance in human cancer: A restraint upon metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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<0.05) at 27 days, in animals carrying the primary implant. Moreover, m-tyr was detected in the serum of mice bearing the RC phenomenon. The tumor growth inhibition was recapitulated in animals inoculated with the primary tumors and injected with m-tyr. Strikingly the RC phenomenon was reversed when secondary implants were injected with phenylalanine, a protective amino acid highly present in primary tumors. In vitro results also showed that exposure of PC3 cells to m-tyr inhibited cell growth and a G0/G1 cell cycle arrest, which was concomitant with alterations in the mRNA expression levels of survivin (apoptosis inhibitor), Ki67 (proliferation marker), Hes1 (transcription factor involved in Notch pathway) and STAT3 (prostate cancer survival factor) (P<0.01). We have further validated the RC phenomenon in two other human cancer models: anaplastic carcinoma of the lung (CALU-6), and nasopharyngeal carcinoma (KB), exhibiting also high levels of m-tyr in serum from nu/nu mice bearing CALU-6 or KB tumors. Altogether, we showcase for the first time that CR is triggered in human solid tumors, that this phenomenon is mediated by m-tyr and provide the molecular mechanisms that drive this process. These results offer an alternative therapeutic avenue in the management of metastatic cancers. Citation Format: Geraldine Gueron, Nicolas 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

Abstract LB-43: Unveiling the molecular significance of HO-1 and muskelin interaction: two masterminds behind the morphology and the adhesive behavior of prostate cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate Cancer (PCa) is the second leading cause of cancer death in American men. The inflammatory tumor microenvironment is a fertile niche that releases reactive oxygen species, which accelerates the malignant transformation and appears as a fine tuner of the adhesive behavior of cells. Heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation, represents an essential event in cellular responses to pro-oxidative and pro-inflammatory insults. As we previously reported that HO-1 over-expression impaired tumor growth and angiogenesis in vivo we sought to assess whether HO-1 could regulate the adhesive properties and the morphology of PCa cells. A bioinformatics enrichment analysis using Metacore, GeneMANIA and DAVID was performed; rendering a significant association of the HO-1 regulated genes with several proteins located in the extracellular space and cell membrane; compartments highly correlated with the adhesive behavior of cells. In an effort to understand the molecular mechanisms underlying HO-1s role in cell morphology regulation we used a proteomics approach to identify HO-1 partners. We performed GST-pull-down assays using lysates from PC3 cells transfected with either GST-tagged HO-1 or the empty vector, and the isolated proteins were subjected to MALDI-TOF/TOF analyses. Our results showed that HO-1 interacts with Muskelin, a nucleocytoplasmic mediator of cellular morphology and adhesiveness. Up-regulation of Muskelin under HO-1 induction in PCa cells was confirmed by confocal microscopy. A high degree of nuclear overlay between HO-1 and Muskelin signals was observed when cells were exposed to hemin, a potent specific inducer of HO-1 or genetically manipulated to over-express HO-1, compared to controls. Interestingly after HO-1 induction, both protein exhibit similar sub-cellular dynamics, relocating from the cell membrane, towards the cell nuclei. Altogether, we have shown for the first time that HO-1 binds and up-regulates Muskelin, a specific factor involved in shaping cellular morphology and adhesive properties, favoring a less aggressive phenotype and further supporting the anti-tumoral function of HO-1in PCa. Citation Format: Geraldine Gueron, Jimena Giudice, Alejandra Paez, Pia Valacco, Noelia Carabelos, Federico Schuster, Javier Cotignola, Felipe Jaworski, Daiana Leonardi, Maria Binaghi, Marcelo Marti, Nora Navone, Elba Vazquez. Unveiling the molecular significance of HO-1 and muskelin interaction: two masterminds behind the morphology and the adhesive behavior of prostate cancer cells. [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 LB-43. doi:10.1158/1538-7445.AM2014-LB-43

Abstract 207: Rearrangements of the E-cadherin/B-catenin-based adherens junctions caused by forced-expression of heme oxygenase 1 (HO-1) in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Prostatic tumor cell plasticity involves cooperative changes in the interactions between tumoral cells. The stable E-cadherin-based Adherence Junctions (AJs) play a pivotal role in the integrity of the epithelium and the maintenance of tissue homeostasis. Heme oxygenase-1 (HO-1) acts as a cell rheostat counteracting oxidative and inflammatory damage. Given that inflammation is critical for the development and progression of PCa, we sought to determine whether HO-1 could regulate the adhesive properties of PCa cells, towards the acquisition of an epithelial-like phenotype. We previously showed that HO-1 over-expression impairs tumor growth and represses angiogenesis in vivo. Here we demonstrate that HO-1 over-expression significantly increased PCa cell adhesion, remodeling the AJs. To better understand the nature of alterations of cell-cell interactions during neoplastic evolution, we examined the expression levels of E-cadherin and B-catenin. Treatment with hemin, a potent and specific inducer of HO-1, increased E-cadherin and b-catenin protein and mRNA levels in PCa cell lines, shown by Western Blot, RT-qPCR and flow cytometry analyses. These epithelial markers were also significantly induced when PCa cells were stably transfected with HO-1. Immunofluorescence, confocal microscopy together with a three-dimensional (3D) acquisition process leading to the collection of image stacks, confirmed the augmented levels of these proteins under forced-expression of HO-1 and also revealed a striking rearrangement of their localization pattern towards the cell membrane. Moreover, quantitative image analysis detected a significant increase in the percentage of cell-to-cell contact among tumoral cells under HO-1 overexpression. The enhanced levels of E-cadherin and B-catenin in PCa cells under hemin treatment coincided with a markedly different morphology compared to untreated cells, observed by bright field microscopy. While untreated cells displayed an elongated and fibroblastic-like shape, with increased number of lamellipodia, HO-1-overexpressing cells showed a spherical cobblestone epithelial shape. These results define a novel role for HO-1 in modulating the cellular morphology and the architecture of cell-to-cell interactions, favoring a less aggressive phenotype and further supporting its anti-tumoral function in PCa. Citation Format: Geraldine Gueron, Belen Elguero, Alejandra Paez, Jimena Giudice, Martin Toscani, Felipe Jaworski, Daiana Leonardi, Federico Coluccio-Leskow, Adriana De Siervi, Nora Navone, Elba Vazquez. Rearrangements of the E-cadherin/B-catenin-based adherens junctions caused by forced-expression of heme oxygenase 1 (HO-1) in prostate cancer. [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 207. doi:10.1158/1538-7445.AM2013-207