Di Vizio Research Articles

Abstract 1382: Activation of the cGAS-STING pathway by large oncosomes induces type I IFN inflammation in mesenchymal stem cells that triggers an immunosuppressive phenotype in neutrophils

Abstract Extracellular vesicles (EVs) have been recognized as key players in cancer progression and metastasis, particularly large oncosomes (LO), secreted by invasive cancer cells, represent a distinct class of EVs with unique features. Previous research by the Di Vizio lab revealed LO's ability to alter recipient cell behavior, induce vascular morphogenesis, and carry cancer-specific biomolecules. Our preliminary studies point to a new mechanism of intercellular communication that involves the release of LO-containing DNA from PC cells. Notably, LO from prostate cancer exhibits enhanced potency in reprogramming bone marrow-derived mesenchymal stem cells (BM-MSCs) compared to exosomes (Exo) as observed by RNAseq. When evaluating the response to LO and Exo of BM-MSC, which are emerging as important drivers of bone metastasis, LO induces a robust inflammatory response, including metastasis-relevant chemokines and transcription factors involved in type I IFN response such as IRF7 and RASD2. Further, REACTOME pathway analysis of the differentially expressed genes revealed enrichment of the cGAS-STING pathway in BM-MSCs treated with LO, indicating a potential link between DNA/RNA sensors and the inflammatory response. BM cells isolated from WT and mutant Sting1gt/J mice demonstrated that up-regulation of IRF3 and IFNA1 genes induced by LO is inhibited when STING is absent. Suggesting that nucleic acid sensors engage in the LO-induced immune response in BM cells. Gene ontology analysis (GO) also identified several chemokines involved in neutrophil chemotaxis. In line with the observation from GO, neutrophil chemotaxis was significantly promoted by PC-derived LO-treated BM-MSC. Interestingly, human blood-derived neutrophils exposed to a conditioned medium from EVs-treated BM-MSC significantly increased the percentage of a population of LOX-1high and CD62Llow/− after 6h of culture. This was observed in concert with increased expression of Arginase1 and IL-10, suggesting their differentiation into polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSC). An experimental orthotopic model using the breast cancer bone metastatic 4T1.2 cell line, with increased capacity to shed LO, demonstrated an increase in the presence of PMN-MDSC in the bone marrow of tumor-bearing mice in addition to a complete lack of lymphocyte populations, indicating their immunosuppressive activity that could lead to a pre-metastatic niche formation. This comprehensive study unveils the molecular pathways underlying the immune response to LO in the bone marrow, shedding light on mechanisms that could influence metastatic progression in prostate cancer. The findings underscore the unique role of LO in modulating the BM microenvironment, providing potential targets for therapeutic intervention and biomarkers for early disease detection. Citation Format: Taylon F. Silva, Diana Kitka, Blandine Victor, Chen Qian, Minhyung Kim, Tatyana Vagner, Giorgia Guerra, Catherine Grasso, Sungyong You, Fayyazz Sutterwala, Caroline Jefferies, Paola de Candia, Michael Freeman, Helen Goodridge, Karen Cavassani, Jlenia Guarnerio, Dolores Di Vizio. Activation of the cGAS-STING pathway by large oncosomes induces type I IFN inflammation in mesenchymal stem cells that triggers an immunosuppressive phenotype in neutrophils [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1382.

Abstract 4302: ONECUT2 activates diverse resistance drivers of androgen receptor-independent heterogeneity in prostate cancer

Abstract Androgen receptor- (AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 targets include the glucocorticoid receptor (GR) and the NE splicing factor SRRM4, which are key drivers of lineage plasticity. Thus, OC2, despite its previously described NEPC driver function, can indirectly activate a portion of the AR cistrome through epigenetic activation of GR. Mechanisms by which OC2 regulates gene expression include promoter binding, enhancement of genome-wide chromatin accessibility, and super-enhancer reprogramming. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC and support enhanced efforts to therapeutically target OC2 as a means of suppressing treatment-resistant disease. Citation Format: Chen Qian, Qian Yang, Mirja Rotinen, Rongrong Huang, Hyoyoung Kim, Brad Gallent, Yiwu Yan, Radu M. Cadaneanu, Baohui Zhang, Salma Kaochar, Stephen J. Freedland, Edwin M. Posadas, Leigh Ellis, Dolores Di Vizio, Colm Morrissey, Peter S. Nelson, Lauren Brady, Ramachandran Murali, Moray J. Campbell, Wei Yang, Beatrice S. Knudsen, Elahe A. Mostaghel, Huihui Ye, Isla P. Garraway, Sungyong You, Michael R. Freeman. ONECUT2 activates diverse resistance drivers of androgen receptor-independent heterogeneity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4302.

Abstract B024: Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression

Abstract Very small nuclear (vsn) circulating tumor cells (CTCs) are detected in a subgroup of metastatic, castration resistant prostate cancer (mCRPC) patients who develop visceral metastasis following intensive androgen receptor suppression. In addition, we have previously observed that nuclear shape instability (characterized by the loss and/or mis-localization of the nuclear envelop protein emerin) is present in human prostate cancer tissues, and in CTCs from patients with metastatic disease. Herein, we investigated the biological and clinical implications of the vsn phenotype in prostate CTCs. Patients with vsnCTCs had poor overall survival and low PSA response to androgen receptor suppression compared with non-vsnCTC patients. The vsnCTCs exhibited a loss/downregulation of emerin (EMD) and we found a direct correlation between emerin loss and nuclear size reduction in CTC from mCRPC patients. Further, in vitro studies revealed that the vsn phenotype was characterized by high invasive capacity, enhanced cellular plasticity, and resistance to AR target therapy. Finally, depletion of EMD in prostate tumor cells resulted in the vsn phenotype. Together, these findings demonstrate that the vsnCTC phenotype is characterized by deregulation of EMD expression, and the presence of these cells result in poor clinical outcomes in prostate cancer. Citation Format: Karen Cavassani, Edwin Posadas, Pai-Chi Teng, Sadaf Pustchi Pustchi, Minhyung Kim, Neil Bhowmick, Sungyong You, Michael Freeman, Leigh Ellis, Dolores Di Vizio, Hsian-Rong Tseng, Leland W. Chung, Susan Yao, Catherine Grasso. Nuclear size and nuclear shape instability in prostate cancer epithelial cells during metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B024.

Oncogene-regulated release of extracellular vesicles.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Abstract P5-01-24: DNA palindromes as novel genomic targets for unbiased, genome-wide, and rapid pan-cancer screening

Abstract Cancer continues to have a significant medical, economic, and psychological burden on patients worldwide. One strategy for reducing this impact involves the early detection of cancer biomarkers. Structural chromosomal aberrations called DNA palindromes have immense potential to be used as early markers in tumorigenesis. Palindromic sequences in the genome are structural genetic phenomena where a sequence of nucleotides is identical to its reverse compliment. Various independent studies have identified this distinctive pattern of genome instability in a wide range of cancers including breast, pancreatic, and ovarian cancers. We have shown previously that DNA palindromes underlie the amplification of ERBB2 in breast cancer. This suggests that DNA palindromes are the candidate breast cancer biomarker. A convenient and sensitive approach for identifying palindromes in breast tumor DNA could be a promising diagnostic test. Palindrome detection from tumor DNA in blood (liquid biopsy) could circumvent the need for invasive biopsies. Here, we develop an unbiased genome-wide technique for detecting DNA palindromes as a genomic target for breast cancer screening in the clinic. Using genome-wide analysis of palindrome formation (GAPF), we are able to enrich for DNA palindromes from samples of genomic DNA isolated from cells and tumor tissues. Following a denaturation step, DNA palindromes naturally form double-stranded DNA with itself because these sequences are physically tethered together and can form energetically favorable base pairs. Non-palindromic DNA remains single-stranded and subsequently removed by single-strand specific nucleases thereby dramatically improving the signal-to-noise ratio. This approach confers advantages over other methods because it simultaneously (1) amplifies target signal and (2) reduces background noise without employing sub-sampling techniques. We first used a cancer cell line Colo320DM containing a known palindromic junction and normal fibroblasts IMR90. By integrating GAPF with next generation sequencing (GAPF-Seq), we mapped sequenced reads to a reference genome and developed an algorithm to demarcate DNA palindromes throughout the genome. We are able to identify the known DNA palindrome in Colo320DM in a 10-fold dilution in IMR90 DNA. We then used GAPF-Seq to identify tumor-specific DNA palindromes in matched pairs of tumor and normal breast cancer tissue. From 100 ng of input DNA, we generated a profile of more than 300 tumor-specific DNA palindromes that were not found in the matched normal tissue. To optimize GAPF for use in cancer screening, we expect to detect DNA palindromes in the presence of 100-fold excess of non-palindromic DNA from as little as 10 ng of input DNA. We will then use matched plasma and buffy coat samples to generate whole-genome palindromic profiles from liquid biopsies. The data can also be used for tracing clonal evolution by comparing individual bins in the palindrome profiles of tumor tissue and plasma cell-free DNA from the same patient. Finally, we will optimize GAPF-Seq for minimal computational processing and data storage requirements. Due to the amplification of palindromes and reduction of background signal, GAPF-Seq may be able to identify tumor-specific breast cancer markers from as few as 1 million reads and 250 MB of data, which would be ideal for wide use in screening for breast tumors in the general population and monitoring disease outcomes with multiple blood samplings. We believe GAPF-Seq addresses a need to develop an unbiased, genome-wide, and rapid approach for breast cancer screening. Citation Format: Michael M. Murata, Lila Mouakkad, Dolores Di Vizio, Armando E. Giuliano, Hisashi Tanaka. DNA palindromes as novel genomic targets for unbiased, genome-wide, and rapid pan-cancer screening [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-24.

Abstract 4448: Machine-learning assisted histopathology (HistoMAP) links nuclear membrane instability to disease progression in prostate cancer

Abstract Introduction: The manifestation of cancer malignancy occurs at the cellular level where individual cells escape tissue confinements and disseminate. Identifying this malignant behavior at a cellular level is necessary in order to deconvolve the tumor heterogeneity, identify disease progression and predict cancer-related morbidity and mortality. To achieve that goal, we established a unique computer-assisted single-cell histopathology analysis of prostate tissue that evaluates nuclear-membrane instability and the associated extracellular vesicle (EV) biogenesis to identify malignant potential and assess future disease progression. In recent years cancer EVs have been identified as important mediators of intercellular communication. The potential for using EVs as a liquid biopsy has promoted research on profiling EVs in biofluids. However, the direct analysis of EV biogenesis in tumor tissue has been largely omitted, even thought identifying such cells is likely to be a specific and sensitive measure of disease malignancy. We have previously demonstrated that highly migratory and metastatic cancer cells shed atypically large EVs, known as large oncosomes (LO, Di Vizio et. al., 2012). LO play distinct functions and contain a specific repertoire of molecules that can be used for detection of tumor-derived cargo in plasma (Minciacchi et. al., 2015). The recent discovery that the biogenesis of large oncosomes (LO) in prostate cancer is associated with nuclear membrane instability (Reis-Sobreiro et. al., 2018) offered an opportunity to investigate such biogenesis in patient specimens. We have developed a machine-learning assisted histopathology (HistoMAP) that quantitatively identifies nuclear-membrane instability in prostate cancer tissue and demonstrate a direct correlation between this molecular biogenesis of vesicles and biochemical recurrence after prostatectomy. Experimental procedures: We developed a multiplex immunofluorescent technique to detect and quantify nuclear-derived LO in formalin fixed paraffin embedded prostatectomy tissues. Using computer-assisted image segmentation and machine-learning we distinguished these LO from all other intracellular compartments and assess malignancy in a cohort of Vanderbilt prostate cancer patients. Results: LO production was significantly elevated in prostate cancer in comparison to benign tissue and particularly evident in lymph node metastases. Moreover, LO production was associated with the risk of future metastatic disease which re-enforced its relevance in cancer progression. Conclusions: Our findings demonstrate that, given knowledge of the EV machinery, EV biogenesis can be detected in patient tissues and provide critical information related to patient disease status and assist with the prediction of future clinical performance. Citation Format: Andries Zijlstra, Tatiana Novitskaya, Dolores Di Vizio, Mariana Reis- Sobreiro, Michael Freeman. Machine-learning assisted histopathology (HistoMAP) links nuclear membrane instability to disease progression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4448.

Abstract A047: ONECUT2 is a targetable master regulator of aggressive variants of castration-resistant prostate cancer

Abstract Background: Treatment of prostate cancer by hormone suppression leads to the appearance of aggressive variants of metastatic castration-resistant prostate cancer (mCRPC) with variable or no dependence on the androgen receptor (AR). Here we identify the neural transcription factor ONECUT2 as a negative regulator of the AR axis, that emerges in aggressive PC variants to control transcriptional networks linked to CRPC and neuroendocrine (NE) differentiation. We further demonstrate that ONECUT2 can be targeted with a small molecule that inhibits mCRPC metastasis in mice. Methods: ONECUT2 was confirmed as a mCRPC-relevant protein and to be targetable by computational modeling and bioinformatics, enforced expression, silencing, microarray, ChIP-Seq, immunohistochemistry, immunofluorescence, quantitative imaging, functional assays, in vivo experiments, and surface plasmon resonance. Results: We have performed a master regulator analysis using 260 mCRPC transcriptome profiles and developed a model transcription factor network for mCRPC that associates ONECUT2 for the first time with metastatic progression. Gene expression profiling of ONECUT2-engineered PC cell lines has allowed us to generate a ONECUT2 activity signature that reveals high positive correlation with pro-neural and aggressive PC signatures, and a negative correlation with AR activation pathways. We find that ONECUT2 is a negative regulator of AR expression and a repressor of its transcriptional program through direct binding to AR target genes. We also find that ONECUT2 is significantly increased in human NEPC and confers NE properties to CRPC through direct downregulation of the NEPC inhibitor FOXA1 and direct upregulation of the NEPC driver PEG10. Finally, we show that ONECUT2 is required for cell growth and survival and that it can be targeted with a small molecule that, by binding to the ONECUT2 C-terminal DNA binding domain, inhibits mCRPC metastasis in mice. Conclusions: OC2 is a master regulator of aggressive mCRPC variants that drives AR-dependent adenocarcinoma toward NEPC differentiation by blocking AR/FOXA1-activity and inducing PEG10. OC2 can be targeted with a drug-like small molecule that inhibits CRPC metastasis in mice. Patients with OC2 active tumors may benefit from OC2 inhibitor therapy. Citation Format: Mirja Rotinen, Sungyong You, Julie Yang, Simon Coetzee, Wen-Chin Huang, Fangjin Huang, Xinlei Pan, Alberto Yáñez, Dennis Hazelett, Chia-Yi Chu, Leland Chung, Stephen Freedland, Dolores Di Vizio, Isla Garraway, Ramachandran Murali, Beatrice Knudsen, Michael Freeman. ONECUT2 is a targetable master regulator of aggressive variants of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A047.

Abstract 4056: Immune regulation by miR-1227 LO

Abstract Immunotherapy offers great promise for the treatment of prostate cancer; however prostate cancer often acquires mechanisms to dampen the tumoricidal immune response. Understanding how prostate cancer dampens the immune system will facilitate the generation of more effective immunotherapies. Here we investigate how large oncosomes (LO)-mediated transfer of miR-1227 to immune cells modulates the immune response. Extracellular vesicle (EV)-encapsulated miRNAs are being increasingly recognized for playing a key role in cancer progression and immune modulation. miR-1227 is a poorly characterized miRNA that is enriched in EV secreted by prostate cancer cells in comparison to non-malignant prostate epithelial cells. miR-1227 expression is increased in migratory glioblastoma cells in comparison to non-migratory glioblastoma cells, and in radiation resistant esophageal cells and colorectal cancer stromal cells indicating that miR-1227 is upregulated in different cancer types. However, the role of miR-1227 in cancer is poorly understood. We demonstrate here that EV obtained from prostate cancer cells overexpressing miR-1227, induce the migration of prostate cancer cells and fibroblasts. A comparative analysis between different EV subtypes indicates that miR-1227 is enriched in LO, a class of EV that are secreted by highly invasive amoeboid-migrating cells. We also show that LO carry more RNA than the more widely studied exosomes indicating that LO may be a more robust source of EV-encapsulated miRNA. RNA sequencing from miR-1227 stably expressing prostate cancer cells and 5 different in silico miRNA target prediction methods were used to identify putative miR-1227 targets. IPA pathway analysis from these predicted targets highlighted several functions related to the immune system, including T cell homeostasis, T cell development, and development of Th17 cells. A more in-depth analysis of the 51 genes involved in T cell homeostasis showed enrichment of genes associated with Th17 and Th1 T cell differentiation. Alterations in Th17 and Th1 differentiation modulates T cell cytokine secretion, which are important mediators of cancer growth and progression. Transient transfection of miR-1227 in immune cells altered the cytokine expression profile, resulting in the upregulation of several cytokines that promote cancer growth including CCL5 and IL6. We tested whether LO can be taken up by activated T cells to directly modulate cytokine secretion. To determine whether miR-1227 can be functionally transferred to recipient cells via LO we isolated LO from prostate cancer cells stably expressing miR-1227 using ultracentrifugation followed by density gradient purification. The isolated LO were used to treat immune cells to show that LO-encapsulated miR-1227 alters cytokine profiles. These results indicate that LO-encapsulated miR-1227 may be involved in cancer immune modulation through targeting specific genes involved in T-cell differentiation. Citation Format: Andrew R. Chin, Dolores Di Vizio, Valentina R. Minciacchi, Mariana Sobreiro, Cristiana Spinelli. Immune regulation by miR-1227 LO [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 4056.

Abstract LB-266: Large oncosomes reprogram prostate fibroblasts toward a pro-angiogenic phenotype

Abstract Introduction: Cancer cells communicate with different cells in the tumor microenvironment, establishing a supportive stroma that sustains tumor development and facilitates the first steps of metastasis. Extracellular vesicles (EVs) have emerged as key functional mediators of this process. Aim of this study was to determine the mechanism of intercellular communication mediated by the atypically large EVs produced by highly migratory and metastatic tumor cells, referred to as large oncosomes (LO), and prostate fibroblasts (NAF). Methods: Filtration, differential centrifugation followed by iodixanol gradient; flow cytometry and confocal imaging; RNA-seq; kinase assay; TF array; luciferase assay; tube formation; siRNA; RT-qPCR. Results: Active AKT1 is significantly more expressed and functional in LO than in exosomes (Exo). Patients with metastatic disease express abundant active AKT1 in plasma LO. Uptake of LO harboring active AKT1 by NAF results in AKT1 and c-MYC activation. Conditioned media from LO-treated NAF, but not from Exo-treated NAF, promoted endothelial morphogenesis. The Dynamin (DNM) inhibitor Dynasore (Dyn) inhibited LO-uptake, as well as MYC activation and tube formation. Transient silencing of DNM2 significantly reduced LO uptake, suggesting that uptake occurs by phagocytosis. LO treatment increased levels of MYC targets in NAF, suggesting that MYC is involved in LO-induced reprogramming of NAF. Accordingly, MYC expression was higher in activated fibroblasts than NAF, and MYC overexpression in NAF induced hyperplasia in normal prostate epithelium in mice, suggesting MYC activation is an early event in cancer development. Summary/Conclusion: Our results indicate that tumor-derived LO induce a novel, c-MYC mediated, pro-tumorigenic reprogramming of fibroblasts that can be reverted by selectively inhibiting LO uptake. Support: National Institutes of Health NCI NIH R00 CA131472; NIH UCLA SPORE in Prostate Cancer award P50 CA092131; Avon Foundation Fund 02-2013-043 (to DDV). Citation Format: Valentina Minciacchi, Cristiana Spinelli, Mariana Reis-Sobreiro, Mandana Zandian, Rosalyn M. Adam, Edwin M. Posadas, Freeman R. Michael, Emanuele Cocucci, Neil Bhowmick, Dolores Di Vizio. Large oncosomes reprogram prostate fibroblasts toward a pro-angiogenic phenotype. [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 LB-266.

Infinitesimal deformation of p-adic differential equations on Berkovich curves

We show that if a differential equation $$\mathscr {F}$$ over a quasi-smooth Berkovich curve X has a certain compatibility condition with respect to an automorphism $$\sigma $$ of X, then $$\mathscr {F}$$ acquires a semi-linear action of $$\sigma $$ (i.e. lifting that on X). The compatibility condition forces the automorphism $$\sigma $$ to be close to the identity of X, so the above construction applies to a certain class of automorphisms called infinitesimal. This generalizes Andre and Di Vizio (Asterisque 1(296):55–111, 2004) and Pulita (Compos. Math. 144(4):867–919, 2008). We also obtain an application to Morita’s p-adic Gamma function, and to related values of p-adic L-functions.

Abstract 4154: Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells

Abstract We have recently established a zoledronic acid-resistant prostate cancer cell line, DU145R80,which exhibits higher invasive capability compared to parental DU145 cells, and epithelial-to-mesenchymal transition (EMT)[Milone MR, Cell Death Dis. 2013]. To investigate the mechanism by which the cells become invasive, we compared DU145R80 and DU145 cells by a proteomic approach, unveiling a signaling network that links the interior of the nucleus to changes in cytoskeleton dynamics and to the extracellular matrix, dictating prostate cancer aggressiveness [Milone MR Oncotarget 2014].Cytoskeletal modifications allow cells to gain a more migratory/invasive behavior and to interact with the surrounding microenvironment triggering different kind of biological phenomena, including the formation of large oncosomes (LO), a bioactive class of extracellular vesicles (EVs) [Di Vizio D, Cancer Res. 2009]. In this study, we found a higher amount of spontaneously shed LO along with an increased gelatinase activity from DU145R80 cells compared to the parental counterpart. By applying low speed and discontinuous gradient ultracentrifugation to both DU145 and DU145R80 cell media, we obtained a pure preparation of LO, floating at a 1.15 g/mL density fraction and positive for LO markers such as as Cav-1, Ck18, GAPDH. Moreover, we treated DU145 parental cells with pure preparations of LO from either DU145 or DU145R80 cells and performed an invasion assay, showing that LO from the resistant, aggressive DU145R80 cell line increase the invading ability of DU145. Treatment of DU145 cells with LO originating from the parental cell line itself did not result in increased invasinevess, suggesting a specific role for EVs from aggressive cells. In addition, in order to investigate LO content, we focused our attention on a cell surface glycoprotein, CUB domain-containing protein 1 (CDCP-1), putatively linked to the network of proteins identified in DU145R80 cells and whose role in cancer is still under debate. Our results demonstrate that CDCP-1 expression is significantly reduced in DU145R80 compared to DU145 cells, suggesting a tumor-protective role for the protein in prostate cancer. We also observed, for the first time, that the CDCP-1 expression pattern displayed by both cell lines was reflected in their derived LO. These data intriguingly suggest that CDCP-1 may change its expression pattern upon modifications in tumor cells properties, playing a different role in different stages of cancer development. Overall, these findings highlight LO as a new biological component in the development of aggressive features by prostate cancer cells, connecting molecular alterations to changes in the ability to interact with the surrounding environment, and suggested new prognostic markers and/or therapeutical targets. Citation Format: Chiara Ciardiello, Valentina R. Minciacchi, Mariana Reis-Sobreiro, Maria R. Milone, Biagio Pucci, Rita Lombardi, Francesca Bruzzese, Dolores Di Vizio, Alfredo Budillon. Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells. [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 4154. doi:10.1158/1538-7445.AM2015-4154

Abstract 4868: Large oncosomes are internalized and functionally modulate transcription factors in recipient cells

Abstract Background and Objective: We recently reported that rapidly migratory, “amoeboid” prostate cancer (PCa) cells shed large (1-10µm diameter), bioactive extracellular vesicles (EV), termed large oncosomes (LO), whose abundance correlates with tumor aggressiveness (Di Vizio et al., Cancer Res. 2009; Di Vizio et al. Am J Pathol. 2012). Increasing evidence supports an important role for EVs in mechanisms of communication between cancer cells and the surrounding microenvironment. EVs can activate signal transduction as well as transfer biomolecules to recipient cells, processes that may promote oncogenesis and/or enhance tumor progression. The aim of this study was to investigate the molecular mechanisms of LO internalization into recipient cells and LO-mediated intercellular communication. Results: LO stimulated the migration of tumor and endothelial cells and contained active MMP2 and MMP9, key proteases involved in tumor cell invasion. Quantitative LC-MS/MS SILAC analysis of LO and exosomes demonstrated enrichment of specific proteins in LO in comparison with exosomes. Fluorescently labeled-LO were internalized into recipient cells and maintained their stability as discrete microvesicles that were localized in the perinuclear space at early time points and into the nucleus at later times. Because of this transfer of LO to the nuclear compartment, we investigated whether exposure to LO altered the activity of transcription factors in recipient cells, hypothesizing a possible novel mechanism of LO-mediated intracellular communication. Interestingly, the degree of internalization, as quantitatively assessed by flow cytometry, varied among different recipient cells, including benign and cancer prostate epithelial cells, immortalized myofibroblasts, cancer associated fibroblasts and endothelial cells. These results suggest cell-specific affinities for target cells and, perhaps, finely regulated mechanisms underlying LO-internalization. Moreover selective inhibition of pathways important in EV internalization indicated that the LO enter cells through an active endocytic process, rather than passively fusing with the plasma membrane of recipient cells. Internalization of LO resulted in altered expression of transcription factors. Conclusions: Our results show for the first time that internalization of intact LO might be necessary for their biological activity in recipient cells. These findings also suggest that the functional role of LO in the tumor microenvironment might be mediated by modulation of transcription factors. Citation Format: Valentina R. Minciacchi, Matteo Morello, Sungyong You, Wei Yang, Mariana Sobreiro, Cristiana Spinelli, Mandana Zandian, Mirja Rotinen, Samantha Morley, Rosalyn Adam, Michael R. Freeman, Dolores Di Vizio. Large oncosomes are internalized and functionally modulate transcription factors in recipient 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 4868. doi:10.1158/1538-7445.AM2014-4868

Abstract 1658: Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment

Abstract The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and silencing of DIAPH3 evokes a transition to an amoeboid phenotype in multiple tumor cell backgrounds. This amoeboid transformation is accompanied by increases in plasma membrane deformations (blebbing), cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the shedding of atypically large (>1μm) extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of bioactive nano-sized EV (exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused exosome shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of exosome production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells (PBMC). DU145-derived EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV across a wide size range (∼70nm to >1um) are produced by amoeboid prostate cancer cells as a result of DIAPH3 loss and/or growth factor stimulation. These EV may condition the tumor microenvironment through multiple mechanisms, including promoting the growth of cancer cells and suppression of tumor-infiltrating immune cells. Citation Format: Jayoung Kim, Samantha Morley, Minh Le, Denis Bedoret, Dale Umetsu, Dolores Di Vizio, Michael Freeman. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment. [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 1658. doi:10.1158/1538-7445.AM2014-1658

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status.

AMP-dependent Kinase Inhibits Oxidative Stress-induced Caveolin-1 Phosphorylation and Endocytosis by Suppressing the Dissociation between c-Abl and Prdx1 Proteins in Endothelial Cells

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.

Abstract 4066: Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior.

Abstract Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis we identified Sh3gl2 (endophilin A1) as a transcript and protein highly enriched in bladder epithelium (urothelium). The gene encoding Sh3gl2 is located on chromosome 9p, a genomic region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles in Oncomine™, as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to non-tumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 15 of 16 human UC cell lines, but preserved in the low grade, well- differentiated cell line RT4. In UC cell lines lacking expression of Sh3gl2, treatment with the demethylating agent 5-aza-deoxycytidine led to upregulation of Sh3gl2 mRNA levels. Methylation-specific restriction analysis and PCR of genomic DNA revealed hypermethylation of a CpG island in the Sh3gl2 promoter in cells lacking Sh3gl2 expression, but no promoter methylation in RT4 cells that express Sh3gl2, suggesting that lack of Sh3gl2 expression results, at least in part, from methylation-induced gene silencing. Stable knockdown of Sh3gl2 in RT4 cells by RNA interference (i) enhanced proliferation and colony formation in vitro; (ii) inhibited EGF-induced EGFR internalization and increased EGFR phosphorylation; (iii) stimulated phosphorylation of Src family kinases and STAT3; (iv) altered sensitivity to chemotherapeutic agents, including the dual-specificity EGFR/ERBB2 inhibitor lapatinib; and (v) promoted formation of RT4 xenografts in subrenal capsule tissue recombination experiments. Together, these findings identify loss of Sh3gl2 as an early event in UC development that promotes disease progression. Sh3gl2 status in UC biopsies may provide a potential marker of risk for UC progression or recurrence and may enable determination of which patients require more aggressive intervention in the form of surgery or chemotherapy in order to improve prognosis. Citation Format: Shyama Majumdar, Edward Gong, Dolores Di Vizio, Jonathan Dreyfuss, David DeGraff, Martin Hager, Peter Park, Joaquim Bellmunt, Robert Matusik, Jonathan Rosenberg, Rosalyn Adam. Loss of Sh3gl2/endophilin A1 is an early event in urothelial carcinoma that regulates malignant behavior. [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 4066. doi:10.1158/1538-7445.AM2013-4066

512 MICROVESICLES SHED FROM DIAPH3-SILENCED, AMOEBOID PROSTATE CANCER CELLS ENHANCE GROWTH OF OTHER TUMOR CELLS AND SUPPRESS PROLIFERATION OF IMMUNE CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research (III)1 Apr 2013512 MICROVESICLES SHED FROM DIAPH3-SILENCED, AMOEBOID PROSTATE CANCER CELLS ENHANCE GROWTH OF OTHER TUMOR CELLS AND SUPPRESS PROLIFERATION OF IMMUNE CELLS Sungyong You, Samantha Morley, Dolores Di Vizio, Kim Jayoung, and Michael Freeman Sungyong YouSungyong You Los Angeles, CA More articles by this author , Samantha MorleySamantha Morley Boston, MA More articles by this author , Dolores Di VizioDolores Di Vizio Los Angeles, CA More articles by this author , Kim JayoungKim Jayoung Los Angeles, CA More articles by this author , and Michael FreemanMichael Freeman Los Angeles, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1906AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastatic castration-resistant prostate cancer (mCRPC) is a leading cause of death in American men, but mechanisms controlling progression to advanced disease are still poorly understood. Genomic loss of the cytoskeletal regulator DIAPH3 occurs frequently in mCRPC (Di Vizio et al., Cancer Res 2009; Hager, Morley et al. EMBO Mol Med 2012). DIAPH3 silencing in prostate cancer and other cell background evokes a transition from a mesenchymal to an amoeboid phenotype, as well as increases in tumor cell migration, invasion and metastasis. The main objective of this study was to identify potential biological functions for microvesicles (MV) produced by prostate cancer cells in which DIAPH3 was silenced. METHODS: The NanoSight platform was used to assess the degree of MV shedding from DIAPH3 knockdown DU145 cells (DIAPH3 KD) and controls. Shed MVs were harvested from conditioned medium using an established ultracentrifugation protocol. Western blot analysis, qRT-PCR, immunofluorescence cell staining, and cell proliferation assays were used for functional assays. RNA profiling was performed to identify MV cargo. RESULTS DIAPH3 KD cells secreted about 2-fold more MVs than control DU145 cells. MV shedding was increased by activation of EGFR-Erk/MAPK signaling by a potent growth factor, HB-EGF, and/or p38MAPK inhibition. MV preparations from DIAPH3 KD cells promoted cell proliferation and invasive behaviors of recipient cells and suppressed proliferation of human immune cells, peripheral blood mononuclear cell, suggesting a potential impact of MV shedding on cancer epithelia as well as the tumor microenvironment. We also found that MVs contained micro RNAs (miRNAs) linked to malignant behavior and disease progression. CONCLUSIONS Our findings suggest that MVs produced in mCRPC by loss of DIAPH3 expression may condition the tumor microenvironment by activation of cancer cells and suppression of tumor infiltrating immune cells. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e210 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sungyong You Los Angeles, CA More articles by this author Samantha Morley Boston, MA More articles by this author Dolores Di Vizio Los Angeles, CA More articles by this author Kim Jayoung Los Angeles, CA More articles by this author Michael Freeman Los Angeles, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cav1 Suppresses Tumor Growth and Metastasis in a Murine Model of Cutaneous SCC through Modulation of MAPK/AP-1 Activation

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

A criterion for the integrality of the Taylor coefficients of mirror maps in several variables

We give a necessary and sufficient condition for the integrality of the Taylor coefficients at the origin of formal power series qi(z)=ziexp(Gi(z)/F(z)), with z=(z1,…,zd) and where F(z) and Gi(z)+log(zi)F(z), i=1,…,d are particular solutions of certain A-systems of differential equations. This criterion is based on the analytical properties of Landau’s function (which is classically associated with sequences of factorial ratios) and it generalizes the criterion in the case of one variable presented in [E. Delaygue, Critère pour l’intégralité des coefficients de Taylor des applications miroir, J. Reine Angew. Math. 662 (2012) 205–252]. One of the techniques used to prove this criterion is a generalization of a version of a theorem of Dwork on formal congruences between formal series, proved by Krattenthaler and Rivoal in [C. Krattenthaler, T. Rivoal, Multivariate p-adic formal congruences and integrality of Taylor coefficients of mirror maps, in: L. Di Vizio, T. Rivoal (Eds.), Théories Galoisiennes et Arithmétiques des Équations Différentielles, in: Séminaire et Congrés, vol. 27, Soc. Math. France, Paris, 2011, pp. 279–307].