Recent developments have broadened our perception of SARS-CoV-2, indicating its capability to affect the body systemically beyond its initial recognition as a mere respiratory pathogen. However, the pathways of its widespread are not well understood. Employing a dual-modality approach, we integrated findings from a Murine Hepatitis Virus (MHV) infection model with corroborative clinical data to investigate the pervasive reach of Coronaviruses. The novel presence of viral particles within red blood cells (RBCs) was demonstrated via high-resolution transmission electron microscopy, with computational modeling elucidating a potential heme-mediated viral entry mechanism via Spike protein affinity. Our data affirm viral localization in RBCs, suggesting heme moieties as facilitators for cellular invasion. Exacerbation of MHV pathology upon hemin administration, contrasted with chloroquine-mediated amelioration, underscoring a heme-centric pathway in disease progression. These observations extend the paradigm of Coronavirus pathogenicity to include hemoprotein interactions. This study casts new light on the systemic invasion capabilities of Coronaviruses, linking RBC hemoproteins with viral virulence. The modulation of disease severity through heme-interacting agents heralds a promising avenue for COVID-19 therapeutics. Our findings propose a paradigm shift in the treatment approach, leveraging the virus-heme interplay as a strategic hinge for intervention.

Abstract About 88% of metastatic prostate cancer (PCa) cases involve bone metastasis. When PCa cells spread to the bone, a metabolic adaptation to the new environment takes place, partly influenced by factors secreted by the homing organ. Thus, we hypothesize that metabolic reprogramming, both in the tumor and bone niche drive PCa bone metastasis. In this work, we carried out an integrative in vitro, in vivo, and bioinformatics approach to identify key regulators governing the metabolic behavior of PCa cells. We performed an indirect co-culture between PCa (PC3) and bone progenitor (MC3T3 or Raw264.7) cells. PCa cells displayed a strong transcriptional (RNA-seq) activation of lipid metabolism, including PPAR-signaling, fat digestion, and PI3K-Akt pathway. Accordingly, treatment with the conditioned media (CM) of the co-culture promoted lipid accumulation in PC3 cells (Bodipy 493/503 staining). To assess the clinical relevance of the in vitro model, we performed an unsupervised clustering analysis using transcriptomics data from human PCa and bone metastatic samples (GSE74685). Results showed that the metabolic genes deregulated in PC3 by the co-culture, could accurately cluster samples in primary tumor or bone metastasis. We next performed a multivariable Cox regression analysis and built a prognostic model, identifying a novel signature of 5 lipid-associated genes, PPARA, VDR, SLC16A1, PAPSS2 and GPX1, associated with a 23-fold higher risk of death in PCa patients (SU2C-PCF dataset). The expression of this signature was validated in a PDX pre-clinical model (MDA PCa PDX series) when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously. In order to identify the central hubs regulating this transcriptional phenotype, we performed an Ingenuity Pathway Analysis (QIAGEN), showcasing that the lipid-related signature deregulated by the bone niche is indeed modulated by the Protein Kinase A (PKA). Accordingly, the expression of these signature was downregulated by PKA inhibition (H89; PKA inhibitor). Moreover, secretome analyses (ESI MS/MS) revealed that bone secreted type I collagen (Col1a1) and fibronectin (Fn1) regulate PKA activity in PCa cells. Further, we investigated whether PKA could modulate critical mechanisms for bone progression, focusing on osteopontin (SPP1), a secreted protein involved in the adhesion and invasion of PCa cells, whose regulatory mechanism in tumor cells is yet to be elucidated. Strikingly, we observed not only that the activation of PKA using Forskolin in PCa cells activated SPP1 expression, but also that PKA inhibition led to a significant impairment of the SPP1 induction triggered by bone soluble factors secreted during the PC3/MC3T3 co-culture. Altogether, we showcase PKA as a central hub of PCa energetic metabolism during bone progression and outline a lipid-associated gene signature that can be targeted to halt metastatic PCa. Citation Format: Pablo Sanchis, Nicolas Anselmino, Estefania Labanca, Agustina Sabater, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Sofia Lage-Vickers, Antonina Mitrofanova, Pia Valacco, Ayelen Toro, Javier Cotignola, Nora Navone, Elba Vazquez, Geraldine Gueron. Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion [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 3064.

Purpose: Metabolic rewiring is critical for the adaptation of tumor cells to the new homing organ. We sought to identify metabolic dysregulations fueling PCa bone metastasis, modulated by bone secreted factors. Methods: By an indirect co-culture system of PCa (PC3) and bone progenitor cells (MC3T3, pre-osteoblasts, or Raw264.7, pre-osteoclasts), we assessed the transcriptome of PC3 cells modulated by soluble factors released by bone precursors. We validated the transcriptional profile of metabolic genes using open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. The bone secretome was profiled in the conditioned media (CM) by ESI-MS/MS. We validated our results using a PDX pre-clinical model comparing gene expression levels in MDA-PCa-183 growing intrafemorally (i.f.) vs. subcutaneously (s.c.). Results: PC3 cells co-cultured with bone progenitors displayed an activation of lipid metabolism, including the PPAR-signaling and fat absorption/digestion pathways. Accordingly, we observed an accumulation of neutral lipids in PC3 cells treated with the CM from the co-culture (Bodipy 493/503 staining). Unsupervised Clustering analysis using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in co-cultured PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, 5 lipid-associated genes, PPARA, VDR, SLC16A1, PAPSS2 and GPX1, were associated with a 23-fold higher risk of death (SU2C-PCF dataset). This expression signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing i.f. vs. s.c. An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PKA inhibition led to a downregulation of these genes. Moreover, GSEA showcased an activation of PKA signaling pathways in MDA-PCa-183 i.f. vs. s.c., reinforcing the fact that the bone environment shapes PCa metabolism. Additionally, secretome and protein-protein interaction analyses revealed soluble factors (Col1a1, Fn1, etc.) secreted by bone cells that could regulate PKA activity. Conclusion: We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. This signature is under the regulation of PKA in response to bone-secreted factors, emerging as a potential target for intervention. Citation Format: Pablo Sanchis, Nicolas Anselmino, Sofia Lage-Vickers, Agustina Sabater, Rosario Lavignolle, Estefania Labanca, Peter Shepherd, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Ayelen Toro, Antonina Mitrofanova, Pia Valacco, Nora Navone, Javier Cotignola, Elba Vazquez, Geraldine Gueron. Activation of PKA Signaling: A Milestone Associated With Bone Progression of Prostate Cancer [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 5.

Purpose: Mortality in prostate cancer (PCa) patients is mostly associated with bone metastasis and aggressive phenotypes promoted by prostate cancer stem cells (PCSCs). We have previously described that heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme catabolism with antioxidant and antiinflammatory properties, presents an antitumoral role in PCa. In this work, we assessed the role of HO-1 as a modulator of stemness and metastasis associated genes in PCa cells conditioned by bone progenitor cells. Methods: An indirect transwell co-culture system of PC3 cells, a PCa cell line, with MC3T3 cells, an osteoblast precursor cell line, was developed as an in vitro model of PCa bone metastasis. Additionally, PC3 cells were pre-treated with hemin, a pharmacological HO-1 inductor. An RNA-seq was performed to analyze the transcriptome of PC3 cells. The expression of a curated list of 87 genes associated with stemness, metastasis and the STAT3 pathway, including PCSC and classic pluripotency markers, together with a gene signature previously described by our group (ADAM15, BCL2L1, LTBR, MBNL2, SPINT1), was assessed. Results were complemented with survival data of PCa patients (TCGA-PRAD, n=565). Results: Our results showed that PC3 cells co-cultured with MC3T3 cells displayed an upregulation of PCSC markers CD44, ALDH1A3 and ALDH1L2, pluripotency markers OCT4 and SOX2, and BCL2L1 and LTBR, two genes included in our signature, compared with non-co-cultured PC3 cells. Further, when comparing co-cultured PC3 cells vs. co-cultured PC3 cells pre-treated with hemin, in the latter group the expression of 71/87 genes associated with stemness, metastasis and STAT3 pathway decreased, including CD44, ALDH1A3, ALDH1L2, OCT4, MBNL2 and ADAM15. Moreover, when evaluating patients’ disease progression free survival, those with high expression of OCT4 showed a lower risk of the event. Conclusion: HO-1 expression modulates stemness and metastasis related genes in PCa, promoting a more differentiated state, counteracting the pro-stemness effect of communication with bone progenitors. This further supports the antitumoral role of HO-1 in PCa pointing out to its potential as a therapeutic target for the disease. Citation Format: Ines Achinelli, Agustina Sabater, Pablo Sanchis, Nicolás Anselmino, Sofía Lage-Vickers, Gaston Pascual, Rocio Seniuk, Pamela Freiria, Javier Cotignola, Elba Vazquez, María Sol Ruiz, Geraldine Gueron, Ayelen Toro. Heme Oxygenase 1 Counteracts Pro-Stemness and Pro-Invasive Effect Triggered by Communication With Bone Progenitors in Prostate Cancer Cells [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 43.

Eighty percent of prostate cancers (PCa) metastasize to bone, showing a significant source of patient morbidity. PCa stem-like cells (PCSCs) are capable to quiescently outlast retaining the ability to proliferate and regenerate, consequently remaining able to develop therapy-resistant tumors and metastatic lesions. We have previously reported that the overexpression of heme oxygenase 1 (HO-1), the rate limiting enzyme in heme degradation, leads to a less aggressive PCa phenotype. However, its effect on metastasis-stemness (MS) remains unknown. In this work, we address the biological significance of HO-1 in association with relevant MS genes for PCa progression. Clonogenic assays performed in PCa cells (PC3 and C4-2B) to assess colony formation, evidenced a reduction on the stem-like properties of tumor cells treated with hemin (FDA approved drug and specific HO-1 inducer). RNA-seq analysis to identify MS genes that could be modulated by HO-1 induction, revealed 32 MS-genes that were modulated in PC3-hemin vs. PC3-control cells. We then mined the Oncomine database (n = 1,128) and found that 15/32 HO-1-modulated MS genes were significantly dysregulated in PCa compared with normal gland. To extend our findings, further computational analyses were conducted, and a custom-made bioinformatics tool (Gene Hunter) was created to analyze gene expression and clinicopathological profiles across multiple publicly available PCa datasets with normal, adjacent, tumoral and metastatic samples (n = 1,629). We found that ADAM15, BCL2L1, LTBR, MBNL2 and SPINT1 are consistently dysregulated across different comparisons, with expression profiles in tumors that could be reverted by hemin treatment, according to our RNA-seq results. We constructed a risk score that efficiently stratified patients in high, intermediate and low-risk of biochemical-relapse groups (p<0.01). We also performed multiple survival analyses including, overall, progression-free, relapse-free and metastasis-free survival data. We found that high MBNL2, a gene previously reported to be related to embryonic stem cell differentiation and to antimetastatic effects in other tumors, was associated with lower risk of relapse, progression and metastasis (p<0.05). Of note, the expression of MBNL2 was downregulated in PCa samples compared with normal gland or normal adjacent tissue. Interestingly MBNL2 expression can be reverted by HO-1 induction in PCa cells, as validated by RT-qPCR. Accordingly, those patients with high expression of MBNL2 showed a decreased risk of biochemical-relapse when they presented high HMOX1 levels (HR = 0.4615, p = 0.0186). Altogether, we highlight the relevance of HO-1 in modulating MS-associated genes in PCa and point out to MBNL2 as a potential druggable target for disease intervention. Citation Format: Agustina A. Sabater, Ayelen R. Toro, Pablo A. Sanchis, Gaston M. Pascual, Rocio A. Seniuk, Elba S. Vazquez, Javier Cotignola, Juan A. Bizzotto, Sofia Lage-Vickers, Geraldine Gueron. HMOX1 modulates tumor stemness and metastatic properties in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2437.

Supplementary methods, Supplementary Figures S1-S3, and Supplementary tables

Abstract Purpose: Mortality in prostate cancer (PCa) patients is mostly associated with bone metastasis and aggressive phenotypes promoted by prostate cancer stem cells (PCSCs). We have previously described that heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme catabolism with antioxidant and antiinflammatory properties, presents an antitumoral role in PCa. In this work, we assessed the role of HO-1 as a modulator of stemness and metastasis associated genes in PCa cells conditioned by bone progenitor cells. Methods: An indirect transwell co-culture system of PC3 cells, a PCa cell line, with MC3T3 cells, an osteoblast precursor cell line, was developed as an in vitro model of PCa bone metastasis. Additionally, PC3 cells were pre-treated with hemin, a pharmacological HO-1 inductor. An RNA-seq was performed to analyze the transcriptome of PC3 cells. The expression of a curated list of 87 genes associated with stemness, metastasis and the STAT3 pathway, including PCSC and classic pluripotency markers, together with a gene signature previously described by our group (ADAM15, BCL2L1, LTBR, MBNL2, SPINT1), was assessed. Results were complemented with survival data of PCa patients (TCGA-PRAD, n=565). Results: Our results showed that PC3 cells co-cultured with MC3T3 cells displayed an upregulation of PCSC markers CD44, ALDH1A3 and ALDH1L2, pluripotency markers OCT4 and SOX2, and BCL2L1 and LTBR, two genes included in our signature, compared with non-co-cultured PC3 cells. Further, when comparing co-cultured PC3 cells vs. co-cultured PC3 cells pre-treated with hemin, in the latter group the expression of 71/87 genes associated with stemness, metastasis and STAT3 pathway decreased, including CD44, ALDH1A3, ALDH1L2, OCT4, MBNL2 and ADAM15. Moreover, when evaluating patients’ disease progression free survival, those with high expression of OCT4 showed a lower risk of the event. Conclusion: HO-1 expression modulates stemness and metastasis related genes in PCa, promoting a more differentiated state, counteracting the pro-stemness effect of communication with bone progenitors. This further supports the antitumoral role of HO-1 in PCa pointing out to its potential as a therapeutic target for the disease. Citation Format: Ines Achinelli, Agustina Sabater, Pablo Sanchis, Nicolás Anselmino, Sofía Lage-Vickers, Gaston Pascual, Rocio Seniuk, Pamela Freiria, Javier Cotignola, Elba Vazquez, María Sol Ruiz, Geraldine Gueron, Ayelen Toro. Heme Oxygenase 1 Counteracts Pro-Stemness and Pro-Invasive Effect Triggered by Communication With Bone Progenitors in Prostate Cancer Cells [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 43.

PURPOSE The arrival of metastatic prostate cancer (PCa) tumor cells to the bone niche requires a metabolic adaptation. We sought to identify metabolic dysregulations fueling PCa metastasis, modulated by bone secreted factors. METHODS By an indirect co-culture system of PCa (PC3) and bone progenitors (MC3T3, pre-osteoblasts, or Raw 264.7, pre-osteoclasts) we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. We validated the transcriptional profile of metabolic genes in open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. Bone secretome was profiled on the conditioned media (CM) by ESI-MS/MS. RESULTS PC3 cells co-cultured with bone progenitors displayed an activation of lipidic categories, including PPAR-signaling and fat absorption/digestion. Principal Component and Unsupervised Clustering analyses using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, four lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, were associated with a shorter survival time (SU2C-PCF dataset), and were independent risk-predictors of death ( P < .05). An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PC3 cells treated with the CM of the co-culture presented a decreased ATP content compared to the treatment with the CM of PC3 grown alone, which was restored upon PKA inhibition. Finally, the secretome analysis revealed soluble factors secreted by bone progenitors (Col1a1, Fn1) which could regulate PKA activity. CONCLUSION We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. Moreover, PKA could regulate this signature in response to bone-secreted factors reprogramming the metabolic phenotype of metastatic cells, emerging as a potential druggable target for this disease.

PURPOSE Prostate cancer stem cells enhance the tumor's capacity to proliferate, metastasize, and to resist therapies. Thus, stemness represents a therapeutic challenge in prostate cancer (PCa). We have previously reported a strong anti-tumoral effect of heme oxygenase 1 (HO-1) in prostate carcinogenesis in vivo and in vitro. However, its association with metastasis-stemness is still poorly elucidated. Thus, we aimed at describing the effects of HO-1 overexpression on metastasis/stemness processes in PCa, by also identifying key HO-1-modulated genes. METHODS We performed clonogenic assays to evaluate the effect of HO-1 pharmacological induction on the stem properties of PC3-cells. We carried out an RNA-seq analysis to assess the transcriptional differences of 144 metastasis/stemness/STAT3-signature genes after HO-1 induction. The clinical significance of HO-1-modulated genes was assessed by comprehensive bioinformatics analyses using public data repositories with gene expression and survival data of PCa patients (Oncomine, n = 1,128; TCGA-PRAD, n = 565). RNA-seq results were validated by RT-qPCR. RESULTS Clonogenic assays results showed a significant reduction in colony formation efficiency after HO-1 pharmacological induction with hemin ( P ≤ .01). Transcriptomics profiling by RNA-seq evidenced a significant modulation of 32 key markers related to metastasis/stemness/STAT3-signature under HO-1 induction. Further, 15 HO-1-modulated genes were differentially expressed in PCa vs. normal prostate gland (Oncomine) and HO-1 induction reverted the expression profile observed for these genes. We next used the TCGA-PRAD dataset which revealed that ADAM15, BCL2L1, LTBR, MBNL2 and SPINT1 had the same expression profile as in Oncomine. Additionally, PCa patients with high MBNL2 expression showed a longer disease progression-free survival, which is interesting as it is upregulated in PC3-cells overexpressing HO-1, as validated by RT-qPCR. CONCLUSION Here, we propose a potential mechanism by which HO-1 could inhibit metastasis/stemness by modulating relevant genes in PCa progression. This study may cast a new light on PCa treatment, highlighting HO-1 modulation of cell plasticity, supporting it as a potential therapeutic target for the disease.

PURPOSE Prostate cancer (PCa) is the second most common type of cancer in men and the sixth leading cause of cancer-related death in men worldwide. The discovery of new therapeutic avenues in PCa, and the development of effective drugs in the era of personalized medicine, would greatly benefit from the field of proteomics. We have previously shown the anti-tumoral role of Heme oxygenase 1 (HO-1) in PCa. METHODS In this work, we undertook a mass spectrometry-based proteomics study to assess whether in PCa cells and under oxidative stress conditions, HO-1 could interact with proteins previously documented to have nuclear localization. RESULTS Among the HO-1 interactors, we identified 11 proteins with nuclear localization. Significant and positive correlation between HMOX1 and six of those genes was observed, using the GSE70770 dataset. Alternatively, HMOX1 and YWHAZ showed negative correlation. High expression of HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1, and HMOX1 was associated with an increased relapse-free survival (RFS) in PCa patients through univariable analyses. Further, PCa patients with high HSPB1/HMOX1, DDB1/HMOX1, and YWHAZ/HMOX1 showed a worse RFS compared with patients with lower ratios. Moreover, by using a prognostic risk score model, a decrease in RFS for patients with higher scores of this signature was observed. However, the only factor significantly associated with a higher risk of relapse was high YWHAZ. HSPB1, DDB1 and YWHAZ independence from PCa clinic-pathological parameters was confirmed through multivariable analyses. Moreover, co-immunoprecipitation analysis in PCa cells ascertained HO-1/14-3-3ζ/δ (YWHAZ encoding gene) interaction. CONCLUSION Herein, we report novel interactions between HO-1 and HSPB1, DDB1 and 14-3-3ζ/δ, highlighting their clinical relevance in PCa.

Prostate cancer (PCa) cells display abnormal expression of proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown the anti-tumoral role of heme oxygenase 1 (HO-1) in this disease. In this work, we undertook a mass spectrometry-based proteomics study to identify HO-1 molecular interactors that might collaborate with its modulatory function in PCa. Among the HO-1 interactors, we identified proteins with nuclear localization. Correlation analyses, using the PCa GSE70770 dataset, showed a significant and positive correlation between HMOX1 and 6 of those genes. Alternatively, HMOX1 and YWHAZ showed a negative correlation. Univariable analyses evidenced that high expression of HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1, and HMOX1 was associated with increased relapse-free survival (RFS) in PCa patients. Further, PCa patients with high HSPB1/HMOX1, DDB1/HMOX1, and YWHAZ/HMOX1 showed a worse RFS compared with patients with lower ratios. Moreover, a decrease in RFS for patients with higher scores of this signature was observed using a prognostic risk score model. However, the only factor significantly associated with a higher risk of relapse was high YWHAZ. Multivariable analyses confirmed HSPB1, DDB1, and YWHAZ independence from PCa clinic-pathological parameters. In parallel, co-immunoprecipitation analysis in PCa cells ascertained HO-1/14-3-3ζ/δ (protein encoded by YWHAZ) interaction. Herein, we describe a novel protein interaction between HO-1 and 14-3-3ζ/δ in PCa and highlight these factors as potential therapeutic targets.

Metastatic prostate cancer (PCa) cells soiling in the bone require a metabolic adaptation. Here, we identified the metabolic genes fueling the seeding of PCa in the bone niche. Using a transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3 or Raw264.7), we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. In a Principal Component Analysis using transcriptomic data from human PCa samples (GSE74685), the altered metabolic genes found in vitro were able to stratify PCa patients in two defined groups: primary PCa and bone metastasis, confirmed by an unsupervised clustering analysis. Thus, the early transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastatic samples. Further, the expression levels of five metabolic genes (VDR, PPARA, SLC16A1, GPX1 and PAPSS2) were independent risk-predictors of death in the SU2C-PCF dataset and a risk score model built using this lipid-associated signature was able to discriminate a subgroup of bone metastatic PCa patients with a 23-fold higher risk of death. This signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously, and appears to be under the regulatory control of the Protein Kinase A (PKA) signaling pathway. Secretome analyses of conditioned media showcased fibronectin and type-1 collagen as critical bone-secreted factors that could regulate tumoral PKA. Overall, we identified a novel lipid gene signature, driving PCa aggressive metastatic disease pointing to PKA as a potential hub to halt progression.

Bone hosts the 88% of Prostate Cancer (PCa) metastases and no curative therapy is currently available for this stage. Arrival of PCa cells to the bone homing organ is accompanied by a metabolic adaptation, which may be mediated by bone secreted factors. Herein, we sought to identify key metabolic genes fueling PCa bone metastasis and soluble factors secreted by bone cells leading to the metabolic rewiring of tumoral cells. By an indirect transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3, pre-osteoblasts; or Raw264.7, pre-osteoclasts) we analyzed the transcriptome (RNA-seq) of PC3 cells modulated by soluble factors released from bone precursors. GSEA showed a strong activation of lipid metabolism, including PPAR and PI3K-Akt pathways, fat absorption and digestion. We then performed a Principal Component Analysis using transcriptomic data from human PCa and bone metastasis samples (GSE74685), showcasing that those metabolic genes that appeared significantly dysregulated in the co-culture model could accurately cluster samples by their tissue of origin in two defined groups: primary PCa and bone metastasis. This result was confirmed by an unsupervised clustering analysis, highlighting that the transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastasis samples. Interestingly, when performing a survival analysis for those genes in the SU2C-PCF dataset, we observed that 4 lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, correlated with a shorter overall survival time, and appeared as independent risk-predictors of death (HR: 4.96, 2.85, 3.93 and 3.67, respectively; P<0.05). These results prompted us to evaluate the communication axis by which the expression of the lipid transcriptomic signature is regulated in PC3 cells co-cultured with bone progenitor cells. We performed an Ingenuity Pathway Analysis (QIAGEN) showing that the tumoral Protein Kinase A (PKA) appears as a master regulator of this signature. Accordingly, when we treated PC3 cells with the conditioned media (CM) of PC3 grown alone or the co-culture, we observed a decreased ATP content in the latter compared with controls, which is restored upon PKA inhibition, confirming the role of this kinase in the metabolic phenotype of co-cultured cells. Finally, secretome analysis (ESI-MS/MS) of CM from the co-cultures displayed relevant soluble factors secreted by bone progenitors (Col1a1, Fn1) which are directly linked to PKA activity. Overall, we identified an early lipid-related gene signature in PCa cells triggered by the dialogue with bone cells, enough to discriminate metastatic human PCa from primary tumors and critical for PCa survival. This signature may respond to released soluble bone factors through tumoral cell PKA activation. Our findings pinpoint new attractive metabolic druggable targets to halt disease progression. Citation Format: Pablo Sanchis, Nicolas Anselmino, Rosario Lavignolle, Agustina Sabater, Estefania Labanca, Juan Bizzotto, Sofia Lage-Vickers, Gaston Pascual, Rocio Seniuk, Ayelen Toro, Nora Navone, Javier Cotignla, Elba Vazquez, Geraldine Gueron. Early metabolic rewiring of prostate cancer cells triggered by bone progenitors defines survival of metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2374.

PURPOSE Prostate cancer (PCa) is a progressive disease involving multiple molecular alterations. The Gleason Grade (GG) is used as an aid for physicians to evaluate the prognosis of men with PCa using samples from prostate tissues. However, some PCa that are histo-pathologically grouped in the same GGs can differ significantly in outcome. The aim of this study was to identify molecular biomarkers that could behave independently from GG and other clinical-pathological variables (age, GG and TMPRSS2-ERG fusion status) that can improve risk prediction of PCa. METHODS An in-depth proteomics analysis (LC/ESI-MS/MS) was performed on human prostate adenocarcinoma and benign prostate hyperplasia (BPH) tissues. Protein candidates enriched in PCa vs BPH were further evaluated in-silico using a custom-made pipeline. For this purpose, seven publicly available transcriptomic PCa datasets (ntotal = 875) were selected, and differential expression analyses were performed in R. A Shiny-based tool was then built to execute the search and to allow visualization across multiple datasets. Further multivariable analyses were performed using clinical-pathological parameters as covariates. RESULTS Proteomics analysis yielded a list of 89 proteins enriched in PCa compared with BPH. 58 showed high expression in PCa datasets and nine of those, showed significant association with higher risk of death. Further, when performing a multivariable Cox proportional-hazard model including age, GG and TMPRSS2-ERG fusion status, PPP1R12B, FBLN5, CRIP2, and POSTN displayed associations with poor prognosis, independently from the other co-variates (HR = 1.5, P = .0084; HR = 1.34, P = .035; HR = 1.36, P = .016 and HR = 1.4, P = .0028, respectively). CONCLUSION PPP1R12B, FBLN5, CRIP2, and POSTN proteins were enriched in PCa human tissues. Our custom pipeline revealed increased expression of these genes across multiple PCa datasets and significant association with increased risk of death, independent from known risk factors and predictors of PCa prognosis. Thus, these factors rise as potential prognostic markers.

Heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation, is involved in the maintenance of cellular homeostasis, exerting a cytoprotective role by its antioxidative and anti-inflammatory functions. HO-1 and its end products, biliverdin, carbon monoxide and free iron (Fe2+), confer cytoprotection against inflammatory and oxidative injury. Additionally, HO-1 exerts antiviral properties against a diverse range of viral infections by interfering with replication or activating the interferon (IFN) pathway. Severe cases of coronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are characterized by systemic hyperinflammation, which, in some cases, leads to severe or fatal symptoms as a consequence of respiratory failure, lung and heart damage, kidney failure, and nervous system complications. This review summarizes the current research on the protective role of HO-1 in inflammatory diseases and against a wide range of viral infections, positioning HO-1 as an attractive target to ameliorate clinical manifestations during COVID-19.

Stromal antigen 2 (STAG2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle-invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n = 119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n = 346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for patients with MIBC. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy-number variations, or cell-cycle distribution. However, unbiased RNA-sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a noncanonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC. Significance: The cohesin component STAG2 regulates cell motility and invasion. STAG2 expression is associated with decreased MIBC survival and may be a useful biomarker to guide bladder cancer treatment.

STAG2 (Stromal Antigen 2) functions in chromatid cohesion, DNA damage repair and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. We have previously found that in MIBC, loss of STAG2 protein expression is associated with better overall survival (n=169; p=0.049) and progression free survival (n=169; p=0.016). Based on these retrospective analyses, we hypothesized that STAG2 promotes an aggressive cell phenotype through its genomic interactions and transcriptomic regulation in MIBC. First, to study the effects of STAG2 on gene expression, we stably knocked down (KD) STAG2 in T24 MIBC cells using two short hairpin RNAs. Scrambled shRNA served as a control in all experiments. Second, using KD and control T24 cell lines, we performed RNA and chromatin-immunoprecipitation sequencing, then integrated these results utilizing the Cistrome analysis algorithm (Wang, S. et al., 2013) to determine how STAG2 regulates gene expression at its genomic binding sites. STAG2 KD led to the differential expression of 2158 genes, with 648 overlapping between the two shRNA cell lines. Through Cistrome analysis, we discovered that genes with increased expression after STAG2 KD were enriched for STAG2 binding sites (p=0.000503), yet genes with decreased expression did not show significant enrichment (p=0.873). This suggests that STAG2 functions as a transcriptional repressor, and subsequently we focused on genes with increased expression after STAG2 KD. We identified the significantly upregulated gene Reelin (RELN) (log2FC=2.89, 1.86; p<1*10-42, 1*10-17 for each shRNA, respectively), which has a well-established role in cell migration and invasion. To investigate this in vitro in MIBC cells, we performed time lapse microscopy and invasion assays to quantitatively determine cell movement over time. Compared to controls, T24 cells with STAG2 KD had reduced displacement (78 vs 114 µm, p<0.05), speed (0.30 vs 0.41 µm/min, p<0.05) and invasion (137 vs 190 cells/field, p<0.001) in vitro. Altogether, our results indicate that STAG2 functions as a transcriptional repressor and promotes movement and invasion of MIBC cells. This may explain why STAG2 protein loss leads to better outcomes for MIBC patients, and points to STAG2 protein expression as a potential prognostic biomarker. In our current work, we are utilizing a large drug screen of FDA-approved agents to identify vulnerabilities of tumor cells lacking STAG2. These studies will help to identify drugs that augment current standard of care and benefit the large proportion of patients with STAG2-mutant tumors. Citation Format: Sarah R. Athans, Nithya Krishnan, Swathi Ramakrishnan, Eduardo Cortes Gomez, Sofia Lage-Vickers, Monika Rak, Zara Kazmierczak, Aimee Stablewski, Kristopher Attwood, Jianmin Wang, Anna Woloszynska. STAG2 acts as a transcriptional repressor and promotes invasion of muscle invasive bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 786.

Interferon gamma (IFN-γ) may be potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 infection. Employing a case-control study from SARS-CoV-2-positive and -negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6, CBL, RUNX3, STAT1, and JAK2 in COVID-19-positive vs. -negative patients. A positive correlation was observed between STAT1/JAK2, which varied alongside the patient’s viral load. Expression of MX1, MX2, ISG15, and OAS1 (four well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19-positive vs. -negative patients. Integrative analyses showcased higher levels of ISGs, which were associated with increased viral load and STAT1/JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly (I:C), a synthetic analog of viral double-stranded RNA; and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A pre-clinical murine model of Coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.

Abstract Cancer is a risk factor for SARS-CoV-2 infection. Recent reports have shown that prostate cancer (PCa) patients undergoing androgen-deprivation therapies (ADT) were partially protected from COVID-19. The human myxovirus resistance gene 1 (MX1) is expressed in many tissues, including prostate, and we have previously demonstrated its antitumoral activity in PCa. This protein participates in the antiviral response and it is an IFN-stimulated gene (ISGs), especially during influenza virus infection. There are ongoing clinical trials for COVID-19 prevention and/or treatment using type I or III interferons. However, IFN administration could enhance a "cytokine-storm" causing a hyper-inflammatory response and contributing to organ failure. In this work, we performed bioinformatics analyses in a case-control study from SARS-CoV-2 positive (n=403) and negative (n=50) patients. We analyzed the response to infection assessing gene expression profiles in nasopharyngeal swabs of key host cell receptors (ACE2, TMPRSS2, BSG/CD147, CTSB, CTSL, ADAM17) and antiviral proteins (MX1, MX2, NRF2, IRF3, HIF1A, HMOX1).SARS-CoV-2 positive cases had higher ACE2, but lower TMPRSS2, BSG/CD147 and CTSB expression. Patient age negatively affected ACE2 expression. MX1 and MX2 were higher in SARS-CoV-2 positive individuals, and negative trends were observed as patients' age increased. Principal Component Analysis determined that ACE2, MX1, MX2 and BSG/CD147 expressions were able to cluster non-COVID-19 and COVID-19 individuals. Multivariable regression showed that MX1 expression significantly increased for each unit of viral load increment.Given that MX1 was differentially expressed between COVID-19 and non-COVID-19 patients, we evaluated MX1 expression in A549 and Calu3 lung cell lines. MX1 was significantly up-regulated upon infection with SARS-CoV-2.Since ADT reduces SARS-CoV-2 infection incidence, we aim to study MX1 regulation by dihydrotestosterone (DHT). We browsed publicly available ChIP-seq experiments evaluating androgen receptor (AR) binding sites in different PCa cell lines under DHT stimulation. Results indicated enriched AR binding sites on the MX1 sequence. Therefore, we treated LNCaP cells with DHT, observing a significant decrease in MX1 mRNA levels. Accordingly, we observed a significant increase of MX1 gene expression in PCa patients after ADT treatment.In summary, our study findings support differences in ACE2, MX1, MX2 and BSG/CD147 expression between COVID-19 and non-COVID-19 patients; and point out to MX1 as a critical responder in SARS-CoV-2 infection. Furthermore, we demonstrated MX1 modulation by ADT. Taking into consideration the fact that PCa patients that underwent ADT were less prone to present the infection, we propose this gene as an alternative druggable target for COVID-19 patients, especially those with PCa as a previous condition. Citation Format: Juan Antonio Bizzotto, Pablo Sanchis, Sofia Lage-Vickers, Rosario Lavignolle, Agustina Sabater, Mercedes Abbate, Ayelen Toro, Florencia Cascardo, Santiago Olszevicki, Nicolas Anselmino, Estefania Labanca, Emiliano Ortiz, Elba Vazquez, Javier Cotignola, Geraldine Gueron. Androgen-deprivation therapy boosts MX1 expression, a silent effector against COVID-19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 710.

Prostate cancer (PCa) that progresses after androgen deprivation therapy (ADT) remains incurable. The underlying mechanisms that account for the ultimate emergence of resistance to ADT, progressing to castrate-resistant prostate cancer (CRPC), include those that reactivate androgen receptor (AR), or those that are entirely independent or cooperate with androgen signaling to underlie PCa progression. The intricacy of metabolic pathways associated with PCa progression spurred us to develop a metabolism-centric analysis to assess the metabolic shift occurring in PCa that progresses with low AR expression. We used PCa patient-derived xenografts (PDXs) to assess the metabolic changes after castration of tumor-bearing mice and subsequently confirmed main findings in human donor tumor that progressed after ADT. We found that relapsed tumors had a significant increase in fatty acids and ketone body (KB) content compared with baseline. We confirmed that critical ketolytic enzymes (ACAT1, OXCT1, BDH1) were dysregulated after castrate-resistant progression. Further, these enzymes are increased in the human donor tissue after progressing to ADT. In an in silico approach, increased ACAT1, OXCT1, BDH1 expression was also observed for a subset of PCa patients that relapsed with low AR and ERG (ETS-related gene) expression. Further, expression of these factors was also associated with decreased time to biochemical relapse and decreased progression-free survival. Our studies reveal the key metabolites fueling castration resistant progression in the context of a partial or complete loss of AR dependence.