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Emerging evidence suggests an important role for SIRT1, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase in cancer development, progression and therapeutic resistance; making it a viable therapeutic target. Here, we examined the impact of resveratrol-mediated pharmacological activation of SIRT1 on the progression of HGPIN lesions (using the Pten(−/−) mouse model) and on prostate tumor development (using an orthotopic model of prostate cancer cells stably silenced for SIRT1). We show that precise SIRT1 modulation could benefit both cancer prevention and treatment. Positive effect of SIRT1 activation can prevent Pten deletion-driven development of HGPIN lesions in mice if resveratrol is administered early (pre-cancer stage) with little to no benefit after the establishment of HGPIN lesions or tumor cell implantation. Mechanistically, our results show that under androgen deprivation conditions, SIRT1 inhibition induces senescence as evidenced by decreased gene signature associated with negative regulators of senescence and increased senescence-associated β-galactosidase activity. Furthermore, pharmacological inhibition of SIRT1 potentiated growth inhibitory effects of clinical androgen receptor blockade agents and radiation. Taken together, our findings provide an explanation for the discrepancy regarding the role of SIRT1 in prostate tumorigenesis. Our results reveal that the bifurcated roles for SIRT1 may occur in stage and context-dependent fashion by functioning in an antitumor role in prevention of early-stage prostate lesion development while promoting tumor development and disease progression post-lesion development. Clinically, these data highlight the importance of precise SIRT1 modulation to provide benefits for cancer prevention and treatment including sensitization to conventional therapeutic approaches.

PDF file - 79KB, Effect of RES on the protein levels of p4E-BP1 and FOXO-3 in prostate cancer cells

PDF file - 94KB, Effect of RES on body weight and food consumption in PTEN knockout mice

PDF file - 55KB

PDF file - 60KB, Apoptosis in SIRT1 knockdown DU145 cells

Supplementary Appendix: List of all supplementary data files; Supplemental Methods: Additional clinical details of Samples, and details of whole exome sequencing, targeted sequencing and in silico structure modeling not included in main article

Purpose: Pheochromocytomas and paragangliomas (PPGL) are genetically heterogeneous tumors of neural crest origin, but the molecular basis of most PPGLs is unknown.Experimental Design: We performed exome or transcriptome sequencing of 43 samples from 41 patients. A validation set of 136 PPGLs was used for amplicon-specific resequencing. In addition, a subset of these tumors was subjected to microarray-based transcription, protein expression, and histone methylation analysis by Western blotting or immunohistochemistry. In vitro analysis of mutants was performed in cell lines.Results: We detected mutations in chromatin-remodeling genes, including histone-methyltransferases, histone-demethylases, and histones in 11 samples from 8 patients (20%). In particular, we characterized a new cancer syndrome involving PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic G34W mutation of the histone 3.3 gene, H3F3A. Furthermore, mutations in kinase genes were detected in samples from 15 patients (37%). Among those, a novel germline kinase domain mutation of MERTK detected in a patient with PPGL and medullary thyroid carcinoma was found to activate signaling downstream of this receptor. Recurrent germline and somatic mutations were also detected in MET, including a familial case and sporadic PPGLs. Importantly, in each of these three genes, mutations were also detected in the validation group. In addition, a somatic oncogenic hotspot FGFR1 mutation was found in a sporadic tumor.Conclusions: This study implicates chromatin-remodeling and kinase variants as frequent genetic events in PPGLs, many of which have no other known germline driver mutation. MERTK, MET, and H3F3A emerge as novel PPGL susceptibility genes. Clin Cancer Res; 22(9); 2301–10. ©2015 AACR.

Supplemental Figure 1: Alternative views of Figure 1 displaying mutations in our cohort; Supplemental Figure 2: Histone 3.3 G34W mutation in patient samples; Supplemental Figure 3: In silico analysis of histone 3.3 and G34W mutant; Supplemental Figure 4: Immunohistochemistry of trimethylated Histone 3; Supplemental Figure 5: Expression profiling of pheochromocytomas and paragangliomas with a H3F3A mutation; Supplemental Figure 6: Familial pheochromocytoma segregating with a MET gene mutation

Table S1: Features of the 41 samples of the next-generation sequencing (NGS) cohort; Table S2: Mutations identified in known pheochromocytoma/paraganglioma-related genes in the NGS cohort; Table S3: Features of the validation cohort of 136 pheochromocytoma/paraganglioma samples; Table S4: Pathway analysis of G34�mutant vs. wt pheochromocytomas and paragangliomas from microarray expression ; Table S5: Main clinical features and MET gene variants found in a validation cohort of 136 pheochromocytomas and paragangliomas

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

This file contains detailed methods for publication as supplementary information.

(S1) List of primer pairs used in qPCR, (S2) list of upregulated genes following NQO1 KD, (S3) list of down-regulated genes following NQO1 KD.

(S1) Basal NQO1 levels in different cancer cells, (S2) Transduction efficiency of NQO1 KD, (S3 and S4) Effects of KD on cellular redox state and AR signaling, (S5) heat maps of differential gene expression with NQO1 KD, (S6) inhibition of NQO1 activity on NFkB-p65 signaling, (S7) Quantification of increased nuclear IKKa following NQO1 KD and (S8) Basal IL-8 message in prostate cancer cells.

NADPH reductase NAD(P)H:quinone oxidoreductase 1 (NQO1) is needed to maintain a cellular pool of antioxidants, and this enzyme may contribute to tumorigenesis on the basis of studies in NQO1-deficient mice. In this work, we sought deeper insights into how NQO1 contributes to prostate carcinogenesis, a setting in which oxidative stress and inflammation are established contributors to disease development and progression. In the TRAMP mouse model of prostate cancer, NQO1 was highly expressed in tumor cells. NQO1 silencing in prostate cancer cells increased levels of nuclear IKKα and NF-κB while decreasing the levels of p53, leading to interactions between NF-κB and p300 that reinforce survival signaling. Gene expression analysis revealed upregulation of a set of immune-associated transcripts associated with inflammation and tumorigenesis in cells in which NQO1 was attenuated, with IL8 confirmed functionally in cell culture as one key NQO1-supported cytokine. Notably, NQO1-silenced prostate cancer cells were more resistant to androgen deprivation. Furthermore, NQO1 inhibition increased migration, including under conditions of androgen deprivation. These results reveal a molecular link between NQO1 expression and proinflammatory cytokine signaling in prostate cancer. Furthermore, our results suggest that altering redox homeostasis through NQO1 inhibition might promote androgen-independent cell survival via opposing effects on NF-κB and p53 function. Cancer Res; 74(19); 5644–55. ©2014 AACR.

Supplementary Notes from Classification of Proliferative Pulmonary Lesions of the Mouse

Previous studies from our laboratory have showed that aerobic exercise significantly reduced the number of aggressive poorly differentiated tumors in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Despite these encouraging data the underlying mechanism of how exercise reduces tumor aggressiveness remains undefined. We aimed to fill this scientific gap by utilizing a transcriptomics approach to identify potential mechanisms by which aerobic exercise suppresses prostate tumor aggressiveness. Methods: Twelve TRAMP mice, 8-10 weeks of age, were equally randomized to exercise or control group. Mice in the exercise group were singularly housed in cages with running wheels for 12 weeks. Mice in the control group maintained normal group housing and activity conditions for 12 weeks. At euthanasia, prostate tumors were excised, weighed and processed for immunohistochemistry and transcriptome analysis. Two independent pathologists, blinded to the interventions, performed histological analysis of the genitourinary mass. Outputs of sequencing data were assessed for quality and accuracy. Counts for all known mRNA, differential expression, and heatmap were prepared. Differential expression was filtered to identify genes that had a ≥2-fold change with an adjusted p Results: No significant difference in genitourinary mass, body mass or tumor free body mass was found between groups. Pathology revealed majority of the tissue from the control group exhibited moderate to poorly differentiated tumors (3/6). On the other hand, none of the animals in the exercise intervention group showed such pathology. Four out of five showed well differentiated tumors including prostatic intraepithelial neoplasia (PIN) lesions in one animal. Transcriptomic analysis coupled with gene set enrichment identified pathways associated with triglyceride catabolic process, lipid homeostasis, lipid metabolic process, triglyceride metabolic process to be most impacted. Differentially expressed genes of interest include haptoglobin (HP) and hormone sensitive lipoprotein lipase (Lipe) were significantly lower in the exercise group. Conclusion: Our preliminary findings provide novel evidence suggesting that exercise suppresses prostate tumor aggressiveness, in part, through transcriptomic modulation and altered cellular pathways associated with intratumoral energy metabolism. This project was supported by the National Center Institute designated Mays Cancer Center at UT Health San Antonio. Citation Format: Darpan I. Patel, Paul Rivas, Yidong Chen, Zhao Lai, Robert L. Reddick, Yuji Ikeno, Rita Ghosh, A. Pratap Kumar. Transcriptomic evaluation of exercise-induced suppression of prostate cancer aggressiveness. [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 4230.

There is an urgent need for innovative strategies such as the discovery of adjuvants that can prevent relapse and improve quality of life for patients treated with radiotherapy. Previously we demonstrated the utility of Nexrutine (Nex) as a neo-adjuvant with radiation. Nex was safe and well tolerated in PCa patients and potentiated radiation response in part through downregulation of ribosomal protein S6K (encoded byRPS6KB1). We now show that RPS6KB1 depleted prostate cancer cells with higher basal levels of γ-H2AX, a marker for DNA double strand breaks (i) are more sensitive to radiation and (ii) form smaller tumors with reduced levels of prostate specific antigen (PSA). Depletion of RPS6KB1 hindered DNA double-strand break repair predominantly through the alternate end-joining pathway, induction of G2/M checkpoint and NFκB pathway activation. Collectively these events led to improved radiation sensitivity. We further identified Berberine (Ber), one of the active constituents of Nex as a potential pharmacological inhibitor of RPS6KB1. In an orthotopic implantation model of C4-2B, treatment with Ber alone or Ber plus radiation decreased PSA levels that was sustained during the course of the experiment. On the other hand animals treated with radiation alone developed recurrent cancer as evidenced by a resurgence of PSA. Animals administered Ber followed by XRT intervention had increased levels of RANTES while there was no change in animals that received XRT followed by Ber. The observed reversal of the Bereffect with the sequence of intervention is statistically significant (p=0.0298). Among animals not subject to XRT, the mean PSA increased in those that did not receive Ber relative to those that did; mean difference=-1.93, 95% CI -3.75 to -0.105, p=0.04 with no significant changes in body weight. Notably,RPS6KB1 mRNA levels increased in tumor samples in patients experiencing biochemical recurrence(BCR). Given that rising PSA following conventional therapeutic approaches such as radiation remain a major clinical challenge, targeting RPS6KB1 signaling with radiation therapy is an attractive strategy to prevent BCR. Supported in part by CPRIT RP190012 (APK). Citation Format: Addanki Pratap Kumar, Alison Clark, Michelle Villarreal, Sridharan Jayamohan, Shih-Bo Huang, Suleman S. Hussain, Xiaoyu Yang, Paul Rivas, Darpan Patel, Bethany L. Pierce, Shreya Tripathy, Pawel Osmulski, Maria Gaczynska, Lai Zhao, Li-Ju Wang, Yidong Chen, Caroline Xavier Paul Ezhilan, Mohan Natarajan, Joel E. Michalek, Robert L. Reddick, Rita Ghosh. Intercepting ribosomal protein S6KB1 signaling: Prevention of prostate cancer recurrence [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 4.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Context Mutations in genes encoding for the succinate dehydrogenase (SDH) complex are linked to hereditary paraganglioma syndromes. Paraganglioma syndrome 3 is associated with mutations inSDHC and typically manifests as benign, nonfunctional head and neck paragangliomas. Design We describe a case of a 51-year-old woman who initially presented with diarrhea and hypertension and was found to have a retroperitoneal mass, which was resected with a pathology consistent with paraganglioma. Five years later, her symptoms recurred, and she was found to have new retroperitoneal lymphadenopathy and lytic lesions in the first lumbar vertebral body and the right iliac crest, which were visualized on CT scan and octreoscan but not on iodine-123-meta-iodobenzylguanidine (123I-MIBG) and bone scans. She had significantly elevated 24-hour urine norepinephrine and dopamine. The patient received external beam radiation and a series of different antineoplastic agents. Her disease progressed, and she eventually expired within 2 years. Genetic testing revealed a heterozygousSDHC c.43C>T, p.Arg15X mutation, which was also detected in her daughter and her grandson, both of whom have no biochemical or imaging evidence of paraganglioma syndrome yet. Conclusion We report a unique case of functional, metastatic abdominal paraganglioma associated withSDHC germline mutation. Our case exemplifies thatSDHC germline mutation has variable penetrance, which may manifest with an aggressive biology that could be missed by a123I-MIBG scan.

NAD[P]H:quinone oxidoreductase 1 (NQO1) regulates cell fate decisions in response to stress. Oxidative stress supports cancer maintenance and progression. Previously we showed that knockdown of NQO1 (NQO1low) prostate cancer cells upregulate pro-inflammatory cytokines and survival under hormone-deprived conditions. Here, we tested the ability of NQO1low cells to form tumors. We found NQO1low cells form aggressive tumors compared with NQO1high cells. Biopsy specimens and circulating tumor cells showed biochemical recurrent prostate cancer was associated with low NQO1. NQO1 silencing was sufficient to induce SMAD-mediated TGFβ signaling and mesenchymal markers. TGFβ treatment decreased NQO1 levels and induced molecular changes similar to NQO1 knockdown cells. Functionally, NQO1 depletion increased migration and sensitivity to oxidative stress. Collectively, this work reveals a possible new gatekeeper role for NQO1 in counteracting cellular plasticity in prostate cancer cells. Further, combining NQO1 with TGFβ signaling molecules may serve as a better signature to predict biochemical recurrence., Thapa et al find that depletion of the antioxidant enzyme NAD[P]H:Quinone Oxidoreductase 1 (NQO1) accelerates prostate tumorigenesis and induces the epithelial-to-mesenchymal transition by activating TGFβ signaling. They also find that low NQO1 is associated with mesenchymal signature and biochemical recurrence in clinical samples.

// Tai Qin 1, 2 , Lindsey Barron 1 , Lu Xia 1, 3 , Haojie Huang 5 , Maria M. Villarreal 4 , John Zwaagstra 9 , Cathy Collins 9 , Junhua Yang 1 , Christian Zwieb 4 , Ravindra Kodali 8 , Cynthia S. Hinck 8 , Sun Kyung Kim 4 , Robert L. Reddick 6 , Chang Shu 2 , Maureen D. O’Connor-McCourt 9 , Andrew P. Hinck 8 , Lu-Zhe Sun 1, 7 1 Department of Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX, USA 2 Department of Vascular Surgery, Second Xiangya Hospital and Xiangya School of Medicine, Central South University, Hunan, China 3 Department of Gynecology and Obstetrics, Xiangya Hospital and Xiangya School of Medicine, Central South University, Hunan, China 4 Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA 5 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA 6 Department of Pathology, University of Texas Health Science Center, San Antonio, TX, USA 7 Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, Texas, USA 8 Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA USA 9 National Research Council Human Health Therapeutics Portfolio, Montreal, Quebec, Canada, Maureen O'Connor-McCourt is currently affiliated with Formation Biologics, Montreal, Quebec, Canada Correspondence to: Lu-Zhe Sun, email: SUNL@uthscsa.edu Andrew P. Hinck, email: ahinck@pitt.edu Keywords: TGF-β trap, RER, tumorigenesis, Pten, prostate cancer Received: August 15, 2016 Accepted: November 07, 2016 Published: November 14, 2016 ABSTRACT The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

Lyme disease (LD) is a systemic disorder caused by Borrelia burgdorferi. Lyme spirochetes encode for a functional 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR EC 1.1.1.88) serving as a rate limiting enzyme of the mevalonate pathway that contribute to components critical for cell wall biogenesis. Statins have been shown to inhibit B. burgdorferi in vitro. Using a mouse model of Lyme disease, we found that statins contribute to reducing bacterial burden and altering the murine immune response to favor clearance of spirochetes.

Purpose: Pheochromocytomas and paragangliomas (PPGL) are genetically heterogeneous tumors of neural crest origin, but the molecular basis of most PPGLs is unknown. Experimental Design: We performed exome or transcriptome sequencing of 43 samples from 41 patients. A validation set of 136 PPGLs was used for amplicon-specific resequencing. In addition, a subset of these tumors was subjected to microarray-based transcription, protein expression, and histone methylation analysis by Western blotting or immunohistochemistry. In vitro analysis of mutants was performed in cell lines. Results: We detected mutations in chromatin-remodeling genes, including histone-methyltransferases, histone-demethylases, and histones in 11 samples from 8 patients (20%). In particular, we characterized a new cancer syndrome involving PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic G34W mutation of the histone 3.3 gene, H3F3A. Furthermore, mutations in kinase genes were detected in samples from 15 patients (37%). Among those, a novel germline kinase domain mutation of MERTK detected in a patient with PPGL and medullary thyroid carcinoma was found to activate signaling downstream of this receptor. Recurrent germline and somatic mutations were also detected in MET, including a familial case and sporadic PPGLs. Importantly, in each of these three genes, mutations were also detected in the validation group. In addition, a somatic oncogenic hotspot FGFR1 mutation was found in a sporadic tumor. Conclusions: This study implicates chromatin-remodeling and kinase variants as frequent genetic events in PPGLs, many of which have no other known germline driver mutation. MERTK, MET, and H3F3A emerge as novel PPGL susceptibility genes. Clin Cancer Res; 22(9); 2301–10. ©2015 AACR.

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O6-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 μmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O6-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma. Cancer Prev Res; 9(3); 245–52. ©2015 AACR.

// Izhar Batth 1, 8 , Huiyoung Yun 2 , Suleman Hussain 2 , Peng Meng 1, 7 , Pawel Osmulski 3 , Tim Hui-Ming Huang 3, 5 , Roble Bedolla 1 , Amanda Profit 4 , Robert Reddick 4 , Addanki Kumar 1, 2, 3, 5, 6 1 Department of Urology, The University of Texas Health Science Center, San Antonio, TX, USA 2 Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX, USA 3 Department of Molecular Medicine, The University of Texas Health Science Center, San Antonio, TX, USA 4 Department of Pathology, The University of Texas Health Science Center, San Antonio, TX, USA 5 Cancer Therapy and Research Center, The University of Texas Health Science Center, San Antonio, TX, USA 6 The University of Texas Health Science Center at San Antonio and South Texas Veterans Health Care System, San Antonio, TX, USA 7 Current address: Life Sciences Division, Lawrence Berkley National Laboratory, Berkley, CA, USA 8 Current address: Department of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Addanki Kumar, e-mail: kumara3@uthscsa.edu Keywords: castrate resistant prostate cancer, apoptosis, FLIP, RON, MST1R Received: November 18, 2015 Accepted: January 29, 2016 Published: February 9, 2016 ABSTRACT Castrate-resistant prostate cancer (CRPC) is the fatal form of prostate cancer. Although reactivation of androgen receptor (AR) occurs following androgen deprivation, the precise mechanism involved is unclear. Here we show that the receptor tyrosine kinase, RON alters mechanical properties of cells to influence epithelial to mesenchymal transition and functions as a transcription factor to differentially regulate AR signaling. RON inhibits AR activation and subset of AR-regulated transcripts in androgen responsive LNCaP cells. However in C4-2B, a castrate-resistant sub-line of LNCaP and AR-negative androgen independent DU145 cells, RON activates subset of AR-regulated transcripts. Expression of AR in PC-3 cells leads to activation of RON under androgen deprivation but not under androgen proficient conditions implicating a role for RON in androgen independence. Consistently, RON expression is significantly elevated in castrate resistant prostate tumors. Taken together our results suggest that RON activation could aid in promoting androgen independence and that inhibition of RON in combination with AR antagonist(s) merits serious consideration as a therapeutic option during hormone deprivation therapy.

The purpose of this investigation was to compare the antitumorigenic effects of the natural product Nexrutine to voluntary wheel running (VWR) in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Forty-five, 10-week old TRAMP mice were randomized to either receive free access to the running wheel, Nexrutine pelleted into chow at 600 mg/kg or no treatment control. Mice were serially sacrificed at weeks 4, 8,12 and 20 weeks. Palpable tumors, body weight, food consumption and running wheel activity were monitored weekly. At necropsy, tumors and serum were harvested and stored for analysis. Serum was used to quantify circulating cytokines in 4 and 20 week time points. Nexrutine supplementation led to a 66% protection against high grade tumors. Exercise resulted in a 60% protection against high grade tumors. Both interventions reduced concentrations of IL-1α. Exercise also significantly lowered concentrations of eotaxin, IL-5, IL-12(p40) and VEGF. While there were no significant differences at baseline, exercise mice had significantly lower IL-5 and VEGF compared to control at the 20 week time point. Nexrutine also significantly reduced circulating IL-9 concentrations. No significant differences were observed when compared to the control group. Immunohistochemistry of tumor sections showed significantly lower expression of pAkt in Nexrutine fed mice with no visible differences for NFκB. In conclusion, both Nexrutine and exercise suppressed tumor growth. Though similar outcomes were seen in this comparative effectiveness study, the mechanisms by which exercise and Nexrutine exert this benefit may focus on different pathways.

Nuclear factor (NF)κB is a transcription factor that controls immune and inflammatory signaling pathways. In skeletal muscle, NFκB has been implicated in the regulation of metabolic processes and tissue mass, yet its affects on mitochondrial function in this tissue are unclear. To investigate the role of NFκB on mitochondrial function and its relationship with muscle mass across the life span, we study a mouse model with muscle-specific NFκB suppression (muscle-specific IκBα super-repressor [MISR] mice). In wild-type mice, there was a natural decline in muscle mass with aging that was accompanied by decreased mitochondrial function and mRNA expression of electron transport chain subunits. NFκB inactivation downregulated expression of PPARGC1A, and upregulated TFEB and PPARGC1B. NFκB inactivation also decreased gastrocnemius (but not soleus) muscle mass in early life (1–6 months old). Lower oxygen consumption rates occurred in gastrocnemius and soleus muscles from young MISR mice, whereas soleus (but not gastrocnemius) muscles from old MISR mice displayed increased oxygen consumption compared to age-matched controls. We conclude that the NFκB pathway plays an important role in muscle development and growth. The extent to which NFκB suppression alters mitochondrial function is age dependent and muscle specific. Finally, mitochondrial function and muscle mass are tightly associated in both genotypes and across the life span.

AIM/HYPOTHESIS: Endothelial dysfunction due to impaired endothelial nitric oxide synthase (eNOS) activity is a key factor implicated in the progression of diabetic nephropathy. Previous studies demonstrated that global deficiency of eNOS in diabetic mice exacerbated renal lesions. In this study we investigated whether targeted expression of eNOS and enhanced bioavailability of nitric oxide in renal microvascular endothelial cells rescues diabetic nephropathy. In transgenic mice, overexpression of eNOS has been shown to protect against tissue injury; however, the effect of targeted expression of eNOS in the kidney microvasculature has not been examined. METHODS: To determine if overexpression of eNOS in endothelial cells of diabetic mice ameliorates renal lesions, transgenic mice selectively expressing eNOS in endothelial cells (eNOSTg) were cross bred with Ins2Akita type-1 (AK) diabetic mice to generate eNOSTg/AK mice. Kidneys from wild type, eNOSTg, AK and eNOSTg/AK mice were assessed for morphology and reactive oxygen species production; each group was analyzed for blood glucose levels, kidney/body weight and urine albumin/creatinine levels. RESULTS: AK and eNOSTg/AK mice were hyperglycemic. eNOSTg mice unexpectedly showed evidence of glomerular injury with segmental mesangiolysis and occasional microaneurysms. As expected, glomeruli of AK mice showed diabetic changes with matrix expansion and early nodules. Notably, overexpression of eNOS in eNOSTg/AK mice led to increased glomerular/endothelial injury that was associated with increased superoxide levels and renal dysfunction. Results indicate for the first time that overexpressing eNOS in endothelial cells cannot ameliorate diabetic lesions, but paradoxically leads to progression of nephropathy likely due to the superoxide generated by the uncoupling of eNOS. CONCLUSION/INTERPRETATION: The eNOSTg/AK model will be useful for elucidating mechanisms that regulate eNOS function and may suggest therapeutic strategies to improve endothelial function and prevent progression of diabetic renal disease.

PPARδ (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPARδ agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPARδ in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPARδ expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPARδ expression in bone marrow cells followed by transplantation of the cells into LDLR −/− mice. We found that knockdown of PPARδ expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPARδ led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPARδ knockdown in hematopoietic cells is able to ameliorate atherosclerosis.

SIRT1 is a NAD+ dependent deacetylase known to regulate a plethora of biological processes through posttranslational regulation of proteins including those that function as tumor promoters and suppressors. In order to define the role of SIRT1 in prostate pathogenesis, we used 2 mouse models: (i) PTEN knockout (PTENKO) mouse model by pharmacological activation of SIRT1 with resveratrol (RES) a known activator of SIRT1, & (ii) orthotopic implantation model with genetic silencing of SIRT1 (SIRT1 shRNA). We also used genetic and pharmacologic inhibition of SIRT1 in cell culture models to understand the mechanism. We tested whether SIRT1 modulation is beneficial when targeted early (before the establishment of prostatic lesions) or late (after the establishment of prostatic lesions) in the PTENKO model. RES intervention was initiated in 4-5-week (early intervention) and 10-15-week-old (late intervention) PTENKO mice. Analyses of samples collected longitudinally during progression revealed that early intervention with RES reduced incidence of high-grade prostate intraepithelial neoplastic lesions (HGPIN) when given for 14 weeks with no significant difference at 7 or at 11 weeks. On the other hand, late intervention after the establishment of HGPIN lesions with RES had no beneficial effect. Importantly, longer treatment duration (28 weeks) resulted in significantly increased incidence of invasive prostate carcinoma. Furthermore, RES had no significant effect on the development of orthotopic prostate tumors following implantation of LNCaP cells in nude mice. In contrast, orthotopic implantation of SIRT1 stably silenced LNCaP cells showed significant impairment in tumor development. Immunohistochemical evaluation showed nuclear localization of SIRT1 in human prostate tumors and was associated with increased risk of biochemical recurrence. Mechanistic investigations revealed (i) suppression of AR signaling in hormone-sensitive LNCaP but not in castration-resistant 22Rv1 cells; (ii) RNAseq coupled with gene ontology enrichment analysis using SIRT1 silenced cells under conditions of androgen stimulation and inhibition identified genes involved in cell cycle checkpoint and senescence as top pathways affected by SIRT1 loss of function. In silicoanalysis shows that the identified SIRT1-regulated targets are associated with disease aggressiveness and poor disease-free survival. Taken together these data demonstrate that (i) SIRT1 plays a contextual role during prostate pathogenesis by functioning as tumor suppressor during early stage and as a tumor promoter during late stage; (ii) SIRT1 inhibition suppresses tumor development and (iii) RES is a better preventive than therapeutic agent. Therefore, our findings offer promising avenues to develop SIRT1-regulated pathways as novel therapeutic targets to inhibit prostate cancer recurrence. Supported by CPRIT Training Grant RP 170345 (SH) and CPRIT RP 150166 (APK) Citation Format: Shih-Bo Huang, Dinesh Thapa, Roble G. Bedolla, Amanda R. Muñoz, Xiaoyu Yang, Paul Rivas, Robert L. Reddick, Hiroshi Miyamoto, Rita Ghosh, Addanki Pratap Kumar. SIRT1 functions as a double-edged sword 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 5082.

Myriad infectious organisms can infect the endocardium, myocardium, and pericardium, including bacteria, fungi, parasites, and viruses. Significant cardiac infections are rare in the general population but are associated with high morbidity and mortality as well as increased risk in certain populations, such as the elderly, those undergoing cardiac instrumentation, and intravenous drug abusers. Diagnostic imaging of cardiac infections plays an important role despite its variable sensitivity and specificity, which are due in part to the nonspecific manifestations of the central inflammatory process of infection and the time of onset with respect to the time of imaging. The primary imaging modality remains echocardiography. However, cardiac computed tomography and magnetic resonance (MR) imaging have emerged as the modalities of choice wherever available, especially for diagnosis of complex infectious complications including abscesses, infected prosthetic material, central lines and instruments, and the cryptic manifestations of viral and parasitic diseases. MR imaging can provide functional, morphologic, and prognostic value in a single examination by allowing characterization of inflammatory changes from the acute to chronic stages, including edema and the patterns and extent of delayed gadolinium enhancement. We review the heterogeneous and diverse group of cardiac infections based on their site of primary cardiac involvement with emphasis on their cross-sectional imaging manifestations. Online supplemental material is available for this article. (©)RSNA, 2016.

Over the last 30 years, little improvement has been made to the 5 year survival rate of pancreatic cancer (PanCA) patients. While survival has increase by 2% over the last several years, the current rate is still less than 8%. This depressing fact demonstrates the importance of developing or improving therapies to more effectively manage this disease. Along these lines, published studies from our laboratory demonstrated the anti-tumorigenic potential of the cork tree bark extract, Nexrutine® (Nx). Nx suppressed growth of pancreatic cancer cells through downregulation of STAT3/NF-κB activation. Subsequent biochemical and molecular investigations revealed palmatine (PMT) (i) as an active constituent of Nx able to suppress the growth of pancreatic cancer cells; (ii) synergizes with gemcitabine (GEM); and (iii) downregulates GLI1, COL1A1 and Survivin. Despite such promising in vitro observations however, the in vivo relevance of PMT is undefined. Furthermore, it is unclear if PMT can recapitulate the biological activities of Nx in vivo. In this investigation, we tested the hypothesis that PMT recapitulates the biological activities of Nx and enhances GEM activity. This hypothesis was tested by comparing the efficacy of Nx and PMT using (i) athymic mice implanted with Capan-2 cells; (ii) a syngenic mouse model using C57BL/6 mice implanted with KPC-GFP-Luc cells; and (iii) a short term ex vivo model utilizing cells isolated from primary pancreatic tumors following surgical resection. Additionally, in vitro experiments were also done to assess the underlying molecular mechanism. Analysis of these data show that both Nx and PMT are well tolerated in vivo and a significant reduction in the levels of serum inflammatory cytokines including IL-6, granulocyte-colony stimulating factor (G-CSF), and CXCL1. Interestingly, animals receiving PMT, but not Nx, showed a trend towards decreased pancreatic tumor weight that was associated with histopathological changes. Investigation into the potential mechanism revealed that Nx and PMT mediated inhibition of STAT3, EP4, Src, TrkA, and RPS6 activities may contribute to the observed growth inhibitory and anti-inflammatory effects. Incredibly, our ex vivo analysis of patient derived PanCA cells demonstrated that both Nx and PMT could inhibit the growth of these cells. Collectively, our data demonstrates PMT recapitulates biological activities of Nx and that there is potential for developing PMT as an agent for clinical management of PanCA. Supported by NCCIH (R01 AT007448; APK) and VA-MERIT Award (I01 BX 000766; APK). Citation Format: Amanda R. Muñoz, Roble Bedolla, Shih-Bo Huang, Xiaou Yang, Paul Rivas, Robert Reddick, Martha Hanes, Glenn Halff, Rita Ghosh, Addanki P. Kumar. Palmatine as a potential pancreatic cancer therapeutic agent [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 5823.

The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2(Akita) spontaneously diabetic (type-1) mice with l-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKβ) inhibitor, salsalate, in the diet regimen in combination with l-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2(Akita) diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with l-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2(Akita) cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities.

The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

With a 5 year survival of less than 8%, the poor prognosis of pancreatic cancer (PanCA) underlines the importance of improving therapies for effective management of this disease. In previous studies, we identified the tumor cell growth inhibitory activities for Nexrutine® (Nx, a bark extract from Phellodendron amurense) and palmatine (PMT) which is a constituent of Nx. These studies revealed an important role for downregulation of KRAS downstream effectors including GLI, STAT3 and NF-κB in mediating growth inhibitory effects. Remarkably, both Nx and PMT exerted synergistic growth inhibitory effects in combination with the conventional chemotherapeutic agent, Gemcitabine (GEM). Despite such promising in vitro observations, the in vivo efficacy of Nx or PMT had not been established. In this investigation, we evaluated the ability of Nx and PMT to (i) prevent the development of pancreatic tumors; and (ii) inhibit the growth of patient derived pancreatic cancer tissue in short term ex vivo cultures. Studies were also conducted to investigate the underlying molecular mechanism using cell culture models. Our results show that both Nx and PMT are well tolerated in vivo as evidenced by a lack of significant changes in the body weights of athymic mice implanted with Capan-2 cells. Interestingly, both Nx and PMT showed a significant decrease in the circulating levels of inflammatory molecules including IL-6 and CXCL1. In this first proof-of-concept study, PMT treated animals demonstrated a trend towards decreased pancreatic tumor weight with associated histopathogical changes. Remarkably, both Nx and PMT inhibited the growth of patient derived pancreatic cancer cells. Mechanistic investigations identified downregulation of STAT3, TrkA, Src, and RPS6 activities as potential contributors to Nx or PMT-induced synergistic growth inhibitory effects with GEM. Further investigation of STAT3 signaling revealed the involvement of the prostaglandin receptor, EP4, in a potential feedback loop with STAT3 and in asserting STAT3-mediated inhibition of autophagy. Taken together these data support potential utility for Nx and/or PMT in the management of PanCA and suggest that these agents use multiple mechanisms to affect growth of pancreatic tumors. Supported by NCCIH (R01 AT007448; APK) and VA-MERIT Award (I01 BX 000766; APK). Citation Format: Amanda R. Munoz, Roble G. Bedolla, Paul Rivas, Divya Chakravarthy, Robert L. Reddick, Martha A. Hanes, Glenn A. Halff, Rita Ghosh, Addanki P. Kumar. Nexrutine and palmatine mediated effects on the STAT3/EP4/IL-6 axis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2017-5268

Radiation Therapy (RT) is a definitive treatment for early-localized prostate cancer (PCA), but is associated with side effects which impair quality of life in addition to development of radioresistance. PI3K/Akt/mTOR signaling is one of the contributors to therapeutic resistance, including radioresistance. Ribosomal protein S6 (rpS6), a downstream effector of PI3K/Akt/mTOR signaling, mediates radioresistance by increasing protein synthesis, cell survival and epithelial mesenchymal transition. Also, increased activation of rpS6 is correlated with poor survival in PCA. Therefore, downregulation of rpS6 could decrease RT induced toxic side effects by sensitizing tumor cells. Based on published evidence demonstrating tumor growth inhibitory activities, we tested if Nexrutine® (Nx), an inexpensive OTC herbal supplement from Phellodendron amurense bark extract, could potentiate RT by inhibiting rpS6 activation. Using clonogenic assays, low dose RT in combination with Nx was found to have similar inhibition of surviving fraction compared to high dose RT in androgen independent PC-3 cells. Isobologram analysis of these data depicted strong synergism. In addition, increased activation of Akt/NFKB signaling molecules was observed in PC-3 cells exposed to radiation. The observed radiation-induced increase in these signaling molecules was either abolished (p-rpS6, p-NFKB and p-p70S6K) or decreased (p-Akt) in cells pretreated with Nx (8 hours) prior to RT. Additionally, Nx pretreatment prolonged the G2/M arrest caused by RT in PC-3 cells. Strikingly, knockdown of rpS6 in PC-3 cells reversed the observed effects of Nx, indicating the importance of rpS6 in mediating these changes. Furthermore, administration of Nx in combination with RT inhibited prostate tumor progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with no prominent toxicity. Notably, immunohistochemical analysis revealed decreased levels of p-mTOR, p70S6K, NFKB, Ki67 and Cyclin D1 in the prostate in combination group of animals compared to monotherapy. Taken together, our data suggest that Nx sensitizes PCA cells by down regulating rpS6 and delays progression to lethal disease. Remarkably, in a recent phase 0/1 study patients (81%) receiving Nx showed decreased PSA levels with no signs of grade 3 toxicity. Thus, Nx shows immense potential for use as an adjuvant in combination with conventional therapy for effective clinical management of PCA. Supported by NCCAM (R01 AT-007448) and VA-MERIT Award (I01 BX 000766; APK) Citation Format: Suleman S. Hussain, Paul Rivas, Roble G. Bedolla, Nikos Papanikolaou, Robert L. Reddick, Brad H. Pollock, Daniel C. Chan, Rita Ghosh, Addanki P. Kumar. Downregulation of ribosomal protein S6 overcomes radioresistance in prostate cancer. [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 1797. doi:10.1158/1538-7445.AM2015-1797