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PDF file - 94K, Fecal colonocytes disorder strength in the control (saline-injected) rats over time.

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RNASeq quality control data

In vitro proliferation of fibroblasts from AA and EA patients. Proliferation of PCa cell lines exposed to CM from AA and EA prostate fibroblasts

Differential expression of selected secreted cytokines and densitometric quantitation

Progress in prostate cancer racial disparity research has been hampered by a lack of appropriate research tools and better understanding of the tumor biology. Recent gene expression studies suggest that the tumor microenvironment (TME) may contribute to racially disparate clinical outcomes in prostate cancer. Analysis of the prostate TME has shown increased reactive stroma associated with chronic inflammatory infiltrates in African-American (AA) compared with European-American (EA) patients with prostate cancer. To better understand stromal drivers of changes in TME, we isolated prostate fibroblasts (PrF) from AA (PrF−AA) and EA (PrF−EA) prostate cancer tissues and studied their functional characteristics. PrF−AA showed increased growth response to androgens FGF2 and platelet-derived growth factor. Compared with PrF−EA, conditioned media from PrF−AA significantly enhanced the proliferation and motility of prostate cancer cell lines. Expression of markers associated with myofibroblast activation (αSMA, vimentin, and tenascin-C) was elevated in PrF−AA. In vivo tumorigenicity of an AA patient–derived prostatic epithelial cell line E006AA was significantly increased in the presence of PrF−AA compared with PrF−EA, and RNA-seq data and cytokine array analysis identified a panel of potential proinflammatory paracrine mediators (BDNF, CHI3L1, DPPIV, FGF7, IL18BP, IL6, and VEGF) to be enriched in PrF−AA. E006AA cell lines showed increased responsiveness to BDNF ligand compared with EA-derived LNCaP and C4-2B cells. Addition of a TrkB-specific antagonist significantly reduced the protumorigenic effects induced by PrF−AA compared with PrF−EA. These findings suggest that fibroblasts in the TME of AA patients may contribute to the health disparity observed in the incidence and progression of prostate cancer tumors.Significance: These findings suggest that stromal cells in the tumor microenvironment of African-American men promote progression of prostate cancer by increasing levels of a specific set of pro-inflammatory molecules compared with European-American men.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6134/F1.large.jpg. Cancer Res; 78(21); 6134–45. ©2018 AACR.

Supplementary Methods

Assessment of collagen deposition and neoangiogenesis in PCa samples from AA and EA patients. Quantitation of IHC staining of stromal markers

IHC staining and quantitation of FAP in AA andEA patient samples

Lipid droplet (LD) accumulation in cancer results from aberrant metabolic reprograming due to increased lipid uptake, diminished lipolysis and/or de novo lipid synthesis. Initially implicated in storage and lipid trafficking in adipocytes, LDs are more recently recognized to fuel key functions associated with carcinogenesis and progression of several cancers, including prostate cancer (PCa). However, the mechanisms controlling LD accumulation in cancer are largely unknown. EPHB2, a tyrosine kinase (TKR) ephrin receptor has been proposed to have tumor suppressor functions in PCa, although the mechanisms responsible for these effects are unclear. Given that dysregulation in TRK signaling can result in glutaminolysis we postulated that EPHB2 might have potential effects on lipid metabolism. Knockdown strategies for EPHB2 were performed in prostate cancer cells to analyze the impact on the net lipid balance, proliferation, triacylglycerol-regulating proteins, effect on LD biogenesis, and intracellular localization of LDs. We found that EPHB2 protein expression in a panel of human-derived prostate cancer cell lines was inversely associated with in vivo cell aggressiveness. EPHB2 silencing increased the proliferation of prostate cancer cells and concurrently induced de novo LD accumulation in both cytoplasmic and nuclear compartments as well as a “shift” on LD size distribution in newly formed lipid-rich organelles. Lipid challenge using oleic acid exacerbated the effects on the LD phenotype. Loss of EPHB2 directly regulated key proteins involved in maintaining lipid homeostasis including, increasing lipogenic DGAT1, DGAT2 and PLIN2 and decreasing lipolytic ATGL and PEDF were observed with EPHB2 silencing. A DGAT1-specific inhibitor abrogated LD accumulation and proliferative effects induced by EPHB2 loss. In conclusion, we highlight a new anti-tumor function of EPHB2 in lipid metabolism through regulation of DGAT1 and ATGL in prostate cancer. Blockade of DGAT1 in EPHB2-deficient tumors appears to be effective in restoring the lipid balance and reducing tumor growth.

The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for β-galactosidase. Senescence-associated β-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE.

Through stromal-epithelial interactions, carcinoma associated fibroblasts (CAF) play a critical role in tumor growth and progression. Activation of erythrophoyetin-producing human hepatocellular (Eph) receptors has been implicated in cancer. Eph receptor interactions with Ephrin ligands lead to bidirectional signals in the recipient and effector cells. The consequences of continuous reverse Ephrin signaling activation in fibroblasts on prostate cancer (PCa) is unknown. When compared to benign prostate fibroblast, CAF displayed higher expression of Ephrin B1, B2, and B3 ligands (EFNB1, EFNB2, and EFNB3). In this study, we found that continuous activation of EFNB1 and EFNB3 in a benign human prostate stromal cell line (BHPrS1) increased the expression of CAF markers and induced a CAF phenotype. BHPrS1EFNB1 and BHPrS1EFNB3 displayed a pro-tumorigenic secretome with multiple effects on neovascularization, collagen deposition, and cancer cell proliferation, overall increasing tumorigenicity of a premalignant prostate epithelial cell line BPH1 and PCa cell line LNCaP, both in vitro and in vivo. Inhibition of Src family kinases (SFK) in BHPrS1EFNB1 and BHPrS1EFNB3 suppressed EFNB-induced ɑ-SMA (Alpha-smooth muscle actin) and TN-C (Tenascin-C) in vitro. Our study suggests that acquisition of CAF characteristics via SFK activation in response to increased EFNB ligands could promote carcinogenesis via modulation of TME in PCa.

Background To assess the feasibility of a novel DNA-based probe panel to detect copy number alterations (CNAs) in prostate tumor DNA and its performance for predicting clinical progression. Methods A probe panel was developed and optimized to measure CNAs in trace amounts of tumor DNA (2 ng) isolated from formalin-fixed paraffin-embedded tissues. Ten genes previously associated with aggressive disease were targeted. The panel's feasibility and performance were assessed in 175 prostate cancer (PCa) patients who underwent radical prostatectomy with a median 10-year follow-up, including 42 men who developed disease progression (either metastasis and/or PCa-specific death). Association with disease progression was tested using univariable and multivariable analyses. Results The probe panel detected CNAs in all 10 genes in tumor DNA isolated from either diagnostic biopsies or surgical specimens. A four-gene model (PTEN/MYC/BRCA2/CDKN1B) had the strongest association with disease progression; 64.3% of progressors and 22.5% of non-progressors had at least one CNA in these four genes, odds ratio (OR) (95% confidence interval) = 6.21 (2.77-13.87), P = 8.48E-06. The association with disease progression remained significant after adjusting for known clinicopathological variables. Among the seven progressors of the 65 patients with clinically low-risk disease, three (42.9%) had at least one CNA in these four genes. Conclusions The probe panel can detect CNAs in trace amounts of tumor DNA from biopsies or surgical tissues at the time of diagnosis or surgery. CNAs independently predict metastatic/lethal cancer, particularly among men with clinically low-risk disease at diagnosis. If validated, this may improve current abilities to assess tumor aggressiveness.

Lipid droplets (LDs) utilize microtubules (MTs) to participate in intracellular trafficking of cargo proteins. Cancer cells accumulate LDs and acidify their tumor microenvironment (TME) by increasing the proton pump V-ATPase. However, it is not known whether these two metabolic changes are mechanistically related or influence LD movement. We postulated that LD density and velocity are progressively increased with tumor aggressiveness and are dependent on V-ATPase and the lipolysis regulator pigment epithelium-derived factor (PEDF). LD density was assessed in human prostate cancer (PCa) specimens across Gleason scores (GS) 6–8. LD distribution and velocity were analyzed in low and highly aggressive tumors using live-cell imaging and in cells exposed to low pH and/or treated with V-ATPase inhibitors. The MT network was disrupted and analyzed by α-tubulin staining. LD density positively correlated with advancing GS in human tumors. Acidification promoted peripheral localization and clustering of LDs. Highly aggressive prostate, breast, and pancreatic cell lines had significantly higher maximum LD velocity (LDVmax) than less aggressive and benign cells. LDVmax was MT-dependent and suppressed by blocking V-ATPase directly or indirectly with PEDF. Upon lowering pH, LDs moved to the cell periphery and carried metalloproteinases. These results suggest that acidification of the TME can alter intracellular LD movement and augment velocity in cancer. Restoration of PEDF or blockade of V-ATPase can normalize LD distribution and decrease velocity. This study identifies V-ATPase and PEDF as new modulators of LD trafficking in the cancer microenvironment. This study investigates lipid droplet (LD) trafficking and LD velocity (LDV) in cancer. LD density correlated with prostate tumor grade and high grade tumors had significantly higher LDV when compared to low grade tumors. Acidifying conditions drove up velocities and LDV was normalized with blockade of H+ proton pump, V-ATPase.

Obesity is a worldwide epidemic associated with increased risk and progression of colon cancer. Here, we aimed to determine the role of adipose triglyceride lipase (ATGL), responsible for intracellular lipid droplet (LD) utilization, in obesity-driven colonic tumorigenesis. In local colon cancer patients, significantly increased ATGL levels in tumor tissue, compared to controls, were augmented in obese individuals. Elevated ATGL levels in human colon cancer cells (CCC) relative to non-transformed were augmented by an obesity mediator, oleic acid (OA). In CCC and colonospheres, enriched in colon cancer stem cells (CCSC), inhibition of ATGL prevented LDs utilization and inhibited OA-stimulated growth through retinoblastoma-mediated cell cycle arrest. Further, transcriptomic analysis of CCC, with inhibited ATGL, revealed targeted pathways driving tumorigenesis, and high-fat-diet obesity facilitated tumorigenic pathways. Inhibition of ATGL in colonospheres revealed targeted pathways in human colonic tumor crypt base cells (enriched in CCSC) derived from colon cancer patients. In CCC and colonospheres, we validated selected transcripts targeted by ATGL inhibition, some with emerging roles in colonic tumorigeneses (ATG2B, PCK2, PGAM1, SPTLC2, IGFBP1, and ABCC3) and others with established roles (MYC and MUC2). These findings demonstrate obesity-promoted, ATGL-mediated colonic tumorigenesis and establish the therapeutic significance of ATGL in obesity-reinforced colon cancer progression.

Obesity is a risk factor for complications in singleton and twin pregnancies; however, there are limited data regarding maternal body mass index (BMI) in the setting of twin-twin transfusion syndrome (TTTS). We hypothesized that increased BMI in TTTS is associated with adverse perinatal outcomes and vascular pathology. A retrospective study of twin reversed arterial perfusion (n = 4), selective intrauterine growth restriction (n = 10) and TTTS (n = 33) was conducted. Treatment included fetoscopic laser photocoagulation (FLP) (n = 35) or Solomon technique (n = 12). Ex vivo placental intravascular injections, immunohistochemistry, and perinatal outcomes were compared by maternal BMI. In pregnancy complicated by TTTS, 16/33 women were obese (BMI > 30 kg/m2) and 11/33 were overweight (BMI 25–29.9 kg/m2). Women who were overweight or obese had an increased rate of premature rupture of membranes (PPROM), cesarean delivery, and/or concomitant co-morbidities when compared to the normal weight group. Duration of neonatal intensive care unit (NICU) admission was longer in neonates of overweight/obese women versus normal weight. Placental examination of FLP sites in the obese group showed larger infarcts, increased adipose triglyceride lipase, and a proangiogenic phenotype. Increased BMI is common in our TTTS cohort and it is associated with higher rate of co-morbidity, PPROM, prolonged NICU stay, and an imbalance of placental metabolic and vascular mediators.

Background & Aims: Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth. Methods: FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3). Results: In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth. Conclusions: We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment. Keywords: Colon Cancer, Inflammation, FOXO3, RNAseq

Autoimmune (AI) diseases can affect many organs; however, the prostate has not been considered to be a primary target of these systemic inflammatory processes. Here, we utilize medical record data, patient samples, and in vivo models to evaluate the impact of inflammation, as seen in AI diseases, on prostate tissue. Human and mouse tissues are used to examine whether systemic targeting of inflammation limits prostatic inflammation and hyperplasia. Evaluation of 112,152 medical records indicates that benign prostatic hyperplasia (BPH) prevalence is significantly higher among patients with AI diseases. Furthermore, treating these patients with tumor necrosis factor (TNF)-antagonists significantly decreases BPH incidence. Single-cell RNA-seq and in vitro assays suggest that macrophage-derived TNF stimulates BPH-derived fibroblast proliferation. TNF blockade significantly reduces epithelial hyperplasia, NFκB activation, and macrophage-mediated inflammation within prostate tissues. Together, these studies show that patients with AI diseases have a heightened susceptibility to BPH and that reducing inflammation with a therapeutic agent can suppress BPH.

Benign prostatic hyperplasia (BPH) is ostensibly linked to autoimmune (AI) diseases, but whether the prostate is a target of systemic inflammation associated with AI conditions is unknown. Prostatic inflammation is linked to fibrosis, hyperplasia, and reduced responses to BPH-related medical therapies. This study was conducted to determine if AI disease correlates with BPH diagnosis and whether systemic targeting of an inflammatory mediator limits prostatic inflammation and hyperplasia. Patient medical records (n=112,152) were evaluated to determine BPH prevalence among different AI diseases. Inflammatory cells from human BPH tissues were analyzed by single-cell (sc)RNA-seq and the tumor necrosis factor (TNF)α-antagonist etanercept was tested in two murine models of prostatic enlargement. BPH prevalence was significantly higher among patients with AI disease compared to unaffected individuals. However, AI patients treated with TNFα-antagonists had a significantly reduced incidence of BPH. Data from scRNA- seq identified macrophages as a dominant source of TNFα and in vitro assays confirmed that TNFα stimulates BPH-derived fibroblast proliferation. In the AI patient cohort and murine models, systemic treatment with TNFα-antagonists decreased prostatic epithelial proliferation, macrophage infiltration, and epithelial NFκB activation compared to control tissues. These studies are the first to show that patients with AI diseases have a heightened susceptibility to BPH and that the TNFα-signaling axis is important for BPH pathogenesis. Macrophage-secreted TNFα may mechanistically drive BPH via chronic activation of the signaling axis and NFκB. TNFα blockade appears to be a promising new pharmacological approach to target inflammation and suppress BPH.One sentence summaryPatient data and mouse models suggest that repurposing tumor necrosis factor alpha blockade reduces inflammation-mediated prostatic hyperplasia.

BackgroundThe association between benign prostatic hyperplasia (BPH) and prostate cancer (PCa) remains controversial, largely due to inherent detection bias in traditional observational studies. The objective of this study is to assess their association using inherited SNPs.MethodsSubjects were White men from the large population-based UK Biobank (UKB). Association between BPH and PCa was tested: 1) phenotypical correlation using chi-square test, 2) genetic correlation (rg) based on 1,126,841 polymorphic SNPs across the genome using linkage disequilibrium score regression (LDSR), and 3) cross-disease genetic associations based on known risk-associated SNPs (15 for BPH and 239 for PCa), individually and cumulatively as measured by genetic risk score (GRS).FindingsAmong 214,717 White men in the UKB, 24,623 (11.47%) and 14,311 (6.67%) had a diagnosis of BPH and PCa, respectively. Diagnoses of these two diseases were significantly correlated, χ2=1862.80, Prg (95% confidence interval (CI))=0.27 (0.15-0.39), P=9.17E-06. In addition, significant cross-disease genetic associations for established risk-associated SNPs were also found. Among the 250 established GWAS-significant SNPs of PCa or BPH, 51 were significantly associated with risk of the other disease at PP=3.04E-7 (χ2-test). Furthermore, significant cross-disease GRS associations were also found; GRSBPH was significantly associated with PCa risk (odds ratio (OR)=1.26 (1.18-1.36), P=1.62E-10), and GRSPCa was significantly associated with BPH risk (OR=1.03 (1.02-1.04), P=8.57E-06). Moreover, GRSBPH was significantly and inversely associated with lethal PCa risk in a PCa case-case analysis (OR=0.58 (0.41-0.81), P=1.57E-03). In contrast, GRSPCa was not significantly associated with lethal PCa (OR=0.99 (0.94-1.04), P=0.79).InterpretationBPH and PCa share common inherited genetics which suggests the phenotypical association of these two diseases in observational studies is not entirely caused by detection bias. This novel finding may have implications in disease etiology and risk stratification.FundingNone.

The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO(3) (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO(3)-treated wild type mice that progressed with time post-injection. NaIO(3)-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.

INTRODUCTION AND OBJECTIVE:Benign prostatic hyperplasia (BPH) is characterized by enlargement of the prostate and often associated with lower urinary tract symptoms. Inflammatory cells are abundant...

Primary culture of human prostate organoids and patient‐derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease., We demonstrate for the first time that induced pluripotent stem cells (iPSCs) can generate human prostate tissue both in vivo and in vitro, overcoming some significant limitations with patient‐derived xenografts and primary culture to provide a complimentary approach. Furthermore, the in vitro model provides a new platform to create a living biobank of prostate cancer organoids derived from gene editing of iPSCs for preclinical drug testing.

Background The tyrosine kinase inhibitors (TKI), imatinib and nilotinib, are used to treat chronic myelogenous leukemia (CML). In three CML patients being monitored for urologic diseases, we observed that switching of TKI therapy affected prostate-specific antigen (PSA) titers. Urologists and other medical professionals need to be aware of the potential side-effects of drugs that patients may be receiving for other indications to modify this important prostate diseases indicator. TKIs may affect PSA titers independent of prostate growth or volume. Materials and methods We followed PSA levels in urology patients who were also undergoing TKI treatment for CML. We determined the effects of nilotinib and imatinib on proliferation, AR and PSA expression in the LNCaP and 22Rv1 prostate cancer (PCa) cell lines using real-time PCR and Western blotting. Results Clinically, nilotinib and dasatinib reversibly reduced PSA titers compared to imatinib. At high doses nilotinib and imatinib both demonstrated antiproliferative effects in the PCa cells. At low doses expression of AR and PSA was decreased by both drugs, at mRNA and protein levels. Nilotinib exerted greater effects at lower doses than imatinib. Conclusions Nilotinib down-regulates serum PSA in patients being treated for non-urological indications, potentially masking a clinical useful marker, we cannot exclude a similar but smaller effect of imatinib. Nilotinib and imatinib both decreased AR and PSA expression in PCa cell lines with the nilotinib effect evident at lower doses. Urologists must appreciate the effects of drugs provided for other diseases on PSA titers and be aware that sudden changes may not reflect underlying prostatic disease.

The N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model is an attractive model system of muscle-invasive bladder cancer (MIBC) as it recapitulates the histology of human tumors in a background with intact immune system. However, it was unknown whether this carcinogen-induced model also mimicked human MIBC at the molecular and mutational level. In our study, we analyzed gene expression and mutational landscape of the BBN model by next-generation sequencing followed by a bioinformatic comparison to human MIBC using data from The Cancer Genome Atlas and other repositories. BBN tumors showed overexpression of markers of basal cancer subtype, and had a high mutation burden with frequent Trp53 (80%), Kmt2d (70%), and Kmt2c (90%) mutations by exome sequencing, similar to human MIBC. Many variants corresponded to human cancer hotspot mutations, supporting their role as driver mutations. We extracted two novel mutational signatures from the BBN mouse genomes. The integrated analysis of mutation frequencies and signatures highlighted the contribution of aberrations to chromatin regulators and genetic instability in the BBN tumors. Together, our study revealed several similarities between human MIBC and the BBN mouse model, providing a strong rationale for its use in molecular and drug discovery studies.

Background Mitochondrial reprogramming has emerged as a hallmark of cancer pathobiology. Although it is believed this reprogramming is essential for cancer cells to thrive, how it supports cancer pathobiology is unclear. We previously generated colonic ρ0 (rho0) cells with reduced mitochondrial energy function and acquired their transcriptional signature. Here, we utilized a bioinformatics approach to identify their changes linked to cancer pathobiology. Methods Human colon cancer HCT116 cells, control and ρ0, were used for qPCR. Bioinformatics analysis: GeneCards, Kaplan-Meier Survival, GENT, cBioPortal. Results The colonic ρ0 transcriptome was linked with proliferation, DNA replication, survival, tumor morphology, and cancer. Among differentially expressed transcripts, 281 were regulators or biomarkers of human colon cancer especially those with inflammatory microsatellite instability (MSI). We identified and validated novel transcripts in ρ0 cells with altered expression in human colon cancer. Among them DGK1, HTR7, FLRT3, and ZBTB18 co-occurred with established regulators of human colon cancer pathobiology. Also, increased levels of DGKI, FLRT3, ZBTB18, and YPEL1 as well as decreased levels of HTR7, and CALML6 were linked to substantially poorer patient survival. Conclusion We identified established and novel regulators in colon cancer pathobiology that are dependent on mitochondrial energy reprogramming and linked to poorer patient survival.

Cigarette smoking is the leading cause of preventable death worldwide and has been linked to the development and progression of cancer. Many cohort studies have described the link between patients with breast cancer and those with long-term smoking history. Despite the claim of correlation, the mechanism by which cigarette smoke alters normal breast epithelial cells and stroma and contributes to tumor cell growth remains undefined. To investigate whether cigarette smoke promotes ductal epithelial cell hyperplasia by stimulating stromal endothelial cell proliferation, we exposed mice to cigarette smoke for 6 months. We observed epithelial proliferation, increased fibrosis, increased vascularity, and mast cell infiltration. This is the first study to look at the in vivo changes in the breast after long-term cigarette smoke exposure and provides a novel insight to understanding how cigarette smoke contributes to early changes that may contribute to tumor formation and progression. In conclusion, this study suggests that cigarette smoke modulates key stromal-epithelial interactions to support increased angiogenesis, desmoplasia, and abnormal ductal epithelial cell growth.

Background: Obesity is an established risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the pathophysiology of how increased adiposity increases the risk for PDAC has not been fully elucidated. Adipose triglyceride lipase (ATGL) is a lipase that catabolizes triglyceride hydrolysis and has been implicated in the development of breast cancer. We hypothesized that overweight patients with PDAC would demonstrate higher tumor ATGL expression compared to non-overweight patients with PDAC. Materials and Methods: Immunohistochemical analysis for ATGL expression was performed on PDAC tissues from 44 patients after Whipple procedure or distal pancreatectomy. Correlation of ATGL expression with clinicopathological features was evaluated. Results: A total of 23/44 (52.2%) PDACs showed low level ATGL immunoreactivity, while 21/44 (47.8%) showed a high level, with moderate to strong positive ATGL immunoreactivity in more than 50% of the tumor cells. Chi-squared testing revealed a statistically significant association between high ATGL expression and both BMI >25 kg/m2 (χ2=5.74, p=0.017) and increased tumor stroma (χ2=19.14, p

Intestinal inflammation is associated with low levels of mucosal ATP, highlighting the importance of mitochondrial function associated with ATP production in the pathophysiology of the disease. In the inflamed colon of humans and mice, we found decreased levels of mitochondrial complex cytochrome c oxidase I/IV and lower ATP levels. Thus, we generated colonic ρ0 cells with reduced mitochondrial function linked to ATP production by selective depletion of mitochondrial DNA. In these cells, RNA sequencing revealed a substantial number of differentially expressed transcripts, among which 240 belonged to inflammatory pathways activated in human inflamed colon and TNF-α-treated cells (false discovery rate < 0.05). TNF-α treatment of colonic ρ0 cells augmented IL-8 expression by 9-fold (P < 0.01) via NF-κB compared to TNF-α-treated control. Moreover, reduced mitochondrial function facilitated TNF-α-mediated NF-κB luciferase promoter activity as a result of lowered inhibitory IκBα (nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor, α), leading to elevated NF-κB. In cells with reduced mitochondrial function, TNF-α facilitated AMPKα2 activation by 8-fold (P < 0.01), which was involved in NF-κB-dependent IL-8 expression. Last, in human and mouse colon, anti-TNF-α treatment restored reduced mitochondria-dependent inflammation. We propose that selective targeting of this novel mechanism provides new treatment opportunities for intestinal inflammation.—Heller, S., Penrose, H. M., Cable, C., Biswas, D., Nakhoul, H., Baddoo, M., Flemington, E., Crawford, S. E., Savkovic, S. D. Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

Obesity, an immense epidemic affecting approximately half a billion adults, has doubled in prevalence in the last several decades. Epidemiological data support that obesity, due to intake of a high-fat, western diet, increases the risk of colon cancer; however, the mechanisms underlying this risk remain unclear. Here, utilizing next generation RNA sequencing, we aimed to determine the high-fat diet (HFD) mediated expression profile in mouse colon and the azoxymethane/dextran sulfate sodium model of colon cancer. Mice on HFD had significantly higher colonic inflammation, tumor burden, and a number of differentially expressed transcripts compared to mice on regular diet (RD). We identified 721 transcripts differentially expressed in mouse HFD colon that were in a shared pattern with colonic tumors (RD and HFD). Importantly, in mouse colon, HFD stimulated an expression signature strikingly similar to human colon cancer, especially those with inflammatory microsatellite instability. Furthermore, pathway analysis of these transcripts demonstrated their association with active inflammation and colon cancer signaling, with leptin and Wnt as the top two transcripts elevated in mouse HFD colon shared with tumors. Moreover, in mouse colon, HFD-stimulated tumorigenic Wnt pathway activation was further validated by upregulation of β-catenin transcriptional targets. Finally, in human colon cancer, upregulation of leptin pathway members was shown with a large network of dysregulated transcripts being linked with worse overall survival.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

Primary culture of human prostate organoids is slow, inefficient and laborious. To overcome this, we demonstrate a new high-throughput model where rapidly proliferating and easily handled induced pluripotent stem cells, for the first time, enable generation of human prostate tissue in vivo and in vitro. Using a co-culture technique with urogenital sinus mesenchyme, we recapitulated the in situ prostate histology, including the stromal compartment and the full spectrum of epithelial differentiation. This approach overcomes major limitations in primary cultures of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. These models provide new opportunities to study prostate development, homeostasis and disease.

Background Prostatic inflammation and various proinflammatory systemic comorbidities, such as diabetes and obesity are associated with human benign prostatic hyperplasia (BPH). There is a paucity of in vivo models reflecting specific aspects of BPH pathogenesis. Our aim was to investigate the nonobese diabetic (NOD) mouse as a potential model for subsequent intervention studies. Materials and methods We used the NOD mouse, a model of autoimmune inflammation leading to type 1 diabetes to examine the effects of systemic inflammation and diabetes on the prostate. We assessed changes in prostatic histology, infiltrating leukocytes, and gene expression associated with aging and diabetic status. Results Both stromal expansion and epithelial hyperplasia were observed in the prostates. Regardless of diabetic status, the degree of prostatic hyperplasia varied. Local inflammation was associated with a more severe prostatic phenotype in both diabetic and nondiabetic mice. Testicular atrophy was noted in diabetic mice, but prostate glands showed persistent focal cell proliferation. In addition, a prostatic intraepithelial neoplasia (PIN)-like phenotype was seen in several diabetic animals with an associated increase in c-Myc and MMP-2 expression. To examine changes in gene and cytokine expression we performed microarray and cytokine array analysis comparing the prostates of diabetic and nondiabetic animals. Microarray analysis revealed several differentially expressed genes including CCL3, CCL12, and TNFS10. Cytokine array analysis revealed increased expression of cytokines and proteases such as LDLR, IL28 A/B, and MMP-2 in diabetic mice. Conclusion Overall, NOD mice provide a model to examine the effects of hyperglycemia and chronic inflammation on the prostate, demonstrating relevance to some of the mechanisms present underlying BPH and potentially the initiation of prostate cancer.

Acyl-CoA:diacylglycerol acyltransferase I (DGAT1) is a key enzyme in lipogenesis which is increased in metabolically active cells to meet nutrient requirements. DGAT1 has been recognized as an anti-obesity target; however, its role in the tumor microenvironment remains unclear. We postulated that, in prostate cancer (PCa) cells, augmented lipogenesis and growth are due to increased DGAT1 expression leading to microtubule-organizing center (MTOC) amplification. Thus, therapeutic targeting of DGAT1 potentially has tumor suppressive activity. We tested whether blocking DGAT1 in PCa cells altered MTOC and lipid signaling. Western blot and immunofluorescence were performed for MTOC and triglyceride mediators. Treatment with a DGAT1 inhibitor was evaluated. We found a stepwise increase in DGAT1 protein levels when comparing normal prostate epithelial cells to PCa cells, LNCaP and PC-3. Lipid droplets, MTOCs, and microtubule-regulating proteins were reduced in tumor cells treated with a DGAT1 inhibitor. Depletion of the non-centrosomal MTOC protein GM130 reduced PCa cell proliferation and migration. Inhibition of DGAT1 reduced tumor growth both in vitro and in vivo, and a negative feedback loop was discovered between DGAT1, PEDF, and GM130. These data identify DGAT1 as a promising new target for suppressing PCa growth by regulating GM130, MTOC number and disrupting microtubule integrity.

The role of tumor heterogeneity in regulating disease progression is poorly understood. We hypothesized that interactions between subpopulations of cancer cells can affect the progression of tumors selecting for a more aggressive phenotype. We developed an in vivo assay based on the immortalized nontumorigenic breast cell line MCF10A and its Ras-transformed derivatives AT1 (mildly tumorigenic) and CA1d (highly tumorigenic). CA1d cells outcompeted MCF10A, forming invasive tumors. AT1 grafts were approximately 1% the size of CA1d tumors when initiated using identical cell numbers. In contrast, CA1d/AT1 mixed tumors were larger than tumors composed of AT1 alone (100-fold) or CA1d (3-fold), suggesting cooperation in tumor growth. One of the mechanisms whereby CA1d and AT1 were found to cooperate was by modulation of TGF-α and TGF-β signaling. Both of these molecules were sufficient to induce changes in AT1 proliferative potential in vitro. Reisolation of AT1 tumor-derived (AT1-TD) cells from these mixed tumors revealed that AT1-TD cells grew in vivo, forming tumors as large as tumorigenic CA1d cells. Cooperation between subpopulations of cancer epithelium is an understudied mechanism of tumor growth and invasion that may have implications on tumor resistance to current therapies.—Franco, O. E., Tyson, D. R., Konvinse, K. C., Udyavar, A. R., Estrada, L., Quaranta, V., Crawford, S. E., Hayward, S. W. Altered TGF-α/β signaling drives cooperation between breast cancer cell populations.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and deformity. OI type VI is unique owing to the mineralization defects observed in patient biopsies. Furthermore, it has been reported to respond less well to standard therapy with bisphosphonates [1]. Others and we have previously identified SERPINF1 mutations in patients with OI type VI. SERPINF1 encodes pigment epithelium derived factor (PEDF), a secreted collagen-binding glycoprotein that is absent in the sera of patients with OI type VI. Serpinf1 null mice show increased osteoid and decreased bone mass, and thus recapitulate the OI type VI phenotype. We tested whether restoration of circulating PEDF in the blood could correct the phenotype of OI type VI in the context of protein replacement. To do so, we utilized a helper-dependent adenoviral vector (HDAd) to express human SERPINF1 in the mouse liver and assessed whether PEDF secreted from the liver was able to rescue the bone phenotype observed in Serpinf1(-/-) mice. We confirmed that expression of SERPINF1 in the liver restored the serum level of PEDF. We also demonstrated that PEDF secreted from the liver was biologically active by showing the expected metabolic effects of increased adiposity and impaired glucose tolerance in Serpinf1(-/-) mice. Interestingly, overexpression of PEDF in vitro increased mineralization with a concomitant increase in the expression of bone gamma-carboxyglutamate protein, alkaline phosphatase and collagen, type I, alpha I, but the increased serum PEDF level did not improve the bone phenotype of Serpinf1(-/-) mice. These results suggest that PEDF may function in a context-dependent and paracrine fashion in bone homeostasis.