Prenatal findings in 11 cases with craniofacial microsomia using the Alberta Congenital Anomalies Surveillance System, 1997-2019.

Craniofacial microsomia (CFM) primarily includes specific head and neck anomalies that co-occur more frequently than expected. The anomalies are usually asymmetric and affect craniofacial features; however, there are frequently additional anomalies of variable severity. Published prenatal findings for CFM are limited. This study contributes 11 cases with CFM and their anomalies identified prenatally. Cases born between January 1, 1997 and December 31, 2019 with CFM were abstracted from the Alberta Congenital Anomalies Surveillance System, which is a population-based program ascertaining congenital anomalies for livebirths, stillbirths, and termination of pregnancies for fetal anomalies. There were 11 cases ascertained with prenatal findings including facial anomalies: one each with left cleft lip, right microtia, and bilateral microphthalmia. Two cases had vertebral anomalies. In addition, anomalies of the kidneys, brain, heart, and radial ray were identified. Six (55%) had a single umbilical artery, five (45%) were small for gestational age, and three (27%) were from a twin pregnancy that were discordant for anomalies. Four (36%) overlapped another proposed recurrent constellations of embryonic malformation condition. This study describes prenatal findings for 11 cases with CFM. Comparable to prior published cases, there were recurring anomalies on prenatal imaging, including anomalies of the brain, eye, heart, kidneys, and radial ray, which may aid in the prenatal diagnosis of CFM.

Perinatal and early life factors and asthma control among preschoolers: a population-based retrospective cohort study.

BACKGROUND: Preventing poor childhood asthma control is crucial for short-term and long-term respiratory health. This study evaluated associations between perinatal and early-life factors and early childhood asthma control. METHODS: This retrospective study used administrative health data from mothers and children born 2010-2012 with a diagnosis of asthma before age 5 years, in Alberta, Canada. The outcome was asthma control within 2 years after diagnosis. Associations between perinatal and early-life factors and risk of partly and uncontrolled asthma were evaluated by multinomial logistic regression. RESULTS: Of 7206 preschoolers with asthma, 52% had controlled, 37% partly controlled and 12% uncontrolled asthma 2 years after diagnosis. Compared with controlled asthma, prenatal antibiotics (adjusted risk ratio (aRR): 1.19; 95% CI 1.06 to 1.33) and smoking (aRR: 1.18; 95% CI 1.02 to 1.37), C-section delivery (aRR: 1.11; 95% CI 1.00 to 1.25), summer birth (aRR: 1.16; 95% CI 1.00 to 1.34) and early-life hospitalisation for respiratory illness (aRR: 2.24; 95% CI 1.81 to 2.76) increased the risk of partly controlled asthma. Gestational diabetes (aRR: 1.41; 95% CI 1.06 to 1.87), C-section delivery (aRR: 1.18; 95% CI 1.00 to 1.39), antibiotics (aRR: 1.32; 95% CI 1.08 to 1.61) and hospitalisation for early-life respiratory illness (aRR: 1.65; 95% CI 1.19 to 2.27) were associated with uncontrolled asthma. CONCLUSION: Maternal perinatal and early-life factors including antibiotics in pregnancy and childhood, gestational diabetes, prenatal smoking, C-section and summertime birth, and hospitalisations for respiratory illness are associated with partly or uncontrolled childhood asthma. These results underline the significance of perinatal health and the lasting effects of early-life experiences on lung development and disease programming.

Craniofacial Microsomia, Associated Congenital Anomalies, and Risk Factors in 63 Cases from the Alberta Congenital Anomalies Surveillance System.

OBJECTIVE: To report associated congenital anomalies with unexplained craniofacial microsomia (CFM) and the phenotypic overlap with other recurrent constellations of embryonic malformations (RCEM), and to assess prenatal and perinatal risk factors. STUDY DESIGN: This is a retrospective cross-sectional study. Cases with CFM, delivered between January 1, 1997, and December 31, 2019, were abstracted from the population-based Alberta Congenital Anomalies Surveillance System. Livebirths, stillbirths, and early fetal losses were reviewed to include all types of pregnancy outcomes along the spectrum of this condition. Prenatal and perinatal risk factors were compared with the Alberta birth population to assess differences between the 2 groups. RESULTS: There were 63 cases with CFM, yielding a frequency of 1 per 16 949. There was a high rate of cases (65%) with anomalies outside the craniofacial and vertebral regions. Congenital heart defects were the most common (33.3%). A single umbilical artery was found in 12.7% of cases. The twin/triplet rate of 12.7% was significantly higher than the Alberta rate of 3.3% (P < .0001). There was an overlap with a second RCEM condition in 9.5% of cases. CONCLUSIONS: Although CFM is primarily a craniofacial condition, the majority of cases have congenital anomalies affecting other systems requiring additional assessments, including an echocardiogram, renal ultrasound examination, and a complete vertebral radiograph. The high rate of an associated single umbilical artery raises the possibility of a related etiological mechanism. Our findings support the proposed concept of RCEM conditions.

Shared Inherited Genetics of Benign Prostatic Hyperplasia and Prostate Cancer.

Background: The association between benign prostatic hyperplasia (BPH) and prostate cancer (PCa) remains controversial, largely due to a detection bias in traditional observational studies. Objective: To assess the association between BPH and PCa using inherited single nucleotide polymorphisms (SNPs). Design setting and participants: The participants were White men from the population-based UK Biobank (UKB). Outcome measurements and statistical analysis: The association between BPH and PCa was tested for (1) phenotypic correlation using chi-square, (2) genetic correlation (r g) based on genome-wide SNPs using linkage disequilibrium score regression, and (3) cross-disease genetic associations based on known risk-associated SNPs (15 for BPH and 239 for PCa), individually and cumulatively using genetic risk score (GRS). Results and limitations: Among 214 717 White men in the UKB, 24 623 (11%) and 14 311 (6.7%) had a diagnosis of BPH and PCa, respectively. Diagnoses of these two diseases were significantly correlated (χ2 = 1862.80, p < 0.001). A significant genetic correlation was found (r g = 0.16; 95% confidence interval 0.03-0.28, p = 0.01). In addition, significant cross-disease genetic associations for established risk-associated SNPs were also found. Among the 250 established genome-wide association study-significant SNPs of PCa or BPH, 49 were significantly associated with the risk of the other disease at p < 0.05, significantly more than expected by chance (N = 12, p < 0.001; 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 < 0.001), and GRSPCa was significantly associated with BPH risk (OR = 1.03 [1.02-1.04], p < 0.001). 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 = 0.002). Only White men were studied. Conclusions: BPH and PCa share common inherited genetics, which suggests that the phenotypic association of these two diseases in observational studies is not entirely caused by the detection bias. Patient summary: For the first time, we found that benign prostatic hyperplasia and prostate cancer are genetically related. This finding may have implications in disease etiology and risk stratification.

Ephrin B Activate Src Family Kinases in Fibroblasts Inducing Stromal Remodeling in Prostate Cancer.

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.

TNF is a potential therapeutic target to suppress prostatic inflammation and hyperplasia in autoimmune disease.

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.

Antagonism Between PEDF and TGF-ß Contributes to Type VI Osteogenesis Imperfecta Bone and Vascular Pathogenesis.

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder of bone and connective tissue, also known as brittle bone disease. Null mutations in SERPINF1, which encodes pigment epithelium-derived factor (PEDF), cause severe type VI OI, characterized by accumulation of unmineralized osteoid and a fish-scale pattern of bone lamellae. Although the potent anti-angiogenic activity of PEDF has been extensively studied, the disease mechanism of type VI OI is not well understood. Using Serpinf1(-/-) mice and primary osteoblasts, we demonstrate that loss of PEDF delays osteoblast maturation as well as extracellular matrix (ECM) mineralization. Barium sulfate perfusion reveals significantly increased vessel density in the tibial periosteum of Serpinf1(-/-) mouse compared with wild-type littermates. The increased bone vascularization in Serpinf1(-/-) mice correlated with increased number of CD31(+)/Endomucin(+) endothelial cells, which are involved in the coupling angiogenesis and osteogenesis. Global transcriptome analysis by RNA-Seq of Serpinf1(-/-) mouse osteoblasts reveals osteogenesis and angiogenesis as the biological processes most impacted by loss of PEDF. Intriguingly, TGF-ß signaling is activated in type VI OI cells, and Serpinf1(-/-) osteoblasts are more sensitive to TGF-ß stimulation than wild-type osteoblasts. TGF-ß stimulation and PEDF deficiency showed additive effects on transcription suppression of osteogenic markers and stimulation of pro-angiogenic factors. Furthermore, PEDF attenuated TGF-ß-induced expression of pro-angiogenic factors. These data suggest that functional antagonism between PEDF and TGF-ß pathways controls osteogenesis and bone vascularization and is implicated in type VI OI pathogenesis. This antagonism may be exploited in developing therapeutics for type VI OI utilizing PEDF and TGF-ß antibody. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Elevated ATGL in colon cancer cells and cancer stem cells promotes metabolic and tumorigenic reprogramming reinforced by obesity.

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.

Loss of ephrin B2 receptor (EPHB2) sets lipid rheostat by regulating proteins DGAT1 and ATGL inducing lipid droplet storage in prostate cancer cells.

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. 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.

Prevalence rates study of selected isolated non-Mendelian congenital anomalies in the Hutterite population of Alberta, 1980-2016.

A study of the prevalence rates for selected isolated non-Mendelian congenital anomalies in the Hutterite Brethren of Alberta, Canada was undertaken to further examine longitudinal data in this isolated community that was last reported in 1985 (Lowry et al., 1985), although there are numerous publications on recessive disorders (Boycott et al., 2008; Triggs-Raine et al., 2016). Cases were ascertained from the Alberta Congenital Anomaly Surveillance System for the years 1997-2016. Since our initial results showed some surprising findings in the Hutterite Brethren, such as zero cases of spina bifida, cleft lip and palate, gastroschisis, and omphalocele, and a significant excess of cases with hypospadias, we extended the study to prior years (1980-1996) for selected anomalies. For the extended study period (1980-2016), there was a significant increased prevalence of hypospadias, tetralogy of Fallot and tricuspid atresia in the Hutterite population, and although not statistically significant, zero cases of cleft lip with cleft palate, gastroschisis and omphalocele were confirmed. Further research is needed to determine the precise effects of rural environmental exposures, lifestyle factors, and genetic associations for selected multifactorial congenital anomalies.

Feasibility and performance of a novel probe panel to detect somatic DNA copy number alterations in clinical specimens for predicting prostate cancer progression.

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.

Propagation of human prostate tissue from induced pluripotent stem cells.

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.

Socioeconomic gradients of adverse birth outcomes and related maternal factors in rural and urban Alberta, Canada: a concentration index approach.

OBJECTIVE: Using a summary measure of health inequalities, this study evaluated the distribution of adverse birth outcomes (ABO) and related maternal risk factors across area-level socioeconomic status (SES) gradients in urban and rural Alberta, Canada. DESIGN: Cross-sectional study using a validated perinatal clinical registry and an area-level SES. SETTING: The study was conducted in Alberta, Canada. Data about ABO and related maternal risk factors were obtained from the Alberta Perinatal Health Program between 2006 and 2012. An area-level SES index derived from census data (2006) was linked to the postal code at delivery. PARTICIPANTS: Women (n=3 30 957) having singleton live births with gestational age ≥22 weeks. PRIMARY AND SECONDARY OUTCOME MEASURES: We estimated concentration indexes to assess inequalities across SES gradients in both rural and urban areas (CIdxR and CIdxU, respectively) for spontaneous preterm birth (PTB), small for gestational age (SGA), large for gestational age (LGA), gestational hypertension, gestational diabetes, smoking and substance use during pregnancy and pre-pregnancy weight >91 kg. RESULTS: The highest health inequalities disfavouring low SES groups were identified for substance abuse and smoking in rural areas (CIdxR-0.38 and -0.23, respectively). Medium inequalities were identified for LGA (CIdxR-0.08), pre-pregnancy weight >91 kg (CIdxR-0.07), substance use (CIdxU-0.15), smoking (CIdxU-0.14), gestational diabetes (CIdxU-0.10) and SGA (CIdxU-0.07). Low inequalities were identified for PTB (CIdxR-0.05; CIdxU-0.05) and gestational diabetes (CIdxR-0.04). Inequalities disfavouring high SES groups were identified for gestational hypertension (CIdxR+0.04), SGA (CIdxR+0.03) and LGA (CIdxU+0.03). CONCLUSIONS: ABO and related maternal risk factors were unequally distributed across the socioeconomic gradient in urban-rural settings, with the greatest concentrations in lower SES groups of rural areas. Future research is needed on underlying mechanisms driving SES gradients in perinatal health across the rural-urban spectrum.

Heterogeneity of human prostate carcinoma-associated fibroblasts implicates a role for subpopulations in myeloid cell recruitment.

BACKGROUND: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment (TME) that can promote tumorigenesis in the prostate. By understanding the mechanism(s) by which CAF contributes to tumor growth, new therapeutic targets for the management of this disease may be identified. These studies determined whether unique sub-populations of human prostate CAF can be identified and functionally characterized. METHODS: Single-cell RNA-seq of primary human prostate CAF followed by unsupervised clustering was utilized to generate cell clusters based on differentially expressed (DE) gene profiles. Potential communication between CAF and immune cells was analyzed using in vivo tissue recombination by combining CAF or normal prostate fibroblasts (NPF) with non-tumorigenic, initiated prostate epithelial BPH-1 cells. Resultant grafts were assessed for inflammatory cell recruitment. RESULTS: Clustering of 3321 CAF allows for visualization of six subpopulations, demonstrating heterogeneity within CAF. Sub-renal capsule recombination assays show that the presence of CAF significantly increases myeloid cell recruitment to resultant tumors. This is supported by significantly increased expression of chemotactic chemokines CCL2 and CXCL12 in large clusters compared to other subpopulations. Bayesian analysis topologies also support differential communication signals between chemokine-related genes of individual clusters. Migration of THP-1 monocyte cells in vitro is stimulated in the presence of CAF conditioned medium (CM) compared with NPF CM. Further in vitro analyses suggest that CAF-derived chemokine CCL2 may be responsible for CAF-stimulated migration of THP-1 cells, since neutralization of this chemokine abrogates migration capacity. CONCLUSIONS: CAF clustering based on DE gene expression supports the concept that clusters have unique functions within the TME, including a role in immune/inflammatory cell recruitment. These data suggest that CCL2 produced by CAF may be involved in the recruitment of inflammatory cells, but may also directly regulate the growth of the tumor. Further studies aimed at characterizing the subpopulation(s) of CAF which promote immune cell recruitment to the TME and/or stimulate prostate cancer growth and progression will be pursued.

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

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.

Lipid droplet velocity is a microenvironmental sensor of aggressive tumors regulated by V-ATPase and PEDF.

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.

Hyperglycemia and T Cell infiltration are associated with stromal and epithelial prostatic hyperplasia in the nonobese diabetic mouse.

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.

DGAT1 Inhibitor Suppresses Prostate Tumor Growth and Migration by Regulating Intracellular Lipids and Non-Centrosomal MTOC Protein GM130.

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.

Loss of Forkhead Box O3 Facilitates Inflammatory Colon Cancer: Transcriptome Profiling of the Immune Landscape and Novel Targets.

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.