Publications by authors named "Susan E Crawford"

104 Publications

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

Lab Invest 2021 Apr 6. Epub 2021 Apr 6.

Department of Surgery, Urology Division, Department of Cancer Biology, NorthShore University HealthSystem, Affiliate of the University of Chicago Pritzker School of Medicine, Evanston, IL, USA.

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.
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http://dx.doi.org/10.1038/s41374-021-00583-9DOI Listing
April 2021

PEDF deficiency increases the susceptibility of rd10 mice to retinal degeneration.

Exp Eye Res 2020 09 25;198:108121. Epub 2020 Jul 25.

Section of Protein Structure and Function, LRCMB-NEI-NIH, Bethesda, MD, USA. Electronic address:

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 (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-treated wild type mice that progressed with time post-injection. NaIO-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.
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http://dx.doi.org/10.1016/j.exer.2020.108121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508969PMC
September 2020

Propagation of human prostate tissue from induced pluripotent stem cells.

Stem Cells Transl Med 2020 07 14;9(7):734-745. Epub 2020 Mar 14.

Translational and Clinical Research Institute, Newcastle University Centre for Cancer, Newcastle University, Newcastle upon Tyne, UK.

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.
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http://dx.doi.org/10.1002/sctm.19-0286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308643PMC
July 2020

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

Prostate 2020 02 25;80(2):173-185. Epub 2019 Nov 25.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois.

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.
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http://dx.doi.org/10.1002/pros.23929DOI Listing
February 2020

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

Lab Chip 2020 01 12;20(1):107-119. Epub 2019 Nov 12.

Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195, USA. and Department of Urology, University of Washington School of Medicine, Seattle, WA 98195, USA.

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.
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http://dx.doi.org/10.1039/c9lc00706gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917835PMC
January 2020

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

Lab Invest 2019 12 13;99(12):1822-1834. Epub 2019 Aug 13.

Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL, 60201, USA.

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.
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http://dx.doi.org/10.1038/s41374-019-0296-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289525PMC
December 2019

Influence of maternal obesity and metabolic and vascular mediators in twin-twin transfusion syndrome.

Reprod Biol 2019 Jun 27;19(2):165-172. Epub 2019 May 27.

Department of Pathology, Saint Louis University School of Medicine, St. Louis, MO, 63103, United States; Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL, 60201, United States. Electronic address:

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/m) and 11/33 were overweight (BMI 25-29.9 kg/m). 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.
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http://dx.doi.org/10.1016/j.repbio.2019.05.002DOI Listing
June 2019

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

Prostate 2019 06 18;79(9):980-993. Epub 2019 Apr 18.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois.

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.
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http://dx.doi.org/10.1002/pros.23809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591734PMC
June 2019

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

Sci Rep 2019 02 28;9(1):3035. Epub 2019 Feb 28.

Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL, 60201, United States.

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.
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http://dx.doi.org/10.1038/s41598-019-39537-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395665PMC
February 2019

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

Cell Mol Gastroenterol Hepatol 2019 13;7(2):391-408. Epub 2018 Oct 13.

Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, Louisiana. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jcmgh.2018.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360252PMC
May 2019

Tyrosine kinase inhibitor therapy prescribed for non-urologic diseases can modify PSA titers in urology patients.

Prostate 2019 02 28;79(3):259-264. Epub 2018 Oct 28.

Department of Surgery, NorthShore University HealthSystem, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, Illinois.

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.
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http://dx.doi.org/10.1002/pros.23730DOI Listing
February 2019

Elevation of Stromal-Derived Mediators of Inflammation Promote Prostate Cancer Progression in African-American Men.

Cancer Res 2018 11 4;78(21):6134-6145. Epub 2018 Sep 4.

Department of Surgery, NorthShore University HealthSystem Research Institute, Evanston, Illinois.

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) and EA (PrF) prostate cancer tissues and studied their functional characteristics. PrF showed increased growth response to androgens FGF2 and platelet-derived growth factor. Compared with PrF, conditioned media from PrF 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 tumorigenicity of an AA patient-derived prostatic epithelial cell line E006AA was significantly increased in the presence of PrF compared with PrF, 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 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 compared with PrF 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. 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. http://cancerres.aacrjournals.org/content/canres/78/21/6134/F1.large.jpg .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3810DOI Listing
November 2018

PEDF regulates plasticity of a novel lipid-MTOC axis in prostate cancer-associated fibroblasts.

J Cell Sci 2018 07 11;131(13). Epub 2018 Jul 11.

Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL 60201, United States

Prostate tumors make metabolic adaptations to ensure adequate energy and amplify cell cycle regulators, such as centrosomes, to sustain their proliferative capacity. It is not known whether cancer-associated fibroblasts (CAFs) undergo metabolic re-programming. We postulated that CAFs augment lipid storage and amplify centrosomal or non-centrosomal microtubule-organizing centers (MTOCs) through a pigment epithelium-derived factor (PEDF)-dependent lipid-MTOC signaling axis. Primary human normal prostate fibroblasts (NFs) and CAFs were evaluated for lipid content, triacylglycerol-regulating proteins, MTOC number and distribution. CAFs were found to store more neutral lipids than NFs. Adipose triglyceride lipase (ATGL) and PEDF were strongly expressed in NFs, whereas CAFs had minimal to undetectable levels of PEDF or ATGL protein. At baseline, CAFs demonstrated MTOC amplification when compared to 1-2 perinuclear MTOCs consistently observed in NFs. Treatment with PEDF or blockade of lipogenesis suppressed lipid content and MTOC number. In summary, our data support that CAFs have acquired a tumor-like phenotype by re-programming lipid metabolism and amplifying MTOCs. Normalization of MTOCs by restoring PEDF or by blocking lipogenesis highlights a previously unrecognized plasticity in centrosomes, which is regulated through a new lipid-MTOC axis.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.213579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051346PMC
July 2018

A Carcinogen-induced mouse model recapitulates the molecular alterations of human muscle invasive bladder cancer.

Oncogene 2018 04 25;37(14):1911-1925. Epub 2018 Jan 25.

Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

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.
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http://dx.doi.org/10.1038/s41388-017-0099-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886988PMC
April 2018

In colonic ρ (rho0) cells reduced mitochondrial function mediates transcriptomic alterations associated with cancer.

Oncoscience 2017 Nov 27;4(11-12):189-198. Epub 2017 Dec 27.

Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, LA 70112, USA.

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.
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http://dx.doi.org/10.18632/oncoscience.386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769983PMC
November 2017

Hormonal and Growth Regulation of Epithelial and Stromal Cells From the Normal and Malignant Endometrium by Pigment Epithelium-Derived Factor.

Endocrinology 2017 09;158(9):2754-2773

Department of Medicine, Division of Translational Medicine, New York University School of Medicine Langone Medical Center, New York, New York 10016.

We discovered that pigment epithelium-derived factor (PEDF)-null mice have endometrial hyperplasia, the precursor to human type I endometrial cancer (ECA), which is etiologically linked to unopposed estrogen (E2), suggesting that this potent antiangiogenic factor might contribute to dysregulated growth and the development of type I ECA. Treatment of both ECA cell lines and primary ECA cells with recombinant PEDF dose dependently decreased cellular proliferation via an autocrine mechanism by blocking cells in G1 and G2 phases of the cell cycle. Consistent with the known opposing effects of E2 and progesterone (Pg) on endometrial proliferation, Pg increases PEDF protein synthesis and release, whereas E2 has the converse effect. Using PEDF luciferase promoter constructs containing two Pg and one E2 response elements, E2 reduced and Pg increased promoter activity due to distal response elements. Furthermore, E2 decreases and Pg increases PEDF secretion into conditioned media (CM) by both normal endometrial stromal fibroblasts (ESFs) and cancer-associated fibroblasts (CAFs), but only CM from ESFs mediated growth-inhibitory activity of primary endometrial epithelial cells (EECs). In addition, in cocultures with primary EECs, Pg-induced growth inhibition is mediated by ESFs, but not CAFs. This is consistent with reduced levels of Pg receptors on CAFs surrounding human malignant glands in vivo. Taken together, the data suggest that PEDF is a hormone-regulated negative autocrine mediator of endometrial proliferation, and that paracrine growth inhibition by soluble factors, possibly PEDF, released by ESFs in response to Pg, but not CAFs, exemplifies a tumor microenvironment that contributes to the pathogenesis of ECA.
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http://dx.doi.org/10.1210/en.2017-00028DOI Listing
September 2017

A genetic variant near GATA3 implicated in inherited susceptibility and etiology of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS).

Prostate 2017 Aug 28;77(11):1213-1220. Epub 2017 Jun 28.

Program for Personalized Cancer Care, NorthShore University HealthSystem, Evanston, Illinois.

Background: Benign prostatic hyperplasia (BPH) and associated lower urinary tract symptoms (LUTS) are common conditions. Little is known about their etiologies except that studies have suggested a substantial heritable component. Our objective is to provide a comprehensive, genome-wide evaluation of inherited risks and possible mechanisms of etiology in BPH.

Methods: We performed a three-stage, genome-wide association study (GWAS) of men from three independent populations, the REduction by DUtasteride of prostate Cancer Events (REDUCE) trial, the CLUE II cohort, and a Finnish hospital-based population. DNA samples were genotyped using the Illumina HumanOmniExpress BeadChip in REDUCE and CLUE II, and using the Sequenom iPLEX system for the confirmation stage in the Finnish population. A logistic regression model was used to evaluate the association between each SNP and BPH/LUTS.

Results: Fourteen SNPs reached P < 5.0 × 10 in the meta-analysis of the two GWASs (CLUE II and REDUCE). A total of 773 SNPs were chosen for the confirmation step in the Finish cohort. Only one SNP (rs17144046) located ∼489 kb downstream of GATA3 remained significant after correction for multiple testing (P < 6.5 × 10 ). This SNP marginally reached the GWAS significance level after performing a meta-analysis of the three stages (P  = 8.89 × 10 ). Expression quantitative trait loci (eQTL) analyses showed that the risk allele (G) of rs17144046 was significantly associated with increased expression of GATA3 (P = 0.017). Reported studies indicated a close correlation between GATA3 and BPH pathogenesis and progression.

Conclusions: Rs17144046 located near GATA3 was significantly associated with BPH/LUTS in three independent populations, but did not reach a stringent GWAS significance level. Genetic variants of GATA3 may play a role in the inherited susceptibility and etiology of BPH/LUTS. Further research in this area is needed.
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http://dx.doi.org/10.1002/pros.23380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565164PMC
August 2017

High-fat diet induced leptin and Wnt expression: RNA-sequencing and pathway analysis of mouse colonic tissue and tumors.

Carcinogenesis 2017 03;38(3):302-311

Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, LA 70112, USA and.

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.
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http://dx.doi.org/10.1093/carcin/bgx001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862315PMC
March 2017

Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

FASEB J 2017 05 9;31(5):2013-2025. Epub 2017 Feb 9.

Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, Louisiana, USA;

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 oxidase I/IV and lower ATP levels. Thus, we generated colonic ρ 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 ρ cells augmented IL-8 expression by 9-fold ( < 0.01) 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 ( < 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.
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http://dx.doi.org/10.1096/fj.201600976RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388551PMC
May 2017

Adipose Triglyceride Lipase (ATGL) Expression Is Associated with Adiposity and Tumor Stromal Proliferation in Patients with Pancreatic Ductal Adenocarcinoma.

Anticancer Res 2017 02;37(2):699-703

Department of Pathology, Saint Louis University School of Medicine, Saint Louis, MO, U.S.A.

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/m (χ=5.74, p=0.017) and increased tumor stroma (χ=19.14, p<0.001). Chi-squared testing failed to reveal a statistically significant association when comparing ATGL expression by lymph node metastasis, histological grade, tumor size, patient age, patient sex and presence of fat invasion.

Conclusion: Our results suggest that increased ATGL expression is associated with increased adiposity and stromal proliferation in patients with PDAC, making it a possible key protein in how obesity increases the risk of PDAC.
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http://dx.doi.org/10.21873/anticanres.11366DOI Listing
February 2017

Altered TGF-α/β signaling drives cooperation between breast cancer cell populations.

FASEB J 2016 10 6;30(10):3441-3452. Epub 2016 Jul 6.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois, USA; Department of Urologic Surgery, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA; and Department of Cancer Biology, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA

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) cells from these mixed tumors revealed that AT1 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.
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http://dx.doi.org/10.1096/fj.201500187RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024699PMC
October 2016

Intestinal inflammation requires FOXO3 and prostaglandin E2-dependent lipogenesis and elevated lipid droplets.

Am J Physiol Gastrointest Liver Physiol 2016 05 11;310(10):G844-54. Epub 2016 Mar 11.

Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, Louisiana;

Intestinal inflammation has been recently characterized by the dysregulation of lipids as metabolic and energy sources, revealing a novel feature of its pathophysiology. Because intracellular lipids, stored in dynamic lipid droplets (LDs), provide energy for cellular needs, we investigated whether they play a role in intestinal inflammation. In the inflamed intestine of mice, elevated LDs were found in colonic and infiltrating immune cells as shown by staining for the LD coat protein PLIN2 and for lipids with BODIPY. In colonic cells, TNF stimulated LD increases by receptor signaling rely on phosphatidylinositol 3-kinase activation. Downstream, TNF triggered a negative regulatory loop between LDs and the transcription factor FOXO3. This was shown in the colon of Foxo3-deficient mice, where elevation in PLIN2 and lipids were further facilitated by inflammation and were more prominent relative to wild-type, whereas, in colonic cells, inhibition of lipogenesis blocked the TNF-mediated loss of FOXO3. Furthermore, blockade of PGE2 synthesis abrogated TNF-stimulated increases in LDs and FOXO3 inactivation. We found in colonic tissue of Foxo3-deficient mice higher levels of cyclooxygenase-2, a mediator of prostaglandin E2 (PGE2) synthesis, supporting involvement of PGE2 in the LD-FOXO3 regulatory loop. Ultimately, TNF-stimulated lipogenesis leading to elevated LDs facilitated NF-κB-mediated increases in IL-8 protein, which is associated with the surface of LDs found in the lumina of the endoplasmic reticulum and Golgi apparatus. This novel immunometabolic mechanism of colonic inflammation involving elevated LDs could provide opportunities for new treatment options.
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http://dx.doi.org/10.1152/ajpgi.00407.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895869PMC
May 2016

Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6.

Biochem Biophys Res Commun 2016 Jan 30;469(3):370-6. Epub 2015 Nov 30.

Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave SL-79, New Orleans, LA 70112, USA. Electronic address:

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression.
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http://dx.doi.org/10.1016/j.bbrc.2015.11.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607009PMC
January 2016

Pigment Epithelium-Derived Factor (PEDF) Inhibits Wnt/-catenin Signaling in the Liver.

Cell Mol Gastroenterol Hepatol 2015 09 4;1(5):535-549.e14. Epub 2015 Jul 4.

Department of Medicine, Yale University School of Medicine, New Haven, Connecticut ; VA CT Healthcare System, West Haven, Connecticut.

Background & Aims: Pigment epithelium-derived factor (PEDF) is a secretory protein that inhibits multiple tumor types. PEDF inhibits the Wnt coreceptor, low-density lipoprotein receptor-related protein 6 (LRP6), in the eye, but whether the tumor-suppressive properties of PEDF occur in organs such as the liver is unknown.

Methods: Wnt-dependent regulation of PEDF was assessed in the absence and presence of the Wnt coreceptor LRP6. Whole genome expression analysis was performed on PEDF knockout (KO) and control livers (7 months). Interrogation of Wnt/-catenin signaling was performed in whole livers and human hepatocellular carcinoma (HCC) cell lines after RNA interference of PEDF and restoration of a PEDF-derived peptide. Western diet feeding for 6 to 8 months was used to evaluate whether the absence of PEDF was permissive for HCC formation (n = 12/group).

Results: PEDF levels increased in response to canonical Wnt3a in an LRP6-dependent manner but were suppressed by noncanonical Wnt5a protein in an LRP6-independent manner. Gene set enrichment analysis (GSEA) of PEDF KO livers revealed induction of pathways associated with experimental and human HCC and a transcriptional profile characterized by Wnt/-catenin activation. Enhanced Wnt/-catenin signaling occurred in KO livers, and PEDF delivery in vivo reduced LRP6 activation. In human HCC cells, RNA interference of PEDF led to increased levels of activated LRP6 and -catenin, and a PEDF 34-mer peptide decreased LRP6 activation and -catenin signaling, and reduced Wnt target genes. PEDF KO mice fed a Western diet developed sporadic well-differentiated HCC. Human HCC specimens demonstrated decreased PEDF staining compared with hepatocytes.

Conclusions: PEDF is an endogenous inhibitor of Wnt/-catenin signaling in the liver.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604042PMC
http://dx.doi.org/10.1016/j.jcmgh.2015.06.006DOI Listing
September 2015

Depletion of polymorphonuclear leukocytes has no effect on preterm delivery in a mouse model of Escherichia coli-induced labor.

Am J Obstet Gynecol 2015 Nov 26;213(5):697.e1-10. Epub 2015 Jul 26.

Department of Obstetrics and Gynecology, NorthShore University HealthSystem, Evanston, IL; Pritzker School of Medicine, University of Chicago, Chicago, IL. Electronic address:

Objective: The objective of the study was to investigate the role of polymorphonuclear leukocytes (PMNs) in a mouse model of Escherichia coli-induced labor.

Study Design: Intraperitoneal injection of rabbit antimouse PMN antiserum or control was performed in CD-1 mice 29 hours and 5 hours prior to laparotomy and intrauterine injection of either killed E coli or phosphate-buffered saline on day 14.5 of pregnancy. Preterm delivery was defined as delivery of at least 1 pup within 48 hours. Circulating leukocyte counts were determined manually or by flow cytometry at the time of surgery and 8, 24, and 48 hours afterward. Maternal and fetal tissues were analyzed in a separate group of animals 8 hours after surgery.

Results: Pretreatment with anti-PMN antiserum significantly decreased the numbers of circulating leukocytes and the proportion of neutrophils among all leukocytes by 70-80% at surgery and at least 8 hours thereafter. Neutrophil depletion significantly reduced 2 markers of neutrophil activation in the uterus and placenta (neutrophil elastase and myeloperoxidase activity) and neutrophil infiltration into gestational tissues in bacterially treated animals to baseline (control) levels but did not affect preterm birth rates. The large E coli-induced increases in uterine inflammatory markers (interleukin-1β, tumor necrosis factor, chemokine ligand-5, cyclooxygenase-2) were not affected or were only minimally affected by neutrophil depletion.

Conclusion: Although PMN antiserum reduces both neutrophil number and activity, it does not diminish sensitivity to bacterially induced delivery or meaningfully alter the expression of inflammatory markers in the mouse model. Preterm birth and inflammation in this model are not likely to depend on neutrophil function.
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http://dx.doi.org/10.1016/j.ajog.2015.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631622PMC
November 2015

Identification of molecular signature of head and neck cancer stem-like cells.

Sci Rep 2015 Jan 15;5:7819. Epub 2015 Jan 15.

1] Department of Pathology, Saint Louis University, Missouri 63104 [2] Saint Louis University Cancer Center, Saint Louis University, Missouri 63104.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer in the world. HNSCC remains difficult to treat, and despite advances in treatment, overall survival rate has modestly improved over the past several years. Poor survival rate is attributed to high frequency of local recurrence and distant metastasis. Cancer stem-like cells (CSCs) have been implicated in tumor recurrence and confer resistance to anti-cancer therapy treatment. In this study, we have characterized genes that are modulated in HNSCC-CSCs and can be targeted in future as potential therapeutics. CSCs were isolated from HNSCC cells (oralspheres) and examined for tumorigenicity in immunodeficient mice. We observed aggressive tumor growth with oralspheres as compared to parental cells. The CSC-derived tumors were grossly extremely vascularized and expressed VEGFR1. We next analyzed the molecular determinant of oralspheres. In addition to CD133 and Nanog, we observed significant higher expression of Notch1 protein in the oralspheres. There was differential expression of angiogenesis and invasive marker genes such as angiopoietin1, integrin β3, MMP9 and THBS1. Interestingly, c-Fos was upregulated in oralspheres as compared to parental cells. Our observations suggest that understanding the molecular determinant of oralspheres will help in developing future therapeutic modalities against treatment resistant HNSCC.
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http://dx.doi.org/10.1038/srep07819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295111PMC
January 2015

Chemopreventive apigenin controls UVB-induced cutaneous proliferation and angiogenesis through HuR and thrombospondin-1.

Oncotarget 2014 Nov;5(22):11413-27

Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1α are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294383PMC
http://dx.doi.org/10.18632/oncotarget.2551DOI Listing
November 2014

MRS measured fatty acid composition of periprostatic adipose tissue correlates with pathological measures of prostate cancer aggressiveness.

J Magn Reson Imaging 2015 Sep 19;42(3):651-7. Epub 2014 Dec 19.

Department of Biomedical Sciences, University of Wisconsin at Milwaukee, Milwaukee, Wisconsin, USA.

Purpose: To investigate the association between magnetic resonance (MR) spectroscopically measured fatty acid composition of periprostatic adipose tissue and pathological markers of prostate cancer aggressiveness.

Materials And Methods: Periprostatic adipose (PPA) and subcutaneous adipose (SQA) tissue from prostate cancer patients undergoing radical prostatectomy were examined ex vivo by proton MR spectroscopy at 14.1T (n = 31). Fractions of monounsaturated, polyunsaturated, total unsaturated, and saturated fatty acids, as well as T2 relaxation times were measured from the spectra. Univariate and multivariate analyses based on receiver operating characteristic (ROC) and support vector machines (SVM) were used to evaluate the association between differential measures of fatty acid levels in the PPA and SQA tissues and Gleason score and extracapsular extension (ECE), which are pathological measures of prostate cancer aggressiveness.

Results: Both pathological markers for aggressive prostate cancer have separable patterns in the MRS features space. The association between ECE and PPA tissue fatty acid composition is linear (area under receiver operating characteristic curve (AROC) and 95% confidence intervals [CIs]: 1.00, [1.00, 1.00]), along the Δ(fM /fS ) measure, and is marked by elevated monounsaturated and reduced saturated fatty acids in the PPA tissue relative to SQA. In contrast, the association between Gleason score and PPA tissue fatty acid composition is nonlinear (classifier AROC and 95% CIs: 0.86, [0.71, 1.00]).

Conclusion: Fatty acid composition is altered in the PPA tissue of patients with aggressive prostate cancer. Ex vivo MR spectroscopy may be a useful tool in studying the altered fatty acid metabolism in prostate cancer.
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http://dx.doi.org/10.1002/jmri.24824DOI Listing
September 2015

Colonic mucosal fatty acid synthase as an early biomarker for colorectal neoplasia: modulation by obesity and gender.

Cancer Epidemiol Biomarkers Prev 2014 Nov 25;23(11):2413-21. Epub 2014 Aug 25.

Section of Gastroenterology, Boston University Medical Center, Boston, Massachusetts.

Background: We have previously reported that colonic pericryptal microvascular blood flow is augmented in the premalignant colonic epithelium, highlighting the increased metabolic demand of the proliferative epithelium as a marker of field carcinogenesis. However, its molecular basis is unexplored. In this study, we assessed the expression of a regulator of the "lipogenic switch," fatty acid synthase (FASN), in early colon carcinogenesis for its potential biomarker utility for concurrent neoplasia.

Methods: FASN expression (IHC) in the colonic epithelium from azoxymethane and polyposis in rat colon (Pirc) models of colorectal cancer was studied. FASN mRNA expression from endoscopically normal rectal mucosa was evaluated and correlated with colonoscopic findings (pathologic confirmation of neoplasia).

Results: FASN expression progressively increased from premalignant to malignant stage in the azoxymethane model (1.9- to 2.5-fold; P < 0.0001) and was also higher in the adenomas compared with adjacent uninvolved mucosa (1.8- to 3.4-fold; P < 0.001) in the Pirc model. Furthermore, FASN was significantly overexpressed in rectal biopsies from patients harboring adenomas compared with those with no adenomas. These effects were accentuated in male (∼2-fold) and obese patients (1.4-fold compared with those with body mass index < 30). Overall, the performance of rectal FASN was excellent (AUROC of 0.81).

Conclusions: FASN is altered in the premalignant colonic mucosa and may serve as a marker for colonic neoplasia present elsewhere. The enhanced effects in men and obesity may have implications for identifying patient subgroups at risk for early-onset neoplasia.

Impact: These findings support the role of rectal FASN expression as a reliable biomarker of colonic neoplasia.
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http://dx.doi.org/10.1158/1055-9965.EPI-14-0026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4470400PMC
November 2014

Elevated neonatal insulin-like growth factor I is associated with fetal hypertrophic cardiomyopathy in diabetic women.

Am J Obstet Gynecol 2014 Sep 9;211(3):290.e1-7. Epub 2014 May 9.

Department of Obstetrics and Gynecology, NorthShore University HealthSystem, Evanston, IL.

Objective: We sought to determine if fetal hypertrophic cardiomyopathy (HCM) or cardiac dysfunction is associated with elevated maternal or neonatal insulin-like growth factor (IGF)-I levels in women with diabetes.

Study Design: In a prospective cohort study, fetal echocardiogram findings at 36 weeks' gestation in women with pregestational diabetes mellitus were compared to those in women without diabetes mellitus. HCM was defined as septal or free wall thickness ≥5 mm and cardiac dysfunction as a modified myocardial performance index ≥0.43. Cord serum IGF-I levels at delivery were measured with enzyme-linked immunosorbent assay. Neonates with abnormal fetal echocardiogram were followed up until resolution or 6 months of life.

Results: In all, 75 participants completed fetal echocardiography (55 diabetics and 20 controls). In the diabetic group, 33 of 55 (60%) had abnormal fetal echocardiograms with cardiac dysfunction in 21 of 55 (38.2%) and HCM in 8 of 55 (14.5%) and both in 4 of 55 (7.3%). At 6 months of age, 1 of 12 (8%) had persistent HCM. None in the comparison group had abnormal findings. There were no significant clinical differences in those diabetic women with normal vs abnormal fetal echocardiograms. However, among diabetic women, mean neonatal IGF-I was significantly higher in fetuses with HCM (80 ± 16 ng/mL) as compared to those without HCM (61 ± 18 ng/mL), (P < .001).

Conclusion: Elevated neonatal IGF-I appears to be associated with fetal HCM in fetuses of diabetic women.
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http://dx.doi.org/10.1016/j.ajog.2014.05.011DOI Listing
September 2014