ERBB3 Overexpression is Enriched in Diverse Patient Populations with Castration-sensitive Prostate Cancer and is Associated with a Unique AR Activity Signature.

PURPOSE: Despite successful clinical management of castration-sensitive prostate cancer (CSPC), the 5-year survival rate for men with castration-resistant prostate cancer is only 32%. Combination treatment strategies to prevent disease recurrence are increasing, albeit in biomarker-unselected patients. Identifying a biomarker in CSPC to stratify patients who will progress on standard-of-care therapy could guide therapeutic strategies. EXPERIMENTAL DESIGN: Targeted deep sequencing was performed for the University of Illinois (UI) cohort (n = 30), and immunostaining was performed on a patient tissue microarray (n = 149). Bioinformatic analyses identified pathways associated with biomarker overexpression (OE) in the UI cohort, consolidated RNA sequencing samples accessed from Database of Genotypes and Phenotypes (n = 664), and GSE209954 (n = 68). Neutralizing antibody patritumab and ectopic HER3 OE were utilized for functional mechanistic experiments. RESULTS: We identified ERBB3 OE in diverse patient populations with CSPC, where it was associated with advanced disease at diagnosis. Bioinformatic analyses showed a positive correlation between ERBB3 expression and the androgen response pathway despite low dihydrotestosterone and stable expression of androgen receptor (AR) transcript in Black/African American men. At the protein level, HER3 expression was negatively correlated with intraprostatic androgen in Black/African American men. Mechanistically, HER3 promoted enzalutamide resistance in prostate cancer cell line models and HER3-targeted therapy resensitized therapy-resistant prostate cancer cell lines to enzalutamide. CONCLUSIONS: In diverse patient populations with CSPC, ERBB3 OE was associated with high AR signaling despite low intraprostatic androgen. Mechanistic studies demonstrated a direct link between HER3 and enzalutamide resistance. ERBB3 OE as a biomarker could thus stratify patients for intensification of therapy in castration-sensitive disease, including targeting HER3 directly to improve sensitivity to AR-targeted therapies.

Multiplex Imaging Reveals Novel Subcellular, Microenvironmental, and Racial Patterns of MRTFA/B Activation in Invasive Breast Cancers and Metastases.

Breast cancer progression and metastasis involve the action of multiple transcription factors in tumors and in the cells of the tumor microenvironment (TME) and understanding how these transcription factors are coordinated can guide novel therapeutic strategies. Myocardin related transcription factors A and B (MRTFA/B) are two related transcription factors that redundantly control cancer cell invasion and metastasis in mouse models of breast cancer, but their roles in human cancer are incompletely understood. Here, we used a combination of multiplexed immunofluorescence and bioinformatics analyses to show that MRTFA/B are concurrently activated in tumor cells, but they show distinct patterns of expression across different histological subtypes and in the TME. Importantly, MRTFA expression was elevated in metastatic tumors of African American patients, who disproportionately die from breast cancer. Interestingly, in contrast to publicly available mRNA expression data, MRTFA was similarly expressed across estrogen receptor (ER) positive and negative breast tumors, while MRTFB expression was highest in ER+ breast tumors. Furthermore, MRTFA was specifically expressed in the perivascular antigen presenting cells (APCs) and its expression correlated with the expression of the immune checkpoint protein V-set immunoregulatory receptor (VSIR). These results provide unique insights into how MRTFA and MRTFB can promote metastasis in human cancer, into the racial disparities of their expression patterns, and their function within the complex breast cancer TME.

Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

C-type lectin receptor expression is a hallmark of neutrophils infiltrating the skin in epidermolysis bullosa acquisita.

Introduction: Inflammatory epidermolysis bullosa acquisita (EBA) is characterized by a neutrophilic response to anti-type VII collagen (COL7) antibodies resulting in the development of skin inflammation and blistering. The antibody transfer model of EBA closely mirrors this EBA phenotype. Methods: To better understand the changes induced in neutrophils upon recruitment from peripheral blood into lesional skin in EBA, we performed single-cell RNA-sequencing of whole blood and skin dissociate to capture minimally perturbed neutrophils and characterize their transcriptome. Results: Through this approach, we identified clear distinctions between circulating activated neutrophils and intradermal neutrophils. Most strikingly, the gene expression of multiple C-type lectin receptors, which have previously been reported to orchestrate host defense against fungi and select bacteria, were markedly dysregulated. After confirming the upregulation of Clec4n, Clec4d, and Clec4e in experimental EBA as well as in lesional skin from patients with inflammatory EBA, we performed functional studies in globally deficient Clec4e-/- and Clec4d-/- mice as well as in neutrophil-specific Clec4n-/- mice. Deficiency in these genes did not reduce disease in the EBA model. Discussion: Collectively, our results suggest that while the upregulation of Clec4n, Clec4d, and Clec4e is a hallmark of activated dermal neutrophil populations, their individual contribution to the pathogenesis of EBA is dispensable.

Machine Learning for Digital Scoring of PRMT6 in Immunohistochemical Labeled Lung Cancer.

Lung cancer is the leading cause of cancer death in the U.S. Therefore, it is imperative to identify novel biomarkers for the early detection and progression of lung cancer. PRMT6 is associated with poor lung cancer prognosis. However, analyzing PRMT6 expression manually in large samples is time-consuming posing a significant limitation for processing this biomarker. To overcome this issue, we trained and validated an automated method for scoring PRMT6 in lung cancer tissues, which can then be used as the standard method in future larger cohorts to explore population-level associations between PRMT6 expression and sociodemographic/clinicopathologic characteristics. We evaluated the ability of a trained artificial intelligence (AI) algorithm to reproduce the PRMT6 immunoreactive scores obtained by pathologists. Our findings showed that tissue segmentation to cancer vs. non-cancer tissues was the most critical parameter, which required training and adjustment of the algorithm to prevent scoring non-cancer tissues or ignoring relevant cancer cells. The trained algorithm showed a high concordance with pathologists with a correlation coefficient of 0.88. The inter-rater agreement was significant, with an intraclass correlation of 0.95 and a scale reliability coefficient of 0.96. In conclusion, we successfully optimized a machine learning algorithm for scoring PRMT6 expression in lung cancer that matches the degree of accuracy of scoring by pathologists.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.

Regulation of SELENOF translation by eIF4a3: Possible role in prostate cancer progression.

The levels of the SELENOF selenoprotein are dramatically reduced in prostate cancer compared to adjacent benign tissue and reducing SELENOF in prostate epithelial cells results in the acquisition of features of the transformed phenotype. It was hypothesized that the aberrant increase in the eiF4a3 translation factor, which has an established role in RNA splicing and the regulation of selenoprotein translation, contributes to the lower levels of SELENOF. Using the available databases, eIF4a3 messenger RNA (mRNA) levels are elevated in prostate cancer compared to normal tissue as is the hypomethylation of the corresponding gene. Using a prostate cancer tissue microarray, we established that eiF4a3 levels are higher in prostate cancer tissue. Ectopic expression of eIF4a3 in prostate cancer cells reduced SELENOF levels and attenuated the readthrough of the UGA codon using a specialized reporter construct designed to examine UGA decoding, with the opposite effects observed using eIF4a3 knock-down constructs. Direct binding of eIF4a3 to the regulatory regions of SELENOF mRNA was established with pull-down experiments. Lastly, we show that an eIF4a3 inhibitor, eIF4a3-IN-2, increases SELENOF levels, UGA readthrough, and reduces binding of eIF4a3 to the SELENOF mRNA 3'-UTR in exposed cells. These data establish eIF4a3 as a likely prostate cancer oncogene and a regulator of SELENOF translation.

α-Mangostin Promotes In Vitro and In Vivo Degradation of Androgen Receptor and AR-V7 Splice Variant in Prostate Cancer Cells.

A major limitation of current prostate cancer pharmacotherapy approaches is the inability of these compounds to target androgen receptor variants or mutants that develop during prostate cancer progression. The demand for novel therapeutics to prevent, slow, and treat prostate cancer is significant because FDA approved anti-androgens are associated with adverse events and can eventually drive drug-resistant prostate cancer. This study evaluated α-mangostin for its novel ability to degrade the androgen receptor and androgen receptor variants. α-Mangostin is one of more than 70 isoprenylated xanthones isolated from Garcinia mangostana that we have been evaluating for their anticancer potential. Prostate cancer cells treated with α-mangostin exhibited decreased levels of wild-type and mutated androgen receptors. Immunoblot, immunoprecipitation, and transfection experiments demonstrated that the androgen receptor was ubiquitinated and subsequently degraded via the proteasome, which we hypothesize occurs with the assistance of BiP, an ER chaperone protein that we have shown to associate with the androgen receptor. We also evaluated α-mangostin for its antitumor activity and promotion of androgen receptor degradation in vivo. In summary, our study demonstrates that androgen receptor degradation occurs through the novel activation of BiP and suggests a new therapeutic approach for prostate cancer.

The basis and design for time-restricted eating compared with daily calorie restriction for weight loss and colorectal cancer risk reduction trial (TRE-CRC trial).

OBJECTIVE: Approximately 42% of American adults are living with obesity, increasing their risk of colorectal cancer (CRC). Efficacious approaches to prevent and treat obesity may reduce CRC incidence. Daily calorie restriction (Cal-R) is the most common approach to treating obesity, yet clinically meaningful weight loss is elusive owing to waning adherence. Time-restricted eating (TRE) consists of consuming foods within a specified time frame, creating a natural calorie deficit. TRE in animals shows cancer protective effects. In humans, TRE is safe and acceptable among adults with obesity, producing ~3% to 5% weight loss and reductions in oxidative stress and insulin resistance. However, TRE has not been tested rigorously for CRC preventive effects. METHODS: The authors describe a 12-month randomized controlled trial of 8-hour TRE (ad libitum 12 PM-8 PM), Cal-R (25% restriction daily), or Control among 255 adults at increased risk for CRC and with obesity. RESULTS: Effects on the following will be examined: 1) body weight, body composition, and adherence; 2) circulating metabolic, inflammation, and oxidative stress biomarkers; 3) colonic mucosal gene expression profiles and tissue microenvironment; and 4) maintenance of benefits on body weight/composition and CRC risk markers. CONCLUSIONS: This study will examine efficacious lifestyle strategies to treat obesity and reduce CRC risk among individuals with obesity.

GATA-3 is a proto-oncogene in T-cell lymphoproliferative neoplasms.

Neoplasms originating from thymic T-cell progenitors and post-thymic mature T-cell subsets account for a minority of lymphoproliferative neoplasms. These T-cell derived neoplasms, while molecularly and genetically heterogeneous, exploit transcription factors and signaling pathways that are critically important in normal T-cell biology, including those implicated in antigen-, costimulatory-, and cytokine-receptor signaling. The transcription factor GATA-3 regulates the growth and proliferation of both immature and mature T cells and has recently been implicated in T-cell neoplasms, including the most common mature T-cell lymphoma observed in much of the Western world. Here we show that GATA-3 is a proto-oncogene across the spectrum of T-cell neoplasms, including those derived from T-cell progenitors and their mature progeny, and further define the transcriptional programs that are GATA-3 dependent, which include therapeutically targetable gene products. The discovery that p300-dependent acetylation regulates GATA-3 mediated transcription by attenuating DNA binding has novel therapeutic implications. As most patients afflicted with GATA-3 driven T-cell neoplasms will succumb to their disease within a few years of diagnosis, these findings suggest opportunities to improve outcomes for these patients.

Notch Signaling Promotes Mature T-Cell Lymphomagenesis.

Peripheral T-cell lymphomas (PTCL) are agressive lymphomas that develop from mature T cells. The most common PTCLs are genetically, molecularly, and clinically diverse and are generally associated with dismal outcomes. While Notch signaling plays a critically important role in both the development of immature T cells and their malignant transformation, its role in PTCL is poorly understood, despite the increasingly appreciated function of Notch in regulating the proliferation and differentiation of mature T cells. Here, we demonstrate that Notch receptors and their Delta-like family ligands (DLL1/DLL4) play a pathogenic role in PTCL. Notch1 activation was observed in common PTCL subtypes, including PTCL-not otherwise specified (NOS). In a large cohort of PTCL-NOS biopsies, Notch1 activation was significantly associated with surrogate markers of proliferation. Complementary genetically engineered mouse models and spontaneous PTCL models were used to functionally examine the role of Notch signaling, and Notch1/Notch2 blockade and pan-Notch blockade using dominant-negative MAML significantly impaired the proliferation of malignant T cells and PTCL progression in these models. Treatment with DLL1/DLL4 blocking antibodies established that Notch signaling is ligand-dependent. Together, these findings reveal a role for ligand-dependent Notch signaling in driving peripheral T-cell lymphomagenesis. SIGNIFICANCE: This work demonstrates that ligand-dependent Notch activation promotes the growth and proliferation of mature T-cell lymphomas, providing new therapeutic strategies for this group of aggressive lymphomas.

Inhibition of CCL28/CCR10-Mediated eNOS Downregulation Improves Skin Wound Healing in the Obesity-Induced Mouse Model of Type 2 Diabetes.

Chronic, nonhealing skin wounds, such as diabetic foot ulcers (DFUs), are common in patients with type 2 diabetes. Here, we investigated the role of chemokine (C-C motif) ligand 28 (CCL28) and its receptor C-C chemokine receptor type 10 (CCR10) in downregulation of endothelial nitric (NO) oxide synthase (eNOS) in association with delayed skin wound healing in the db/db mouse model of type 2 diabetes. We observed reduced eNOS expression and elevated CCL28/CCR10 levels in dorsal skin of db/db mice and subdermal leg biopsy specimens from human subjects with type 2 diabetes. Further interrogation revealed that overexpression of CCR10 reduced eNOS expression, NO bioavailability, and tube formation of human dermal microvascular endothelial cells (HDMVECs) in vitro, which was recapitulated in mouse dorsal skin. In addition, incubation of HDMVECs with CCL28 led to internalization of the CCR10/eNOS complex and colocalization with lysosome-associated membrane protein 1. Finally, topical application of myristoylated CCR10 binding domain 7 amino acid (Myr-CBD7) peptide prevented CCR10-eNOS interaction and subsequent eNOS downregulation, enhanced eNOS/NO levels, eNOS/VEGF-R2+ microvessel density, and blood perfusion, reduced inflammatory cytokine levels, and importantly, decreased wound healing time in db/db mice. Thus, endothelial cell CCR10 activation in genetically obese mice with type 2 diabetes promotes eNOS depletion and endothelial dysfunction, and targeted disruption of CCR10/eNOS interaction improves wound healing.

Advanced glycation end-products (AGEs) are lower in prostate tumor tissue and inversely related to proportion of West African ancestry.

BACKGROUND: The metabolism of normal prostate relies on glycolysis, with prostate cancer having reduced glycolysis and increased aerobic metabolism. Advanced glycation end products (AGEs) accumulate in tissues as a result of age and glycolytic rate. Differential AGE levels were recently observed in prostate cancer tissues. Herein we sought to quantify AGEs in benign and cancer prostate tissue in a diverse cohort of patients. METHODS: Levels of the AGE Nε-(carboxylethyl)lysine (CML) were quantified by immunohistochemistry (IHC) in a tissue microarray which consisted of 3 cores from tumor and 2 cores from benign areas from 118 patients (87 African American and 31 European American). Ancestry informative markers for African Ancestry were available for 79 patients. Epithelial and stromal areas were quantified separately using an E-cadherin mask. CML levels were compared with clinical grade group and ancestry by mixed linear effect models. Age, prostate-specific antigen (PSA) levels, body mass index (BMI), and hemoglobin A1C were included as covariates. RESULTS: CML levels were lower in areas of the tumor, for both epithelium and surrounding stroma, compared with benign, but did not significantly change with tumor grade group. Age, PSA levels, BMI, and hemoglobin A1C did not associate with CML levels. CML levels were inversely associated with the percentage of African Ancestry in all tissues. CONCLUSIONS: The low CML levels in cancer may reflect the reduced glycolytic state of the tissue. The inverse relationship between African Ancestry and CML levels in both benign and cancer areas suggests a state of reduced glycolysis. It is yet to be determined whether altered glycolysis and CML levels are bystanders or drivers of carcinogenesis.

Targeting Lymphoma-associated Macrophage Expansion via CSF1R/JAK Inhibition is a Therapeutic Vulnerability in Peripheral T-cell Lymphomas.

The reciprocal relationship between malignant T cells and lymphoma-associated macrophages (LAM) within the tumor microenvironment (TME) is unique, as LAMs are well poised to provide ligands for antigen, costimulatory, and cytokine receptors that promote T-cell lymphoma growth. Conversely, malignant T cells promote the functional polarization and homeostatic survival of LAM. Therefore, we sought to determine the extent to which LAMs are a therapeutic vulnerability in these lymphomas, and to identify effective therapeutic strategies for their depletion. We utilized complementary genetically engineered mouse models and primary peripheral T-cell lymphoma (PTCL) specimens to quantify LAM expansion and proliferation. A high-throughput screen was performed to identify targeted agents that effectively deplete LAM within the context of PTCL. We observed that LAMs are dominant constituents of the TME in PTCL. Furthermore, their dominance was explained, at least in part, by their proliferation and expansion in response to PTCL-derived cytokines. Importantly, LAMs are a true dependency in these lymphomas, as their depletion significantly impaired PTCL progression. These findings were extrapolated to a large cohort of human PTCL specimens where LAM proliferation was observed. A high-throughput screen demonstrated that PTCL-derived cytokines led to relative resistance to CSF1R selective inhibitors, and culminated in the identification of dual CSF1R/JAK inhibition as a novel therapeutic strategy to deplete LAM in these aggressive lymphomas. Malignant T cells promote the expansion and proliferation of LAM, which are a bone fide dependency in these lymphomas, and are effectively depleted with a dual CSF1R/JAK inhibitor. Significance: LAMs are a therapeutic vulnerability, as their depletion impairs T-cell lymphoma disease progression. Pacritinib, a dual CSF1R/JAK inhibitor, effectively impaired LAM viability and expansion, prolonged survival in preclinical T-cell lymphoma models, and is currently being investigated as a novel therapeutic approach in these lymphomas.

Conventional versus drug-eluting embolic transarterial chemoembolization with doxorubicin: comparative drug delivery, pharmacokinetics, and treatment response in a rabbit VX2 tumor model.

The purpose of this study was to compare intra-tumoral drug delivery, pharmacokinetics, and treatment response after doxorubicin (DOX) conventional (c-) versus drug-eluting embolic (DEE-) transarterial chemoembolization (TACE) in a rabbit VX2 liver tumor model. Twenty-four rabbits with solitary liver tumors underwent c-TACE (n = 12) (1:2 water-in-oil emulsion, 0.6 mL volume, 2 mg DOX) or DEE-TACE (n = 12) (130,000 70-150 µm 2 mg DOX-loaded microspheres). Systemic, intra-tumoral, and liver DOX levels were measured using mass spectrometry up to 7-day post-procedure. Intra-tumoral DOX distribution was quantified using fluorescence imaging. Percent tumor necrosis was quantified by a pathologist blinded to treatment group. Lobar TACE was successfully performed in all cases. Peak concentration (CMAX, µg/mL) for plasma, tumor tissue, and liver were 0.666, 4.232, and 0.270 for c-TACE versus 0.103, 8.988, and 0.610 for DEE-TACE. Area under the concentration versus time curve (AUC, µg/mL ∗ min) for plasma, tumor tissue, and liver were 18.3, 27,078.8, and 1339.1 for c-TACE versus 16.4, 26,204.8, and 1969.6 for DEE-TACE. A single dose of intra-tumoral DOX maintained cytotoxic levels through 7-day post-procedure for both TACE varieties, with a half-life of 1.8 (c-TACE) and 0.8 (DEE-TACE) days. Tumor-to-normal liver DOX ratio was high (c-TACE, 20.2; DEE-TACE, 13.3). c-TACE achieved significantly higher DOX coverage of tumor vs. DEE-TACE (10.8% vs. 2.3%; P = 0.003). Percent tumor necrosis was similar (39% vs. 37%; P = 0.806). In conclusion, in a rabbit VX2 liver tumor model, both c-TACE and DEE-TACE achieved tumoricidal intra-tumoral DOX levels and high tumor-to-normal liver drug ratios, though c-TACE resulted in significantly greater tumor coverage.

Loss of SELENOF Induces the Transformed Phenotype in Human Immortalized Prostate Epithelial Cells.

SELENOF is a member of the class of selenoproteins in which the amino acid selenocysteine is co-translationally inserted into the elongating peptide in response to an in-frame UGA codon located in the 3'-untranslated (3'-UTR) region of the SELENOF mRNA. Polymorphisms in the 3'-UTR are associated with an increased risk of dying from prostate cancer and these variations are functional and 10 times more frequent in the genomes of African American men. SELENOF is dramatically reduced in prostate cancer compared to benign adjacent regions. Using a prostate cancer tissue microarray, it was previously established that the reduction of SELENOF in the cancers from African American men was significantly greater than in cancers from Caucasian men. When SELENOF levels in human prostate immortalized epithelial cells were reduced with an shRNA construct, those cells acquired the ability to grow in soft agar, increased the ability to migrate in a scratch assay and acquired features of energy metabolism associated with prostate cancer. These results support a role of SELENOF loss in prostate cancer progression and further indicate that SELENOF loss and genotype may contribute to the disparity in prostate cancer mortality experienced by African American men.

Integration of genomics and transcriptomics predicts diabetic retinopathy susceptibility genes.

We determined differential gene expression in response to high glucose in lymphoblastoid cell lines derived from matched individuals with type 1 diabetes with and without retinopathy. Those genes exhibiting the largest difference in glucose response were assessed for association with diabetic retinopathy in a genome-wide association study meta-analysis. Expression quantitative trait loci (eQTLs) of the glucose response genes were tested for association with diabetic retinopathy. We detected an enrichment of the eQTLs from the glucose response genes among small association p-values and identified folliculin (FLCN) as a susceptibility gene for diabetic retinopathy. Expression of FLCN in response to glucose was greater in individuals with diabetic retinopathy. Independent cohorts of individuals with diabetes revealed an association of FLCN eQTLs with diabetic retinopathy. Mendelian randomization confirmed a direct positive effect of increased FLCN expression on retinopathy. Integrating genetic association with gene expression implicated FLCN as a disease gene for diabetic retinopathy.

One of the side effects of diabetes is loss of vision from diabetic retinopathy, which is caused by injury to the light sensing tissue in the eye, the retina. Almost all individuals with diabetes develop diabetic retinopathy to some extent, and it is the leading cause of irreversible vision loss in working-age adults in the United States. How long a person has been living with diabetes, the extent of increased blood sugars and genetics all contribute to the risk and severity of diabetic retinopathy. Unfortunately, virtually no genes associated with diabetic retinopathy have yet been identified. When a gene is activated, it produces messenger molecules known as mRNA that are used by cells as instructions to produce proteins. The analysis of mRNA molecules, as well as genes themselves, can reveal the role of certain genes in disease. The studies of all genes and their associated mRNAs are respectively called genomics and transcriptomics. Genomics reveals what genes are present, while transcriptomics shows how active genes are in different cells. Skol et al. developed methods to study genomics and transcriptomics together to help discover genes that cause diabetic retinopathy. Genes involved in how cells respond to high blood sugar were first identified using cells grown in the lab. By comparing the activity of these genes in people with and without retinopathy the study identified genes associated with an increased risk of retinopathy in diabetes. In people with retinopathy, the activity of the folliculin gene (FLCN) increased more in response to high blood sugar. This was further verified with independent groups of people and using computer models to estimate the effect of different versions of the folliculin gene. The methods used here could be applied to understand complex genetics in other diseases. The results provide new understanding of the effects of diabetes. They may also help in the development of new treatments for diabetic retinopathy, which are likely to improve on the current approach of using laser surgery or injections into the eye.

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells.

Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.

Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

AKT activation by N-cadherin regulates beta-catenin signaling and neuronal differentiation during cortical development.

BACKGROUND: During cerebral cortical development, neural precursor-precursor interactions in the ventricular zone neurogenic niche coordinate signaling pathways that regulate proliferation and differentiation. Previous studies with shRNA knockdown approaches indicated that N-cadherin adhesion between cortical precursors regulates ß-catenin signaling, but the underlying mechanisms remained poorly understood. RESULTS: Here, with conditional knockout approaches, we find further supporting evidence that N-cadherin maintains ß-catenin signaling during cortical development. Using shRNA to N-cadherin and dominant negative N-cadherin overexpression in cell culture, we find that N-cadherin regulates Wnt-stimulated ß-catenin signaling in a cell-autonomous fashion. Knockdown or inhibition of N-cadherin with function-blocking antibodies leads to reduced activation of the Wnt co-receptor LRP6. We also find that N-cadherin regulates ß-catenin via AKT, as reduction of N-cadherin causes decreased AKT activation and reduced phosphorylation of AKT targets GSK3ß and ß-catenin. Inhibition of AKT signaling in neural precursors in vivo leads to reduced ß-catenin-dependent transcriptional activation, increased migration from the ventricular zone, premature neuronal differentiation, and increased apoptotic cell death. CONCLUSIONS: These results show that N-cadherin regulates ß-catenin signaling through both Wnt and AKT, and suggest a previously unrecognized role for AKT in neuronal differentiation and cell survival during cortical development.