Cholesterol Pathway Inhibition Induces TGF-ß Signaling to Promote Basal Differentiation in Pancreatic Cancer.

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by KrasG12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor ß signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.

Remodeling of the Collagen Matrix in Aging Skin Promotes Melanoma Metastasis and Affects Immune Cell Motility.

Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins, and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3-D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effect on the migration of T cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected regulatory T-cell recruitment. These data suggest that although age-related physical changes in the ECM can promote tumor cell motility, they may adversely affect the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older patients with melanoma. SIGNIFICANCE: These data shed light on the mechanochemical interactions that occur between aged skin, tumor, and immune cell populations, which may affect tumor metastasis and immune cell infiltration, with implications for the efficacy of current therapies for melanoma.See related commentary by Marie and Merlino, p. 19.This article is highlighted in the In This Issue feature, p. 1.

Patient-derived Models Reveal Impact of the Tumor Microenvironment on Therapeutic Response.

BACKGROUND: Androgen deprivation therapy is a first-line treatment for disseminated prostate cancer (PCa). However, virtually all tumors become resistant and recur as castration-resistant PCa, which has no durable cure. One major hurdle in the development of more effective therapies is the lack of preclinical models that adequately recapitulate the heterogeneity of PCa, significantly hindering the ability to accurately predict therapeutic response. OBJECTIVE: To leverage the ex vivo culture method termed patient-derived explant (PDE) to examine the impact of PCa therapeutics on a patient-by-patient basis. DESIGN SETTING AND PARTICIPANTS: Fresh PCa tissue from patients who underwent radical prostatectomy was cultured as PDEs to examine therapeutic response. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The impact of genomic and chemical perturbations in PDEs was assessed using various parameters (eg, AR levels, Ki67 staining, and desmoplastic indices). RESULTS AND LIMITATIONS: PDE maintained the integrity of the native tumor microenvironment (TME), tumor tissue morphology, viability, and endogenous hormone signaling. Tumor cells in this model system exhibited de novo proliferative capacity. Examination of the native TME in the PDE revealed a first-in-field insight into patient-specific desmoplastic stromal indices and predicted responsiveness to AR-directed therapeutics. CONCLUSIONS: The PDE model allows for a comprehensive evaluation of individual tumors in their native TME to ultimately develop more effective therapeutic regimens tailored to individuals. Discernment of novel stromal markers may provide a basis for applying precision medicine in treating advanced PCa, which would have a transformative effect on patient outcomes. PATIENT SUMMARY: In this study, an innovative model system was used to more effectively mimic human disease. The patient-derived explant (PDE) system can be used to predict therapeutic response and identify novel targets in advanced disease. Thus, the PDE will be an asset for the development of novel metrics for the implementation of precision medicine in prostate cancer.The patient-derived explant (PDE) model allows for a comprehensive evaluation of individual human tumors in their native tumor microenvironment (TME). TME analysis revealed first-in-field insight into predicted tumor responsiveness to AR-directed therapeutics through evaluation of patient-specific desmoplastic stromal indices.

Matrix-regulated integrin αvß5 maintains α5ß1-dependent desmoplastic traits prognostic of neoplastic recurrence.

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5ß1-integrin. Mechanistically, we established that TGFß is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvß5-integrin redistribution of pFAK-independent active α5ß1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5ß1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool.

Travel and implications for the elimination of trachoma in ethiopia.

PURPOSE: Trachoma is the leading infectious cause of blindness. The World Health Organization has set a goal of reducing the trachoma disease burden to a level where it is no longer a public health concern by the year 2020. Some investigators feel that local elimination of ocular chlamydia infection is possible, but little has been done to study the likelihood of reintroduction of infection from neighboring areas. Mass administration of azithromycin has been shown to dramatically reduce the prevalence of infection in many villages in central Ethiopia. However, after treatment is discontinued, infection returns. Reintroduction of infection could occur from the few remaining infected cases in a treated community or from outside the community. People traveling between villages might be responsible thus complicating the elimination of trachoma. METHODS: We conducted a survey to assess the travel pattern of the Gurage zone residents in Ethiopia. Seven hundred and seventeen households with at least one child aged 1-5 years in 48 villages were surveyed to collect the details of travel in 1 month prior to the survey. RESULTS: Seventy-eight percent of the surveyed households had at least one traveler, with the majority being women. Pre-school children, the main reservoir of clinically active infection, rarely traveled. Most travel was to the market or to school, and most for less than 1 day. CONCLUSIONS: Travel routinely takes place in these villages. Trachoma control programs in this area might consider treating areas with the same markets and schools in the same period to increase the efficacy of mass treatment.

Detection of Chlamydia trachomatis ocular infection in trachoma-endemic communities by rRNA amplification.

PURPOSE: Trachoma remains the leading infectious cause of blindness worldwide. The World Health Organization (WHO) recommends mass antibiotic distributions in its strategy to eliminate blinding trachoma. To determine the most effective antibiotic treatment strategy, it is essential to have a diagnostic test that can correctly measure the true status of ocular Chlamydia trachomatis infection in individuals, particularly after treatment. A newer ribosomal ribonucleic acid (rRNA)-based amplification test was compared with the current DNA-based polymerase chain reaction (PCR) for the detection of C. trachomatis. METHODS: An rRNA-based assay and PCR were performed on swab specimens taken from the right upper tarsal conjunctiva of 240 children aged 1 to 5 years living among 16 endemic villages in the Gurage Zone, Ethiopia. RESULTS: The rRNA-based test detected ocular C. trachomatis infection in 142 (59%) subjects compared with 67 (28%) detected by PCR (McNemar's test, P < 0.0001). The rRNA-based test gave positive results for all subjects who were positive by PCR and detected infection in 75 (31%) additional subjects. CONCLUSIONS: The rRNA-based test appears to have significantly greater sensitivity than PCR for the detection of ocular C. trachomatis infection in children in trachoma-endemic villages. The increased sensitivity of the rRNA-based test may be due to its ability to detect low levels of C. trachomatis infection in individuals, which can occur especially after antibiotic treatment. Data from past studies in which PCR was used to assess the prevalence of infectious trachoma after community-wide antibiotic treatments could have underestimated the true prevalence of infection.

Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway.

The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

Basal Igalpha/Igbeta signals trigger the coordinated initiation of pre-B cell antigen receptor-dependent processes.

The pro-B to pre-B transition during B cell development is dependent upon surface expression of a signaling competent pre-B cell Ag receptor (pre-BCR). Although the mature form of the BCR requires ligand-induced aggregation to trigger responses, the requirement for ligand-induced pre-BCR aggregation in promoting B cell development remains a matter of significant debate. In this study, we used transmission electron microscopy on murine primary pro-B cells and pre-B cells to analyze the aggregation state of the pre-BCR. Although aggregation can be induced and visualized following cross-linking by Abs to the pre-BCR complex, our analyses indicate that the pre-BCR is expressed on the surface of resting cells primarily in a nonaggregated state. To evaluate the degree to which basal signals mediated through nonaggregated pre-BCR complexes can promote pre-BCR-dependent processes, we used a surrogate pre-BCR consisting of the cytoplasmic regions of Igalpha/Igbeta that is targeted to the inner leaflet of the plasma membrane of primary pro-B cells. We observed enhanced proliferation in the presence of low IL-7, suppression of V(H)(D)J(H) recombination, and induced kappa light (L) chain recombination and cytoplasmic kappa L chain protein expression. Interestingly, Igalpha/Igbeta-mediated allelic exclusion was restricted to the B cell lineage as we observed normal TCRalphabeta expression on CD8-expressing splenocytes. This study directly demonstrates that basal signaling initiated through Igalpha/Igbeta-containing complexes facilitates the coordinated control of differentiation events that are associated with the pre-BCR-dependent transition through the pro-B to pre-B checkpoint. Furthermore, these results argue that pre-BCR aggregation is not a requirement for pre-BCR function.

Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation.

The mammalian Bin1/Amphiphysin II gene encodes an assortment of alternatively spliced adapter proteins that exhibit markedly divergent expression and subcellular localization profiles. Bin1 proteins have been implicated in a variety of different cellular processes, including endocytosis, actin cytoskeletal organization, transcription, and stress responses. To gain insight into the physiological functions of the Bin1 gene, we have disrupted it by homologous recombination in the mouse. Bin1 loss had no discernible impact on either endocytosis or phagocytosis in mouse embryo-derived fibroblasts and macrophages, respectively. Similarly, actin cytoskeletal organization, proliferation, and apoptosis in embryo fibroblasts were all unaffected by Bin1 loss. In vivo, however, Bin1 loss resulted in perinatal lethality. Bin1 has been reported to affect muscle cell differentiation and T-tubule formation. No striking histological abnormalities were evident in skeletal muscle of Bin1 null embryos, but severe ventricular cardiomyopathy was observed in these embryos. Ultrastructurally, myofibrils in ventricular cardiomyocytes of Bin1 null embryos were severely disorganized. These results define a developmentally critical role for the Bin1 gene in cardiac muscle development.

Platelet factor 4 binds to low-density lipoprotein receptors and disrupts the endocytic machinery, resulting in retention of low-density lipoprotein on the cell surface.

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R-dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 microg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.