Endorepellin remodels the endothelial transcriptome toward a pro-autophagic and pro-mitophagic gene signature.

Regulation of autophagy by proteolytically cleaved fragments of heparan sulfate proteoglycans is a novel and current research focus in tumor biology. Endorepellin is the C-terminal angiostatic fragment of the heparan sulfate proteoglycan perlecan and induces autophagy in endothelial cells. To further investigate this property, we used NanoString, a digital PCR platform for measuring pre-defined transcripts in biological samples to analyze a custom subset of 95 autophagy-related genes in human umbilical vein endothelial cells treated with ultrapure human recombinant endorepellin. We discovered an endorepellin-evoked pro-autophagic and pro-mitophagic gene expression signatures, which included two coordinately up-regulated mitochondrial-associated genes encoding the E3 ubiquitin protein ligase Parkin and the tumor suppressor mitostatin. Induction of both proteins required the tyrosine kinase activity of vascular endothelial growth factor receptor 2 (VEGFR2). Furthermore, we discovered that endorepellin evoked mitochondrial depolarization in endothelial cells via a specific interaction between its two proximal LG1/2 domains and VEGFR2. We also found that following loss of membrane potential, mitostatin and parkin interact and that mitostatin associates with the established Parkin receptor mitofusin-2. In conclusion, we have identified a critical role for endorepellin in remodeling the autophagic transcriptome and influencing mitochondrial homeostasis

The metastasis suppressor KISS1 lacks antimetastatic activity in the C8161.9 xenograft model of melanoma.

The objective of this study was to use the established xenograft model of human melanoma (C8161.9) to test a pharmacological approach to the effect of the metastasis suppressor KISS1. A KISS1 analog was used to inhibit the metastatic development of C8161.9 cells in nude mice. Further experiments were performed to test the validity of the C8161.9 model and test the connection between KISS1 expression and loss of metastatic potential. New clones of C8161.9 cells were obtained, with or without KISS1 expression, and were tested for metastasis formation. The absence of benefit in survival with the KISS1 analog compared with PBS prompted us to revisit the C8161.9 model. We found that the cells expressing KISS1, used in the previous study and obtained by transfection and single-cell cloning, were defective for both formation of orthotopic tumors and metastases. In mixing experiments, these cells could not suppress orthotopic tumor growth of KISS1-negative C8161.9 cells, suggesting that the suppression of metastasis by C8161.9-KISS1 cells may be intrinsic to the selected clone rather than related to KISS1 expression. Isolation of clones from parental C8161.9 cells in soft agar yielded cell populations that phenotypically and genotypically mimicked the KISS1-positive clone. In addition, new clones expressing KISS1 did not show any decrease in metastatic growth. These data demonstrate the heterogeneity of cell types in the C8161.9 cell line and the high risk of artifact linked to single-cell selection. A different xenograft model will be necessary to evaluate the use of KISS1 analogs as antimetastatic therapy

Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma.

Intratumoral heterogeneity (ITH) is a prominent feature of kidney cancer. It is not known whether it has utility in finding associations between protein expression and clinical parameters. We used ITH that is detected by immunohistochemistry (IHC) to aid the association analysis between the loss of SWI/SNF components and clinical parameters.160 ccRCC tumors (40 per tumor stage) were used to generate tissue microarray (TMA). Four foci from different regions of each tumor were selected. IHC was performed against PBRM1, ARID1A, SETD2, SMARCA4, and SMARCA2. Statistical analyses were performed to correlate biomarker losses with patho-clinical parameters. Categorical variables were compared between groups using Fisher\u27s exact tests. Univariate and multivariable analyses were used to correlate biomarker changes and patient survivals. Multivariable analyses were performed by constructing decision trees using the classification and regression trees (CART) methodology. IHC detected widespread ITH in ccRCC tumors. The statistical analysis of the Truncal loss (root loss) found additional correlations between biomarker losses and tumor stages than the traditional Loss in tumor (total) . Losses of SMARCA4 or SMARCA2 significantly improved prognosis for overall survival (OS). Losses of PBRM1, ARID1A or SETD2 had the opposite effect. Thus Truncal Loss analysis revealed hidden links between protein losses and patient survival in ccRCC

Heat Shock Response in CHO Mammalian Cells Is Controlled by a Nonlinear Stochastic Process

In many biological systems, the interactions that describe the coupling between different units in a genetic network are nonlinear and stochastic. We study the interplay between stochasticity and nonlinearity using the responses of Chinese hamster ovary (CHO) mammalian cells to different temperature shocks. The experimental data show that the mean value response of a cell population can be described by a mathematical expression (empirical law) which is valid for a large range of heat shock conditions. A nonlinear stochastic theoretical model was developed that explains the empirical law for the mean response. Moreover, the theoretical model predicts a specific biological probability distribution of responses for a cell population. The prediction was experimentally confirmed by measurements at the single-cell level. The computational approach can be used to study other nonlinear stochastic biological phenomena

Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model

Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal) and Homo sapiens (epithelial) tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties

A survey of physician receptivity to molecular diagnostic testing and readiness to act on results for early-stage colon cancer patients.

BACKGROUND: We sought to assess physician interest in molecular prognosic testing for patients with early stage colon cancer, and identify factors associated with the likelihood of test adoption. METHODS: We identified physicians who care for patients with early-stage (pN0) colon cancer patients, mailed them a survey, and analyzed survey responses to assess clinician receptivity to the use of a new molecular test (GUCY2C) that identifies patients at risk for recurrence, and clinician readiness to act on abnormal test results. RESULTS: Of 104 eligible potential respondents, 41 completed and returned the survey. Among responding physicians, 56 % were receptive to using the new prognostic test. Multivariable analyses showed that physicians in academic medical centers were significantly more receptive to molecular test use than those in non-academic settings. Forty-one percent of respondents were ready to act on abnormal molecular test results. Physicians who viewed current staging methods as inaccurate and were confident in their capacity to incorporate molecular testing in practice were more likely to say they would act on abnormal test results. CONCLUSIONS: Physician receptivity to molecular diagnostic testing for early-stage colon cancer patients is likely to be influenced by practice setting and perceptions related to delivering quality care to patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01972737

Regulation of Human Chemokine Receptors CXCR4: Role of Phosphorylation in Desensitization and Internalization

Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (ΔCyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing ΔCyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of ΔCyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in ΔCyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in ΔCyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and ΔCyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipaze Cβ3 in Wt and ΔCyto CXCR4 cells. Both SDF- 1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of ΔCyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase Cβ3

Characterization and Clinical Significance of EIF1AX Mutations and Co-Mutations in Cytologically Indeterminate Thyroid Nodules: A 5-Year Retrospective Analysis.

OBJECTIVE: Mutations in the EIF1AX gene have been recently detected in a small percentage of benign and malignant thyroid lesions. We sought to investigate the prevalence and clinical significance of EIF1AX mutations and co-mutations in cytologically indeterminate thyroid nodules at our institution. MATERIALS AND METHODS: A 5-year retrospective analysis was performed on thyroid nodules with a cytologic diagnosis of Bethesda category III or IV, which had undergone testing by our in-house next generation sequencing panel. Surgically resected nodules with EIF1AX mutations were identified, and mutation type and presence of co-mutations were correlated with histopathologic diagnosis. RESULTS: 41/904 (4.5%) cases overall and 26/229 (11.4%) surgically resected nodules harbored an EIF1AX mutation. The most common histologic diagnoses were follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. 11/26 (42.3%) of nodules had isolated EIF1AX mutation. Comutations were found in RAS (12/26; 46.2%), TERT (5/26; 19.2%) and TP53 (2/26; 7.7%). EIF1AX mutation alone conferred a 36.4% risk of malignancy (ROM) and 54.5% ROM or noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), while the ROM was significantly higher in nodules with concurrent RAS (71.4%), TERT, TP53 and RAS+TERT (100%) mutations. CONCLUSION: EIF1AX mutations occur in benign and malignant follicular thyroid neoplasms. In our cohort, the majority of mutations occurred at the splice acceptor site between exons 5 and 6. Importantly, the coexistence of EIF1AX mutations with other driver pathogenic mutations in RAS, TERT and TP53 conferred a 100% ROM or NIFTP, indicating that such nodules require surgical removal

A novel role for drebrin in regulating progranulin bioactivity in bladder cancer.

We recently established a critical role for the growth factor progranulin in bladder cancer insofar as progranulin promotes urothelial cancer cell motility and contributes, as an autocrine growth factor, to the transformed phenotype by modulating invasion and anchorage-independent growth. In addition, progranulin expression is upregulated in invasive bladder cancer tissues compared to normal controls. However, the molecular mechanisms of progranulin action in bladder cancer have not been fully elucidated. In this study, we searched for novel progranulin-interacting proteins using pull-down assays with recombinant progranulin and proteomics. We discovered that drebrin, an F-actin binding protein, bound progranulin in urothelial cancer cells. We characterized drebrin function in urothelial cancer cell lines and showed that drebrin is critical for progranulin-dependent activation of the Akt and MAPK pathways and modulates motility, invasion and anchorage-independent growth. In addition, drebrin regulates tumor formation in vivo and its expression is upregulated in bladder cancer tissues compared to normal tissue controls. Our data are translationally relevant as indicate that drebrin exerts an essential functional role in the regulation of progranulin action and may constitute a novel target for therapeutic intervention in bladder tumors. In addition, drebrin may serve as novel biomarker for bladder cancer

Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer.

Whereas VHL inactivation is a primary event in clear cell renal cell carcinoma (ccRCC), the precise mechanism(s) of how this interacts with the secondary mutations in tumor suppressor genes, including PBRM1, KDM5C/JARID1C, SETD2, and/or BAP1, remains unclear. Gene expression analyses reveal that VHL, PBRM1, or KDM5C share a common regulation of interferon response expression signature. Loss of HIF2α, PBRM1, or KDM5C in VHL-/-cells reduces the expression of interferon stimulated gene factor 3 (ISGF3), a transcription factor that regulates the interferon signature. Moreover, loss of SETD2 or BAP1 also reduces the ISGF3 level. Finally, ISGF3 is strongly tumor-suppressive in a xenograft model as its loss significantly enhances tumor growth. Conversely, reactivation of ISGF3 retards tumor growth by PBRM1-deficient ccRCC cells. Thus after VHL inactivation, HIF induces ISGF3, which is reversed by the loss of secondary tumor suppressors, suggesting that this is a key negative feedback loop in ccRCC. © 2018, Liao et al