The Role of Metastasis Suppressor CD82 in the Deactivation of the c-Met Signaling Pathway in Prostate Cancer

BACKGROUND AND PURPOSE: Prostate cancer is the second leading cause of cancer deaths among men in the United States. Metastasis plays a major role in patient prognosis and treatment options. One of the factors that influences metastasis is the activation of the c-Met signaling pathway. Activation of the growth factor c-Met results in cytoskeletal rearrangement, migration, and invasion. Previous studies have shown CD82 to act as an active metastatic suppressor in normal, healthy cells and is downregulated in various forms of cancer. When CD82 is re-expressed in cancer cells, the cells no longer express metastatic characteristics. The purpose of this study was to determine what effect, if any, re-expression of CD82 in prostate cancer cells had on c- Met mediated migratory characteristics. METHODS AND MATERIALS: Two prostate cancer cell lines were utilized for this project. PC3-29 cells have been engineered with a vector that expresses CD82 and PC3-5V carry an empty vector and were used as a control in this study. ANALYSES: Expression levels of CD82, c-Met, related migratory proteins, and the activation state of c-Met were determined via western blot analysis. Visualization of cytoskeletal changes was done by staining F-actin fibers and focal adhesions. RESULTS: Re-expression of CD82 in PC3-29 cells led to a decrease in c-Met activation. CD82 prevented the formation of F-actin fibers and focal adhesions needed for metastasis. CONCLUSIONS: CD82 directly impacts the activation of c-Met by preventing the phosphorylation of Rac1, inhibiting the cytoskeletal changes needed for metastasis to occur

Sept8/SEPTIN8 involvement in cellular structure and kidney damage is identified by genetic mapping and a novel human tubule hypoxic model.

Chronic kidney disease (CKD), which can ultimately progress to kidney failure, is influenced by genetics and the environment. Genes identified in human genome wide association studies (GWAS) explain only a small proportion of the heritable variation and lack functional validation, indicating the need for additional model systems. Outbred heterogeneous stock (HS) rats have been used for genetic fine-mapping of complex traits, but have not previously been used for CKD traits. We performed GWAS for urinary protein excretion (UPE) and CKD related serum biochemistries in 245 male HS rats. Quantitative trait loci (QTL) were identified using a linear mixed effect model that tested for association with imputed genotypes. Candidate genes were identified using bioinformatics tools and targeted RNAseq followed by testing in a novel in vitro model of human tubule, hypoxia-induced damage. We identified two QTL for UPE and five for serum biochemistries. Protein modeling identified a missense variant within Septin 8 (Sept8) as a candidate for UPE. Sept8/SEPTIN8 expression increased in HS rats with elevated UPE and tubulointerstitial injury and in the in vitro hypoxia model. SEPTIN8 is detected within proximal tubule cells in human kidney samples and localizes with acetyl-alpha tubulin in the culture system. After hypoxia, SEPTIN8 staining becomes diffuse and appears to relocalize with actin. These data suggest a role of SEPTIN8 in cellular organization and structure in response to environmental stress. This study demonstrates that integration of a rat genetic model with an environmentally induced tubule damage system identifies Sept8/SEPTIN8 and informs novel aspects of the complex gene by environmental interactions contributing to CKD risk

Role of Metastasis Suppressor CD82 in the Deactivation of the C-Met Signaling Pathway in Prostate Cancer

PURPOSE: Prostate cancer is the second leading cause of cancer deaths among men in the United States. Metastasis plays a major role in patient prognosis and treatment options. One of the factors that has been proven to influence metastasis is the activation of the c-Met signaling pathway. Activation of the growth factor c-Met results in cytoskeletal rearrangement, migration and invasion. Previous studies have shown CD82 to act as an active metastatic suppressor in normal, healthy cells, and is downregulated in various forms of cancer. When CD82 is re-expressed in cancer cells, the cells no longer express metastatic characteristics. The purpose of this study was to determine what effect, if any, re-expression of CD82 in prostate cancer cells had on c-Met mediated migratory characteristics. METHODS AND MATERIALS: Two prostate cancer cell lines were utilized for this project. PC3-29 cells have been engineered with a vector that expresses CD82 and PC3-5V carry an empty vector and used as a control in this study. ANALYSES: Expression levels of CD82, c-Met, and related migratory proteins and activation state of c-Met were determined via western blot analysis. Visualization of cytoskeletal changes was done by immunostaining F-actin fibers and focal adhesions. RESULTS: Re-expression of CD82 in PC3-29 cells led to a decrease in c-Met activation. CD82 prevented the formation of F-actin fibers and focal adhesions needed for metastasis. CONCLUSIONS: CD82 directly impacts the activation levels of c-Met by preventing the phosphorylation of downstream effector molecules, halting the cytoskeletal changes needed for metastasis to occur

Harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor induces caspase-mediated apoptosis in neuroblastoma

Background: Neuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system. Harmine is a tricyclic β-carboline alkaloid isolated from the harmal plant that exhibits both cytostatic and cytotoxic effects. Harmine is capable of blocking the activities of dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins and mitogen-activated protein kinase. These kinases promote proliferation and inhibit apoptosis. Methods: Four human NB cell lines were used to study the effects of harmine treatment: SKNBE and KELLY (MYCN-amplified) as well as SKNAS and SKNFI (MYCN non-amplified). The anti-cancer properties of harmine were examined by RealTime-Glo MT cell viability assays, caspase activity assays, PARP cleavage using Western blot analysis, and flow cytometry-based Annexin V detection. A molecular interaction model of harmine bound to the DYRK2 family kinase was generated by computational docking using X-ray structures. NB tumors from human patients were profiled for DYRK mRNA expression patterns and clinical correlations using the R2 platform. Results: The IC50 values for harmine after 72h treatment were 169.6, 170.8, and 791.7μM for SKNBE, KELLY, and SKNFI, respectively. Exposure of these NB cell lines to 100μM of harmine resulted in caspase-3/7 and caspase-9 activation as well as caspase-mediated PARP cleavage and Annexin V-positive stained cells, as early as 24h after treatment, clearly suggesting apoptosis induction, especially in MYCN-amplified cell lines. Elevated DYRK2 mRNA levels correlated with poor prognosis in a large cohort of NB tumors. Conclusion: Harmine is a known inhibitor of DYRK family kinases. It can induce apoptosis in NB cell lines, which led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that DYRK inhibition by harmine and the subsequent triggering of caspase-mediated apoptosis might present a novel approach to NB therapy

Correlation (Pearson's coefficient) between BDNF levels and LDAEP or psychometric ratings.

<p>HAMD, Hamilton Depression Rating Scale; HAMA, Hamilton Anxiety Scale; BIS, Barratt Impulsiveness Scale; BHS, Beck Hopelessness Scale; BSS, Beck Scale for Suicidal Ideation.</p

New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape

The eukaryotic translation initiation factor 5A1 (eIF5A1) and 5A2 (eIF5A2) are important proteins in a variety of physiological and pathophysiological processes and their function has been linked to neurodevelopmental disorders, cancer, and virology. Here, we report two new genome-edited mouse models, generated using a CRISPR-Cas9 approach, in which the amino acid residue lysine 50 is replaced with arginine 50 (K50R) in eIF5A1 or in the closely related eIF5A2 protein. This mutation prevents the spermidine-dependent post-translational formation of hypusine, a unique lysine derivative that is necessary for activation of eIF5A1 and eIF5A2. Mouse brain lysates from homozygous eif5a2-K50R mutant mice (eif5a2K50R/K50R) confirmed the absence of hypusine formation of eIF5A2, and metabolomic analysis of primary mouse dermal fibroblasts revealed significant alterations in the metabolite landscape compared to controls including increased levels of tryptophan, kyrunenine, pyridoxine, NAD, riboflavin, FAD, pantothenate, and CoA. Further supported by new publicly available bioinformatics data, these new mouse models represent excellent in vivo models to study hypusine-dependent biological processes, hypusination-related disorders caused by eIF5A1 and eIF5A2 gene aberrations or mRNA expression dysregulation, as well as several major human cancer types and potential therapies

Genomic, transcriptomic, and protein landscape profile of CFTR and cystic fibrosis

Cystic Fibrosis (CF) is caused most often by removal of amino acid 508 (Phe508del, deltaF508) within CFTR, yet dozens of additional CFTR variants are known to give rise to CF and many variants in the genome are known to contribute to CF pathology. To address CFTR coding variants, we developed a sequence-to-structure-to-dynamic matrix for all amino acids of CFTR using 233 vertebrate species, CFTR structure within a lipid membrane, and 20 ns of molecular dynamic simulation to assess known variants from the CFTR1, CFTR2, ClinVar, TOPmed, gnomAD, and COSMIC databases. Surprisingly, we identify 18 variants of uncertain significance within CFTR from diverse populations that are heritable and a likely cause of CF that have been understudied due to nonexistence in Caucasian populations. In addition, 15 sites within the genome are known to modulate CF pathology, where we have identified one genome region (chr11:34754985-34836401) that contributes to CF through modulation of expression of a noncoding RNA in epithelial cells. These 15 sites are just the beginning of understanding comodifiers of CF, where utilization of eQTLs suggests many additional genomics of CFTR expressing cells that can be influenced by genomic background of CFTR variants. This work highlights that many additional insights of CF genetics are needed, particularly as pharmaceutical interventions increase in the coming years