RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo.

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet ß cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

NIAM-deficient mice are predisposed to the development of proliferative lesions including B-cell lymphomas.

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAM(m/m)) mice homozygous for a ß-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM(+/m) heterozygous animals developed lesions. In the spleen, NIAM(m/m) mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

RABL6A promotes G1-S phase progression and pancreatic neuroendocrine tumor cell proliferation in an Rb1-dependent manner.

Mechanisms of neuroendocrine tumor (NET) proliferation are poorly understood, and therapies that effectively control NET progression and metastatic disease are limited. We found amplification of a putative oncogene, RABL6A, in primary human pancreatic NETs (PNET) that correlated with high-level RABL6A protein expression. Consistent with those results, stable silencing of RABL6A in cultured BON-1 PNET cells revealed that it is essential for their proliferation and survival. Cells lacking RABL6A predominantly arrested in G1 phase with a moderate mitotic block. Pathway analysis of microarray data suggested activation of the p53 and retinoblastoma (Rb1) tumor-suppressor pathways in the arrested cells. Loss of p53 had no effect on the RABL6A knockdown phenotype, indicating that RABL6A functions independent of p53 in this setting. By comparison, Rb1 inactivation partially restored G1 to S phase progression in RABL6A-knockdown cells, although it was insufficient to override the mitotic arrest and cell death caused by RABL6A loss. Thus, RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET proliferation and development. This work identifies RABL6A as a novel negative regulator of Rb1 that is essential for PNET proliferation and survival. We suggest RABL6A is a new potential biomarker and target for anticancer therapy in PNET patients.

RABL6A, a novel RAB-like protein, controls centrosome amplification and chromosome instability in primary fibroblasts.

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53-/- MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.

RABL6A Promotes Oxaliplatin Resistance in Tumor Cells and Is a New Marker of Survival for Resected Pancreatic Ductal Adenocarcinoma Patients.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early recurrence following pancreatectomy, rapid progression, and chemoresistance. Novel prognostic and predictive biomarkers are urgently needed to both stratify patients for clinical trials and select patients for adjuvant therapy regimens. This study sought to determine the biological significance of RABL6A (RAB, member RAS oncogene family-like protein 6 isoform A), a novel pancreatic protein, in PDAC. Analyses of RABL6A protein expression in PDAC specimens from 73 patients who underwent pancreatic resection showed that RABL6A levels are altered in 74% of tumors relative to adjacent benign ductal epithelium. Undetectable RABL6A expression, found in 7% (5/73) of patients, correlated with improved overall survival (range 41 to 118 months with 3/5 patients still living), while patients with RABL6A expression had a worse outcome (range 3.3 to 100 months, median survival 20.3 months) (P = 0.0134). In agreement with those findings, RABL6A expression was increased in pancreatic cancer cell lines compared to normal pancreatic epithelial cells, and its knockdown inhibited pancreatic cancer cell proliferation and induced apoptosis. Moreover, RABL6A depletion selectively sensitized cells to oxaliplatin-induced arrest and death. This work reveals that RABL6A promotes the proliferation, survival, and oxaliplatin resistance of PDAC cells, whereas its loss is associated with extended survival in patients with resected PDAC. Such data suggest RABL6A is a novel biomarker of PDAC and potential target for anticancer therapy.

Thomsen or Becker myotonia? A novel autosomal recessive nonsense mutation in the CLCN1 gene associated with a mild phenotype.

We describe a large Brazilian consanguineous kindred with 3 clinically affected patients with a Thomsen myotonia phenotype. They carry a novel homozygous nonsense mutation in the CLCN1 gene (K248X). None of the 6 heterozygote carriers show any sign of myotonia on clinical evaluation or electromyography. These findings confirm the autosomal recessive inheritance of the novel mutation in this family, as well as the occurrence of phenotypic variability in the autosomal recessive forms of myotonia.

Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.

Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of dysferlin in the heart remains to be determined. Here, we demonstrate that dysferlin is also involved in cardiomyocyte membrane repair and that dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in dysferlin-null mice and increases Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and dysferlin leads to early onset cardiomyopathy. Our results suggest that dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of dysferlin.

Screening for mutations in the RYR1 gene in families with malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic predisposition associated with a susceptibility to volatile anesthetics and depolarizing muscle relaxants that lead to a fulminant anesthetic crisis with hyperthermia, skeletal muscle rigidity, respiratory and metabolic acidosis, and muscle rhabdomyolysis. Malignant hyperthermia crises are caused by an abnormal regulation of the calcium release mechanism, which reflects the consequences of disturbed skeletal muscle calcium homeostasis. We screened 64 individuals of 27 unrelated families for the most frequently described mutations associated with MH in the genes RYR1 and CACNL1A3. We identified only one family with the Arg614Cys mutation but with a discordant segregating pattern to the in vitro contracture test (IVCT). To elucidate which other mechanism could lead to susceptibility in the members of this family, we tested it for further MH susceptibility loci. The same haplotype was shown to segregate with the individuals carrying the Arg614Cys mutation in chromosome 19; however, the other susceptible and equivocal individuals do not share this haplotype. Markers for the susceptible locus in chromosome regions 17q, 7q, 3q, and 5p did not segregate with the IVCT phenotype in the susceptible individuals, suggesting that the positivity of the IVCT could be attributable to other ambient factors.