Urrutia, Raúl

Noonan syndrome (NS; MIM 163950) is an autosomal dominant disorder and a member of a family of developmental disorders termed “RASopathies,” which are caused mainly by gain-of-function mutations in genes encoding RAS/MAPK signaling pathway proteins. Whole exome sequencing (WES) and trio-based genomic triangulation of a 15-year-old female with a clinical diagnosis of NS and concomitant cardiac hypertrophy and her unaffected parents identified a de novo variant in MRAS -encoded RAS-related protein 3 as the cause of her disease. Mutation analysis using in silico mutation prediction tools and molecular dynamics simulations predicted the identified variant, p.Gly23Val-MRAS, to be damaging to normal protein function and adversely affect effector interaction regions and the GTP-binding site. Subsequent ectopic expression experiments revealed a 40-fold increase in MRAS activation for p.Gly23Val-MRAS compared with WT-MRAS. Additional biochemical assays demonstrated enhanced activation of both RAS/MAPK pathway signaling and downstream gene expression in cells expressing p.Gly23Val-MRAS. Mutational analysis of MRAS in a cohort of 109 unrelated patients with phenotype-positive/genotype-negative NS and cardiac hypertrophy yielded another patient with a sporadic de novo MRAS variant (p.Thr68Ile, c.203C>T). Herein, we describe the discovery of mutations in MRAS in patients with NS and cardiac hypertrophy, establishing MRAS as the newest NS with cardiac hypertrophy-susceptibility gene.

AbstractBackgroundKleefstra Syndrome (KS) (MIM# 610253) is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase‐1 (EHMT1, GLP). EHMT1 (MIM# 607001) encodes a histone methyltransferase that heterodimerizes with EHMT2 (also known as G9a, MIM# 604599), which together are responsible for mono‐ and dimethylation of H3 lysine 9 (H3K9me1 and ‐me2), resulting in transcriptional repression of target genes.MethodsThis report describes an 18‐year‐old woman with intellectual disability, severely limited speech, hypotonia, microcephaly, and facial dysmorphisms, who was found to have a novel de novo single‐base frameshift deletion in EHMT1.ResultsFunctional studies using patient fibroblasts showed decreased H3K9me2 compared to wild‐type control cells, thus providing a rapid confirmatory test that complements molecular studies.ConclusionWhole exome sequencing revealed a novel frameshift deletion in EHMT1 after a lengthy diagnostic odyssey in this patient. Functional testing using this patient's fibroblasts provides proof‐of‐concept for the analysis of variants of uncertain significance that are predicted to impact EHMT1 enzymatic activity.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular findings of aneurysm and/or dissection of cerebral, thoracic, or abdominal arteries and skeletal findings. We report a case of a novel pathogenic variant in TGFBR2 and phenotype consistent with classic LDS. The proband was a 10-year-old presenting to the genetics clinic with an enlarged aortic root (Z-scores 5-6), pectus excavatum, and congenital contractures of the right 2nd and 3rd digit. Molecular testing of TGFBR2 was sent to a commercial laboratory and demonstrated a novel, likely pathogenic, variant in exon 4, c.1061T>C, p.(L354P). Molecular modeling reveals alteration of local protein structure as a result of this pathogenic variant. This pathogenic variant has not been previously reported in LDS and thus expands the pathogenic variant spectrum of this condition.

Key Clinical MessageA de novo GFAP variant, p.R376W, was identified in a child presenting with hypotonia, developmental delay, and abnormal brain MRI. Following the 2015 ACMG variant classification guidelines and the functional studies showing protein aggregate formation in vitro, p.R376W should be classified as a pathogenic variant, causative for Alexander disease.

Abstract Introduction: Liver metastasis of pancreatic or colorectal tumors is a major cause of cancer-related death worldwide. When cancer cells reach the liver parenchyma and make a direct contact with the surrounding extracellular matrix (ECM), β1-integrin-FAK signaling is activated allowing the cells attach, migration, and proliferate. Thus, the β1-integrin-FAK signaling axis is key for cancer cells to colonize the liver. However, mechanisms governing β1-integrin activation and how this pathway promotes liver metastasis remain incompletely understood. Aim: We sought to test the hypothesis that vasodilator-stimulated phosphoprotein (VASP), an actin binding protein, regulates the β1-integrin-FAK signaling axis, thereby, promoting liver metastasis of GI cancer. Methods: We employed lentiviruses encoding different VASP shRNAs to knockdown (KD) VASP in human GI cancer cells. We used a Matrigel-based 3-dimensional (3D) cell culture model and intrasplenic tumor injection mouse model to assess the role of VASP for cancer cells in vitro and in vivo. Immunofluorescence (IF) was used to study localization of VASP and active β1-integrin (using Huts-4 antibody), and Western blot analysis (WB) was used to quantitate protein levels of active β1-integrin and YAP1/TAZ. Additionally, we performed VASP immunohistochemistry (IHC) to analyze VASP immunoreactivity in 116 colorectal or pancreatic adenocarcinomas of patients. Results: In the Matrigel-based 3D-culture model, a single cancer cell gave arise to a 3D-cancer spheroid after being cultured for 6-8 days. Using KM12L4, HCT116, or HT29 colorectal cancer cells, and L3.6 or MIA PaCa-1 pancreatic cancer cells, we found that VASP KD reduced the sizes of the 3D-cancer spheroids grown on Matrigel (P<0.05 by t-test for each cell line). IF and WB show that VASP KD reduced levels of Huts-4 (active β1-integrin) in KM12L4, HCT116 colorectal cancer cells and in L3.6, MIA PaCa-1 pancreatic cancer cells. VASP KD also reduced the protein levels of P-FAK and YAP1/TAZ in the 4 cell lines tested. Additionally, YAP1/TAZ protein levels were reduced by PF-228, a FAK inhibitor that blocks the β1-integrin-FAK signaling. In the intrasplenic tumor implanation mouse model, VASP KD suppressed liver metastasis of GI cancer as well as reduced Huts-4 and YAP1 signals of their liver metastases in mice (P<0.05 by t-test). IHC of patient's samples reveals that VASP protein levels were upregulated in cancer cells in 81% of colorectal tumors (51/63) and pancreatic tumors (43/53), with higher VASP levels being associated with liver metastasis of colorectal cancer (P<0.01 by Fisher exact test). Conclusions: VASP promotes ECM-mediated β1-integrin-FAK-YAP1/TAZ signaling axis of GI cancer. We propose that VASP and the β1-integrin-FAK-YAP1/TAZ signaling axis represent potential new therapeutic targets for liver metastasis of pancreatic or colorectal cancer. Citation Format: Xiaoyu Xiang, Jinhua Piao, Selvaraj Muthusamy, Lei Wang, Yibin Deng, Shengbing Huang, Raul Urrutia, Jinping Lai, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes β1-integrin-FAK-YAP1/TAZ signaling axis that is required for liver metastasis of GI cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1633.

Krüppel-like factor (KLF)-10 is an important transcriptional regulator of TGF-β1signaling in both CD8+and CD4+T lymphocytes. In the present study, we demonstrate a novel role for KLF10 in the regulation of TGFβRII expression with functional relevance in macrophage differentiation and activation. We first show that transfer of KLF10−/−bone marrow-derived macrophages into wild-type (WT) mice leads to exacerbation of experimental colitis. At the cell biological level, using two phenotypic strategies, we show that KLF10-deficient mice have an altered colonic macrophage phenotype with higher frequency of proinflammatory LyC6+MHCII+cells and a reciprocal decrease of the anti-inflammatory LyC6−MHCII+subset. Additionally, the anti-inflammatory CD11b+CX3CR1hisubset of colonic macrophages is significantly decreased in KLF10−/−compared with WT mice under inflammatory conditions. Molecularly, CD11b+colonic macrophages from KLF10−/−mice exhibit a proinflammatory cytokine profile with increased production of TNF-α and lower production of IL-10 in response to LPS stimulation. Because KLF10 is a transcription factor, we explored how this protein may regulate macrophage function. Consequently, we analyzed the expression of TGFβRII expression in colonic macrophages and found that, in the absence of KLF10, macrophages express lower levels of TGFβRII and display an attenuated Smad-2 phosphorylation following TGF-β1stimulation. We further show that KLF10 directly binds to the TGFβRII promoter in macrophages, leading to enhanced gene expression through histone H3 acetylation. Collectively, our data reveal a critical role for KLF10 in the epigenetic regulation of TGFβRII expression in macrophages and the acquisition of a “regulatory” phenotype that contributes to intestinal mucosal homeostasis.

Abstract Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering “druggable” molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL17 functions during this transition are currently unclear. In this study, we demonstrate that IL17 induces the expression of REG3β, a well-known mediator of pancreatitis, during acinar-to-ductal metaplasia and in early pancreatic intraepithelial neoplasia (PanIN) lesions. Furthermore, we found that REG3β promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3β in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3β. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiologic role for REG3β during pancreatitis and PDAC initiation. Cancer Res; 75(22); 4852–62. ©2015 AACR.