Ming-Hai Wang

Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas : generation of different splicing RON variants and their oncogenic potential

The RON receptor tyrosine kinase is a member of the MET proto-oncogene family that has been implicated in regulating motile-invasive phenotypes in certain types of epithelial cancers. The purpose of this study was to determine if RON expression is altered in primary human colorectal adenocarcinomas. Results from immunohistochemical staining showed that RON is highly expressed in the majority of colorectal adenocarcinomas (29/49 cases). Accumulated RON is also constitutively active with autophosphorylation in tyrosine residues. Moreover, three splicing variants of RON, namely RONΔ165, RONΔ160, and RONΔ155 were detected and cloned from two primary colon cancer samples. These RON variants were generated by deletions in different regions in extracellular domains of the RON β chain. Functional studies showed that expression of RONΔ160 or RONΔ155 in Martin–Darby canine kidney cells resulted in increased cell dissociation (scatter-like activity). RON variants, RONΔ160 and RONΔ155, also exerted the ability to induce multiple focus formation and sustain anchorage-independent growth of transfected NIH3T3 cells. Moreover, NIH3T3 cells expressing RONΔ160 or RONΔ155 formed tumors in athymic nude mice and colonized in the lungs. These data suggest that RON expression is altered in certain primary colon cancers. Abnormal accumulation of RON variants may play a role in the progression of certain colorectal cancers in vivo.

Identification of a novel splicing product of the RON receptor tyrosine kinase in human colorectal carcinoma cells

The RON receptor tyrosine kinase is a 180 kDa heterodimeric protein composed of a 40 kDa alpha chain and a 145 kDa beta chain with intrinsic tyrosine kinase activity. Activation of RON causes cell dissociation, motility and invasion of extracellular matrices, suggesting that RON might be involved in tumor metastasis. We report here the cloning of a novel splice variant of RON in human colorectal carcinoma cell line HT-29. This RON variant is first produced as a single chain precursor with a molecular mass of 160 kDa. Proteolytic cleavage results in a 40 kDa alpha chain and a short form of the beta chain with a molecular mass of 125 kDa. The altered receptor is synthesized from a transcript differing from the full-length RON mRNA by an in-frame deletion of 109 amino acids in the extracellular domain of the RON beta chain. The consequence of the deletion is constitutive activation of the protein with autophosphorylation. Expression of the RON variant in colon epithelial CoTr cells results in increased cell migration and invasion of extracellular matrices. These data suggest that generation of the activated splice variant of RON may contribute to the invasive phenotype of human colorectal carcinomas in vivo.

Targeted expression of the receptor tyrosine kinase RON in distal lung epithelial cells results in multiple tumor formation: oncogenic potential of RON in vivo

RON, a member of the MET proto-oncogene family, has been implicated in the progression of certain epithelial cancers. The purpose of this study was to determine the oncogenic potential of RON in vivo in lung epithelial cells. Transgenic mice were established using surfactant protein C promoter to express human RON in the distal lung epithelial cells. These mice were born normal but developed multiple lung tumors with distinct morphology and growth patterns. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules located mostly in the peripheral portions of the lung. A transition from early adenomas to later adenocarcinomas was observed. Morphologically, tumors were characterized as cuboidal epithelial cells with a type II cell phenotype, grew along the alveolar walls, and projected into the alveolar septa. RON was highly expressed and constitutively activated in tumors. These results indicate that overexpression of human wild-type RON causes the formation of lung tumors with unique biological characteristics in vivo. This model provides opportunities to study the role of RON in the pathogenesis of lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.

Activation of the RON receptor tyrosine kinase protects murine macrophages from apoptotic death induced by bacterial lipopolysaccharide

RON is a receptor tyrosine kinase activated by macrophage‐stimulating protein. We demonstrate here that RON activation inhibits LPS‐induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS‐induced production of nitric oxide (NO), a molecule responsible for LPS‐induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO‐induced apoptotic death of Raw264.7 cells and inhibited LPS‐induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)‐3 kinase, prevents the inhibitory effect of RON on LPS‐induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI‐3 kinase p85 subunit. These data suggest that by activating PI‐3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.

Wnt/β-catenin activation and macrophage induction during liver cancer development following steatosis

Obesity confers an independent risk for carcinogenesis. In the liver, steatosis often proceeds cancer formation; however, the mechanisms by which steatosis promotes carcinogenesis is unknown. We hypothesize that steatosis alters the microenvironment to promote proliferation of tumor initiating cells (TICs) and carcinogenesis. We used several liver cancer models to address the mechanisms underlying the role of obesity in cancer and verified these findings in patient populations. Using bioinformatics analysis and verified by biochemical assays, we identified that hepatosteatosis resulting from either Pten deletion or transgenic expression of HCV core/NS5A proteins, promotes the activation of Wnt/β-catenin. We verified that high fat diet lipid accumulation is also capable of inducing Wnt/β-catenin. Caloric restriction inhibits hepatosteatosis, reduces Wnt/β-catenin activation and blocks the expansion of TICs leading to complete inhibition of tumorigenesis without affecting the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) loss regulated protein kinase B (AKT) activation. Pharmacological inhibition or loss of the Wnt/β-catenin signal represses TIC growth in vitro, and decreases the accumulation of TICs in vivo. In human liver cancers, ontology analysis of gene set enrichment analysis (GSEA)-defined Wnt signature genes indicates that Wnt signaling is significantly induced in tumor samples compared with healthy livers. Indeed, Wnt signature genes predict 90% of tumors in a cohort of 558 patient samples. Selective depletion of macrophages leads to reduction of Wnt and suppresses tumor development, suggesting infiltrating macrophages as a key source for steatosis-induced Wnt expression. These data established Wnt/β-catenin as a novel signal produced by infiltrating macrophages induced by steatosis that promotes growth of tumor progenitor cells, underlying the increased risk of liver tumor development in obese individuals.