Based on the results of our recent experiments, we hypothesize that one major function of Met protooncogene in the liver is to inhibit apoptosis in hepatocytes. We believe that one mechanism by which Met elicits an anti-apoptotic effect is via an interaction between Met and the death promoting cell surface receptor Fas, the net result of which is sequestration of Fas by Met and inhibition of the initiation of the Fas-mediated apoptotic pathway. Another novel mechanism by which Met may modulate apoptosis involves direct inhibition of the executioners of the apoptotic pathway, namely the effector caspases (casepase- 3 and caspase-7), by a substrate suicide mechanism employing the cytoplasmic c-terminal tail of the Met protein. These interactions squelch the apoptotic command and thus promote cell survival.
In Aim 1, we will determine the nature of the Met-Fas interaction and will map the structural domains in Met and Fas that are required for their association. We will address the biological relevance of Fas sequestration by Met and its contribution to hepatocyte survival, liver development, hepatic homeostasis and transformation using transgenic and knock out mouse models.
In Aim 2, we will test the hypothesis that Met is a key target of the destructive action of the effector caspases (such as caspase-3) during apoptosis. This hypothesis is based on our findings that Met is essential for cell survival and because a perfect caspase cleavage site is present in Met's tyrosine kinase activation domain. Importantly, germline and sporadic mutations in this tyrosine kinase activation domain (in the putative caspase cleavage site that we have identified) have been reported. We will investigate the hypothesis that these mutations make Met refractory to caspase cleavage thus causing cells to be resistant to apoptosis and contributing to malignant transformation.
In Aim 3, we propose to test the hypothesis that Met protein has evolved a novel mechanism to elude/inhibit the executioners of the apoptotic pathway, namely the effector caspases. We propose that caspase inhibition by Met is achieved through a substrate suicide mechanism involving the intracellular cytoplasmic end of the Met molecule. In this region, we have discovered the octapeptide sequence DNADDEVD. Since this peptide sequence harbors tandem perfect effector caspase cleavage sites (i.e. for caspase-3), we propose that during apoptosis this peptide sequence is cleaved by these caspases resulting in the formation of the tetrapeptide DEVD, a well-known potent inhibitor of caspase-3 and caspase-7.
In Aim 4, we will test the hypothesis that the met gene is induced in response to cellular stress to protect cells from apoptosis and is a target of upregulation of NF-kappaB, a transcription factor known to be essential for hepatocyte survival during embryonic development.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Chemical Pathology Study Section (CPA)
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Spalholz, Barbara A
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University of Pittsburgh
Schools of Medicine
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Seneviratne, Danushka; Ma, Jihong; Tan, Xinping et al. (2015) Genomic instability causes HGF gene activation in colon cancer cells, promoting their resistance to necroptosis. Gastroenterology 148:181-191.e17
Ma, Jihong; Zou, Chunbin; Guo, Lida et al. (2014) Novel Death Defying Domain in Met entraps the active site of caspase-3 and blocks apoptosis in hepatocytes. Hepatology 59:2010-21
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Wang, Xue; DeFrances, Marie C; Dai, Yu et al. (2002) A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 9:411-21