My research focuses on the study of controlling cancer growth, particularly hepatocellular carcinoma (HCC), by silencing over-expressed apoptosis inhibitors SPIK (serine protease inhibitor Kazal) in cancer cells. HCC is one of the most common causes of cancer death in the world. Though many factors may contribute to the pathogenesis of HCC, approximately 80% of HCC incidences are associated with chronic viral hepatitis such as HBV or HCV infection. The molecular mechanism linking HBV/HCV to HCC is still unclear, but the chronic inflammation associated with viral hepatitis is assumed to play an important role in most cases. Recent studies suggest that in chronic viral hepatitis, the body’s immune system cannot clear the infected cells. Most likely, this failure stems from an inability to induce apoptosis in these infected cells. For example, persistent HBV/HCV infection suppresses CTL and NK cell-induced apoptosis. The non-cleared, infected cells can then gradually accumulate genetic changes and  subsequently become transformed into HCC. This failure of the immune-mediated removal of malignant cells through apoptosis may be due to the up-regulation of apoptosis inhibitors in these cells. Our recent findings support this concept by showing that HBV/HCV infection can up-regulate expression of an apoptosis inhibitor SPIK, resulting in resistance of the infected cell to SPDCA. SPIK, also known as TATI (tumor associated trypsin inhibitor) and PSTI (pancreas secretory trypsin inhibitor), is a small protein. It was first discovered in the pancreas as an inhibitor of autoactivation of trypsinogen. The expression of SPIK in normal tissues is limited or inactivated outside of the pancreas, however, expression of SPIK is elevated in numerous cancers such as colorectal tumors, renal-cell carcinoma, gastric carcinoma, intrahepatic cholangiocarcinoma (ICC), and HCC. The role of SPIK in cancer formation and development is still unknown, but it is known that SPIK can be activated as a reactant during hepatitis or liver inflammation. For example, SPIK was activated in rat liver cells to counter turpentine-induced liver inflammation. SPIK is also activated during human viral hepatitis in response to inflammatory cytokines. Over-expression of SPIK was found in HBV/HCV infected human livers, and even higher expression of SPIK was found in HBV/HCV associated HCC tissue. Interestingly, the level of SPIK in patients has been correlated with the progression of HCC. Moreover, the high expression of SPIK is closely related with early recurrence of HCC in patients following surgical resection. Because recurrence of cancer often implies an inability to clear lingering oncogenetic cells by the immune-system, early recurrence of HCC in patients with high levels of SPIK raises the possibility that over-expression of SPIK might interfere with the immune-elimination of lingering oncogenetic cells. This hypothesis is supported by the fact that SPIK can bind GzmA and inhibit GzmA induced cellular apoptosis (our preliminary evidences). GzmA is a cytotoxic serine protease secreted by activated CTL and NK cells to kill target cells during immune-clearance. GzmA has also been shown to be especially important in the challenge of viral infection. For example, GzmA deficient mice showed a compromised ability to maintain the mousepox virus ectromelia and herpes simplex neuronal infections even though GzmB and perforin were competently expressed. Because GzmA-induced apoptosis is an important part of CTL and NK cell mediated immune-surveillance, it is likely that suppression or blockage of GzmA-induced apoptosis would result in the escape of infected or malignant cells from immune-clearance.
The second field in which I am interested is hepatitis B virus (HBV), a major human pathogen responsible for acute and chronic liver disease. It is estimated that more than 350 million people are chronically infected with HBV worldwide and one third of these individuals will progress to serious liver diseases such as liver cirrhosis, chronic liver failure, and hepatocellular carcinoma (HCC) that result in an estimated one million deaths annually. Therefore, there is a clear need for understanding HBV infection and introduction of safe and effective therapies for chronic hepatitis B virus. A major limitation to thoroughly studying HBV and exploration of new anti-HBV drugs has been the lack of suitable cell-lines that are susceptible to HBV infection. Human hepatocyte primary cultures, while susceptible to HBV infection, are viable only for a very limited amount of time following the explants. Alternatively, hepatoma cell lines such as HepG2, which are capable of supporting viral replication and producing progeny viruses, generally do not allow the infection of serum derived HBV to happen. Therefore, our first effort is to establish a cell line that is permissible to HBV infection.
We have previously reported that, limited digestion of human and woodchuck hepatitis viruses with the Staphylococcal protease V8 rendered the viruses “infectious” in cultured HepG2 cells. The infected HepG2 cells were shown to produce progeny viruses. The reason that V8 protease treatment induces viral infectivity is not clear. Our studies suggest that the proteolysis of V8 has exposed a region that contains a hydrophobic motif associated with membrane fusion that probably results in virus-cell membrane fusion, permitting viral genome entry into the cytosol, and then triggering HBV infection. The fusogenesis of this motif was supported by the fact that synthetic peptides corresponding to this motif induced fusion of bio-membranes, and of HepG2 cells from without in vitro (our unpublished observations). Other evidence supporting this assumption comes from the fact that a similar motif with the identical sequence has been found in the other viral envelope polypeptides associated with fusion, including Human Immunodeficiency Virus (HIV) and influenza virus. Accordingly, the resistance of HepG2 cells to HBV infection is a possible result of the lack of a proteolysis that induces virus-cell fusion. The observation that supports this hypothesis is the finding of the over-expressing of a kind of protease inhibitor named serine protease inhibitor (SPI) in HepG2 cells. The probable mechanism is that the SPI, by inactivating vital proteases in the virus-cell fusion process, prevents infecting and gives immunity to the HepG2 cells. Further support for this hypothesis is provided when an anti-sense oligo was used to down regulate the SPIK gene expression led to a potential HBV infection of HepG2 cells in vitro. Logically following this, a permanent cell line that can be infected by HBV could be established by silencing the over-expressed SPI in the HepG2 cell line. These studies are currently ongoing.
Selected Research Publications:
"Hepatitis B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal, Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis"
Lamontagne J, Pinkerton M, Block, TM, and X Lu
Journal of Virology, 84: 907-917, 2010.
"Tumor-associated protein SPIK/TATI suppresses serine protease dependent cell apoptosis"
Lu X, Lamontagne J, Lu F, and TM Block
Apoptosis, 13:483-94, 2008.
"Novel autoregulatory function of hepatitis B virus M protein on surface gene expression"
Huang T-J, Lu C-C, Tsai J-C, Yao W-J, Lu X, Lai M-D, Liu H-S, and A-L Shiau
Journal of Biological Chemistry, 280: 27742-27754, 2005.
"A phage with high affinity for hepatitis B surface antigen for detection of HBsAg"
Lu X, Weiss P, and TM Block
Journal of Virological Methods, 119(1): 51-54, 2004.
"Study of the early steps of the Hepatitis B Virus life cycle"
Lu X, and TM Block
International Journal of Medical Sciences 1(1): 21-33, 2004.
"The alkylated imino sugar, n- (n-nonyl)-deoxygalactonojirimycin, reduces the amount of hepatitis B virus (HBV) nucleocapsid in tissue culture"
Lu X, Tran T, Semick E, and TM Block
Journal of Virology, 77(22): 11933-11940, 2003.
"Limited proteolysis induces woodchuck hepatitis virus infectivity for human HepG2 cells"
Lu X, Hazboun T, and TM Block
Virus Research, 73(1): 27-40, 2001.
"The hepatitis B virus MHBs antigen is selectively sensitive to glucosidase mediated processing in the endoplasmic reticulum"
Lu X, Lu Y, Geschwindt R, Dwek RA, and TM Block
DNA and Cell Biology, 20(10): 647-656, 2001.
"Treatment of Hepatitis virus in chronically infected woodchucks with folding and trafficking inhibitor"
Block, TM, Lu X, Mehta A, Blumburg BS, Tennant B, Ebling M, Korba B, Lansky D, Jacob GS, and R Dwek
Nature Medicine, 4(5): 610-614, 1998.
"Aberrant trafficking of hepatitis b virus glycoproteins in which N-glycan processing is inhibited"
Lu X, Metha A, Dwek DM, Blumberg BS, and TM Block
PNAS USA, 94: 2380-2385, 1997.
"Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion"
Mehta A, Lu X, Block TM, Blumberg BS, and R Dwek
PNAS USA, 94 1822-1827, 1997.
"Protease-induced infectivity of hepatitis B virus for a human hepatoma cell line"
Lu X, Block TM, and WH Gerlich
Journal of Virology, 70: 2277-2285, 1996.
"Evidence the N-linked glycosylation is necessary for hepatitis B virus secretion"
Lu X, Metha A, Dwek R, Butters T, and TM Block
Virology, 213: 660-665, 1995.
"The secretion of human hepatitis B virus is inhibited by the imino sugar, N-butyldeoxynojirimycin"
Block TM, Lu X, Platt FM, Foster GR, Gerlich WH, Blumberg BS, and RA Dwek
PNAS USA, 91: 2235-2239, 1994.
"Post-translational alterations in transmenbrance topology of the hepatitis B virus large envelope protein"
Bruss V, Lu X, Thomssen R, and WH Gerlich
EMBO Journal, 13: 2273-2279, 1994.
|