Hepatocellular carcinoma (HCC) is the second most deadly cancer worldwide, which can result from the progression of fatty liver disease (NAFLD) and steatohepatitis (NASH) to tumorigenesis. NAFLD is an increasing problem in the Western world with the rapid increase in obesity. It is the most commonly diagnosed condition in patients with liver disorders, and is present in 20-30% of adults in the United States. NASH is a more severe form of NAFLD characterized by inflammation and fibrosis, which can advance to cirrhosis and HCC. Our most recent study to elucidate the molecular mechanisms of HCC showed that two molecules, Shp2 and Pten, have a synergistic effect to prevent hepatocarcinogenesis. The deletion of Shp2 and Pten led to severely fatty livers and early-onset NASH and tumors. As mitochondria are the major site for fatty acid metabolism and source of ROS, we looked further to address how mitochondrial function is disrupted in this NASH model and whether mitochondrial dysfunction is likely contributing to the progression of NASH to HCC. We quantified the mitochondria and examined mitochondrial functions, including structure, integrity, motility, and gene expression. We show that mitochondrial structure and integrity were compromised in Pten knockout and Shp2/Pten double-knockout mice. Mitochondrial numbers were dramatically decreased in double-knockout mice. We also found that mitochondrial metabolic pathways are dysregulated in Pten knockout mice. These results demonstrate that dysfunctional mitochondria may contribute to the progression of NASH to HCC, and may be a good therapeutic target.

Hepatocellular carcinoma (HCC) is quickly rising to become one of the deadliest cancers worldwide. Simultaneously inhibiting the Ras and NF-κB pathways in hepatocytes surprisingly causes liver tumorigenesis and circadian clock disruption in hepatocytes. However, it is unknown if there is clock crosstalk between hepatocytes and specific non-parenchymal cell populations. To study clock disruption in this HCC mouse model, I used single-cell RNA sequencing to characterize cell populations and study clock gene expression. My analysis revealed that compared to the wild type, the DKO model had a lower proportion of B cells, but a higher proportion of macrophages and neutrophils. I also found that circadian clock gene expression in non-parenchymal cells differ between cell populations. There appears to be higher expression of Dbp, Per1, and Per2 in stellate cells and cholangiocytes, Dbp in endothelial cells, Cry1 and Per1 in neutrophils, and Per1 in macrophages. To a lesser extent, clock gene expression in non-parenchymal cells also differs between wild type and DKO. Cell-cell interaction analysis revealed that in communication from hepatocytes to NPC populations, there were the addition of the Lgals9 - Ptprc and Lgals9 - Cd44 interactions in the DKO model. In communication from NPCs to hepatocytes, the DKO lacked the H2-K1 - Cd8a/Cd8b1 interaction, but had enhanced FN1 - Sdc4 interactions. These results suggest that hepatocytes may be communicating to specific NPC populations differently regarding the circadian clock, and further studies may help us uncover new mechanisms of clock crosstalk between hepatic cell types and their relation to liver tumorigenesis.

Liver is the major metabolic organ. We analyzed single cell RNA-sequencing (scRNA-seq) data collected from mouse liver to study its postnatal development and tumorigenesis. The postnatal development and maturation of liver are inadequately understood. We analyzed 52,834 single cell transcriptomes collected at postnatal day 1, 3, 7, 21 and 56. We observed unexpectedly high levels of hepatocyte heterogeneity in the developing liver and progressive construction of the zonated metabolic functions from pericentral to periportal hepatocytes, which was orchestrated with development of sinusoid endothelial, stellate and Kupffer cells. Trajectory and gene regulatory analyses captured 36 transcription factors, including a circadian regulator Bhlhe40, in programming liver development. Remarkably, we identified a special group of macrophages enriched at day 7 with a hybrid phenotype of macrophages and endothelial cells, which may regulate sinusoidal construction and regulatory T (Treg) cell function. For tumorigenesis, we show that Myc-driven hepatocellular carcinoma (HCC) was dramatically aggravated in mice with hepatocyte-specific Ptpn11/Shp2 deletion. However, Myc-induced tumors developed selectively from the rare Shp2-positive hepatocytes in Shp2-deficent liver, and Myc-driven oncogenesis depended on an intact Ras-Erk signaling ensued by Shp2 to sustain Myc stability. Despite a stringent requirement of Shp2 cell-autonomously, Shp2 deletion induced an immune-suppressive environment, resulting in defective clearance of tumor-initiating cells and aggressive tumor progression. The basal Wnt/beta-catenin signaling was upregulated in Shp2-deficient liver, which was further augmented by Myc transfection. Ablating Ctnnb1 suppressed Myc-induced HCC in Shp2-deficient livers, revealing an essential role of beta-catenin. Consistently, Myc overexpression and CTNNB1 mutations were frequently co-detected in HCC patients with poor prognosis. These data elucidate complex mechanisms of liver tumorigenesis driven by cell-intrinsic oncogenic signaling in cooperation with environmental tumor-promoting factors generated by disrupting the specific oncogenic pathway.

Shp2 is an SH2-tyrosine phosphatase acting downstream of receptor tyrosine kinases (RTKs) as a positive regulator of signal transduction. Despite its proto-oncogenic role, recent data demonstrated a liver tumor-suppressing role for Shp2, as ablating Shp2 in hepatocytes aggravated hepatocellular carcinoma (HCC) induced by chemical carcinogen. I further investigated the possible multi-faceted roles of Shp2 by examining the effect of hepatocyte specific ablation of Shp2 on oncogene-induced autochthonous liver tumor formation. Despite the induction of hepatic oxidative and metabolic stresses, Shp2 deletion in hepatocytes suppressed hepatocarcinogenesis driven by overexpression of oncoproteins Met/β-catenin or Met/PI3K-p110α. Mechanistically, Shp2 loss inhibited proliferative signaling from oncogenic pathways and triggered cell senescence following exogenous expression of the oncogenes. Further, I demonstrated that the catalytic activity of Shp2 was essential for relay of oncogenic signals from RTK in HCC and that chemical inhibition of Shp2 robustly suppressed HCC driven by RTK. However, in contrast to a tumor-promoting hepatic niche generated by genetic deletion of Shp2 in hepatocytes, pharmacological Shp2 inhibition had a tumor-suppressing effect on liver metastasized tumor progression. Mechanistically, the Shp2 inhibitor enhanced an innate anti-tumor immunity by downregulating inflammatory cytokines, suppressing the CCR5 signaling axis and upregulating interferon-β secretion. Collectively, this dissertation study dissected multi-faceted roles of Shp2 in hepatocarcinogenesis, as well as provided preclinical evidence of anti-tumor activity of Shp2 pharmacological inhibition through both cell-autonomous and nonautonomous mechanisms.

Despite ever-rising mortality due to hepatocellular carcinoma (HCC), efforts to identify effective chemotherapeutics have languished. A major source of this difficulty may be the underappreciated complexity of oncogenic signaling mechanisms that drive liver tumorigenesis. The Ras/ERK and NF-κB pathways have received extensive attention in the cell signaling and cancer research fields in the past few decades. These pathways play critical roles in cell survival and proliferation, and have been demonstrated to drive oncogenesis when excessively or constitutively activated. However, studies in animal models have revealed tumor-suppressive functions in the liver, as deleting Shp2 or Ikkβ in hepatocytes, which promote Ras/ERK and NF-κB signaling, respectively, ironically aggravated HCC development induced by the chemical carcinogen diethylnitrosamine (DEN). Since parallel pathways might work antagonistically or cooperatively, our lab has been interested in taking a genetic approach to generate compound mutant mouse lines, which often yield unanticipated results and provide a fresh view on pathway cross-talk. The goal of my dissertation work was to dissect molecular and cellular mechanisms of hepatocarcinogenesis by creating a mutant mouse model with both Shp2 and Ikkβ deleted in hepatocytes. This allowed us to examine the functional interaction of these pathways in stressed and unstressed livers. My experimental results showed that dual Shp2 and Ikkβ deletion (DKO) dramatically accelerated DEN-induced tumorigenesis in the liver compared to either single knockout, evidently due to more extensive liver damages and metabolic disorders. More surprisingly, this dual deletion resulted in spontaneous development of HCC. Although multiple hepatic factors contributed to the pathogenic process, Shp2- and Ikkβ- deficient hepatocytes were characterized by disrupted expression of circadian clock genes. Circadian disruption has been linked to liver tumorigenesis in humans and in mice, suggesting a mechanism underlying spontaneous hepatocarcinogenesis in the DKO livers. In support of this mechanism, human HCCs with dysregulated circadian gene expression displayed downregulation of Ras/ERK and NF-κB signaling and poor prognosis. These data indicate that the ground state of the two central signaling pathways, previously known to mediate proliferative and oncogenic signaling, sustains tumor suppressive circadian homeostasis in the mammalian liver. Disruption of this signaling network results in spontaneous hepatocarcinogenesis.

Age-related macular degeneration (AMD) is a major cause of central vision loss among the elderly. Genome-wide association studies (GWAS) have implicated polymorphisms in complement factor H (CFH) and high-temperature requirement A1 (HTRA1) in progression to advanced AMD. The complement factor H plays a key regulator role in complement system of the innate immune defense. CFH is also found to have high affinity binding with oxidized phospholipids (oxPLs), which shields oxPLs from inducing inflammation in the eye. On the other hand, the HTRA1 is a serine protease that is identified to be an extracellular matrix homeostasis regulator and TGF-β1 pathway inhibitor. Despite the extensive studies on the two proteins’ respective properties and involved pathways, the mechanism between the genetic risk factors and AMD disease pathology remains to be explored. My research project investigates the cellular interactions and molecular mechanisms of CFH and HTRA1 in elevated oxidative stress environment. In this study, recombinant CFH and HTRA1 variants were produced for the experiments. The inhibitory effect of CFH and HTRA1 in modulating the uptake of oxidized low-density lipoprotein (oxLDL) into the retinal pigment epithelium cells (RPE) is demonstrated by Oil Red O staining. The CFH risk variant was identified to exhibit competitive binding against HTRA1 interaction with lipoproteins via competitive ELISA. Furthermore, CD36 mRNA expression is found to be significantly heightened with the administration of HTRA1 or oxidative stress. However, the CD36 protein level analysis via immunocytochemistry and western blot demonstrates reduction of CD36 protein expression by proteolytic active HTRA1. We also found that CD36 protein expression is either preserved or enhanced with the addition of CFH variants, which we speculate to protect CD36 against proteolytic cleavage by HTRA1. This preliminary evidence suggests that CFH and HTRA1 have an antagonistic effect on inhibiting oxidized phospholipids uptake in RPE cells. This finding provides a new aspect of complement independent role of CFH in lipid metabolism which may be explored as a therapeutic treatment to AMD.

Hepatocellular Carcinoma is a complex cancer with little available targeted therapeutics, in part due to the recently uncovered paradoxical roles of previously defined proto-oncogenes. Our lab and others have identified overexpression and ablation of several proto-oncogenes sensitizes the liver towards HCC development. Previously, our lab demonstrated hepatocyte deletion of Shp2/Ptpn11 in mice (SKO) ablates HCC driven by cMET/Δ90-β-catenin or cMET/PIK3CA mutant yet greatly accelerates chemical carcinogen-induced HCC. To clarify the complex mechanisms of Shp2 function in HCC, we examined the contribution of the proto-oncogene Myc, which acts downstream of Shp2. The aim of my dissertation research is to elucidate the novel murine model of Myc-driven tumorigenesis in SKO background, examining cell-intrinsicand extrinsic factors driving tumor development. Original work in this dissertation shows that Myc-driven hepatocellular carcinoma (HCC) is dramatically aggravated in mice with hepatocyte-specific Ptpn11/Shp2 deletion. Using single-cell RNA-sequencing coupled with functional assays, we show Shp2-knockout livers exhibit defective clearance of tumor-initiating cells by altering macrophage polarization and reducing macrophage phagocytic activity, resulting in an immune-suppressive environment. We uncover that Myc-induced tumors selectively develop from the rare Shp2-positive hepatocytes in Shp2-deficent liver. Myc-driven oncogenesis depends on intact Ras-Erk signaling, driven by Shp2, to sustain Myc stability. Basal Wnt/β-catenin signaling is upregulated in Shp2-deficient liver, which is further augmented by Myc transfection. Ablating Ctnnb1 suppresses Myc-induced HCC in Shp2-deficient livers, revealing an essential role of β- catenin in assisting in Myc-driven HCC. Consistently, Myc overexpression and CTNNB1 mutations are frequently co-detected in HCC patients with poor prognosis. Single cell RNA sequencing and functional in vivo / in vitro data together reveal the complex mechanisms of liver tumorigenesis driven by cell-intrinsic oncogenic signaling of Myc, supported by intrinsic Shp2 and β-catenin signaling, in cooperation with a tumor promoting microenvironment generated by disruption of Shp2 proto-oncogenic signaling.