William Puszyk

Gestational Diabetes Mellitus Impairs Nrf2-Mediated Adaptive Antioxidant Defenses and Redox Signaling in Fetal Endothelial Cells In Utero

In utero exposure to gestational diabetes mellitus (GDM) is associated with an increased risk of type 2 diabetes and cardiovascular disease in later life, yet the underlying mechanisms remain to be elucidated. We examined the effects of GDM on the proteome, redox status, and nuclear factor erythroid 2–related factor 2 (Nrf2)-mediated antioxidant gene expression in human fetal endothelial cells. Proteomic analysis revealed that proteins involved in redox homeostasis were significantly altered in GDM and associated with increased mitochondrial superoxide generation, protein oxidation, DNA damage, and diminished glutathione (GSH) synthesis. In GDM cells, the lipid peroxidation product 4-hydroxynonenal (HNE) failed to induce nuclear Nrf2 accumulation and mRNA and/or protein expression of Nrf2 and its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), Bach1, cystine/glutamate transporter, and glutamate cysteine ligase. Although methylation of CpG islands in Nrf2 or NQO1 promoters was unaltered by GDM, decreased DJ-1 and increased phosphorylated glycogen synthase kinase 3β levels may account for impaired Nrf2 signaling. HNE-induced increases in GSH and NQO1 levels were abrogated by Nrf2 small interfering RNA in normal cells, and overexpression of Nrf2 in GDM cells partially restored NQO1 induction. Dysregulation of Nrf2 in fetal endothelium may contribute to the increased risk of type 2 diabetes and cardiovascular disease in offspring.

Epigenetic Upregulation of HGF and c-Met Drives Metastasis in Hepatocellular Carcinoma

Hepatocyte growth factor (HGF) and its receptor, c-Met, are important regulators of growth and differentiation of healthy hepatocytes. However, upregulation of HGF and c-Met have been associated with tumor progression and metastasis in hepatocellular carcinoma (HCC). Hematogenous dissemination is the most common route for cancer metastasis, but the role of HGF and c-Met in circulating tumor cells (CTCs) is unknown. We have isolated and established a circulating tumor cell line from the peripheral blood of a mouse HCC model. Our studies show that these CTCs have increased expression of HGF and c-Met in comparison to the primary tumor cells. The CTCs display phenotypic evidence of epithelial-mesenchymal transition (EMT) and the EMT appears to be inducible by HGF. Epigenetic analysis of the c-Met promoter identified significant loss of DNA methylation in CTCs which correlated with overexpression of c-Met and increased expression of HGF. Six specific CpG sites of c-Met promoter demethylation were identified. CTCs show significantly increased tumorigenicity and metastatic potential in a novel orthotopic syngeneic model of metastatic HCC. We conclude that during hematogenous dissemination in HCC, CTCs undergo EMT under the influence of increased HGF. This process also involves up regulation of c-Met via promoter demethylation at 6 CpG sites. Consequently, targeting HGF and c-Met expression by CTCs may be a novel non-invasive approach with potential clinical applications in HCC management.

The epigenetic regulator PLZF represses L1 retrotransposition in germ and progenitor cells

Germ cells and adult stem cells maintain tissue homeostasis through a finely tuned program of responses to both physiological and stress‐related signals. PLZF (Promyelocytic Leukemia Zinc Finger protein), a member of the POK family of transcription factors, acts as an epigenetic regulator of stem cell maintenance in germ cells and haematopoietic stem cells. We identified L1 retrotransposons as the primary targets of PLZF. PLZF‐mediated DNA methylation induces silencing of the full‐length L1 gene and inhibits L1 retrotransposition. Furthermore, PLZF causes the formation of barrier‐type boundaries by acting on inserted truncated L1 sequences in protein coding genes. Cell stress releases PLZF‐mediated repression, resulting in L1 activation/retrotransposition and impaired spermatogenesis and myelopoiesis. These results reveal a novel mechanism of action by which, PLZF represses retrotransposons, safeguarding normal progenitor homeostasis.

A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy

AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

OPA1 downregulation is involved in sorafenib-induced apoptosis in hepatocellular carcinoma

Sorafenib has been used to treat advanced hepatocellular carcinoma (HCC), but the underlying molecular mechanisms remain controversial and why some patients do not respond to this therapy is poorly understood. In this study, we show that sorafenib triggers cell growth inhibition and apoptosis in HCC cells by directly targeting the mitochondria. Treatment with sorafenib induces rapid mitochondrial fragmentation, which is associated with the deregulation of mitochondria fusion-related protein optic atrophy 1 (OPA1). Exposure of cells or isolated mitochondria to sorafenib substantially induces cytochrome c release. Our data indicate that siRNA-mediated OPA1 knockdown significantly sensitizes HCC cells to sorafenib-induced apoptosis. Furthermore, sorafenib has no apparent apoptotic toxicity to normal human primary hepatocytes. Sorafenib inhibits HCC xenograft tumor growth in vivo and murine xenograft tumor tissue analysis reveals mitochondria fusion protein. OPA1 expression levels are strongly downregulated by sorafenib treatment. Western blotting evaluation of patient HCC with matched non-tumor tissue samples demonstrates that OPA1 expression is decreased in up to 40% of HCC patients. Taken together, we have shown that sorafenib suppresses the tumorigenesis of HCC through the induction of mitochondrial injury via OPA1. Our results provide new insights into the pathogenesis of HCC and suggest that OPA1 is a novel therapeutic target in patients with HCC.

Linking metabolism and epigenetic regulation in development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common form of cancer globally and is rarely curable once detected. The 5-year survival rate of patients diagnosed with late-stage HCC may be as low as 27%. HCC is a cancer largely driven by epigenetic changes that arise from exposure to exogenous environmental factors rather than coding sequence mutations. The liver is susceptible to effects from Hepatitis C and Hepatitis B viruses, exposure to aflatoxin and continuous excessive consumption of alcohol. The liver is a highly metabolic organ balancing many vital biochemical processes; exposure to any of the above environmental factors is associated with loss of liver function and is a major risk factor for the development of HCC. Emerging studies aim to examine the underlying metabolic processes that are abrogated in cancer and lead to the altered flux and availability of key metabolites important for epigenetic processes. Metabolites have been shown to act as substrates for many canonical epigenetic regulators. These enzymes are responsible for regulating histone modification, DNA methylation and micro RNA expression. By studying the impact of altered liver metabolism, we may better understand the long-term epigenetic processes, which lead to the development and progression of HCC.

Focal adhesion kinase inhibitor PF573228 and death receptor 5 agonist lexatumumab synergistically induce apoptosis in pancreatic carcinoma

Pancreatic cancer has one of the lowest survival rates of all cancers. The mechanism underlying chemo-resistance of pancreatic cancer is not well understood. Our previous article reported that small molecule YM155 induced apoptosis in pancreatic cancer cells via activation of death receptor 5. In this study, we aim to continuously address death receptor 5–mediated apoptosis in chemo-resistant pancreatic carcinoma. We found that in comparison to paired pancreatic cancer tissues and adjacent normal tissues, five of the six cancer tissues had downregulated death receptor 5 and upregulated Bcl-xL. Mono treatment with lexatumumab was not sufficient to induce apoptosis in pancreatic cancer cells, whereas focal adhesion kinase inhibitor PF573228 significantly sensitized lexatumumab-induced apoptosis. Western blotting analysis revealed that lexatumumab and PF573228 combination treatment increased death receptor 5 but decreased Bcl-xL expression. Interestingly, pre-treatment with Bcl-xL inhibitor ABT263 reversed the insensitivity of panc-1 cells to lexatumumab or PF573228-induced apoptosis. Specific small interfering RNA-mediated gene silencing of Bcl-xL effectively sensitized pancreatic cancer cells to lexatumumab or PF573228-induced apoptosis. Furthermore, lexatumumab and PF573228 combination was shown to exhibit significant xenograft pancreatic tumor growth inhibition in SCID mice. Our data provide fundamental evidence to support the notion that lexatumumab and PF573228 co-treatment could be a potentially effective regime for patients with pancreatic cancer.

Small Molecule Inhibitor YM155-Mediated Activation of Death Receptor 5 Is Crucial for Chemotherapy-Induced Apoptosis in Pancreatic Carcinoma

Despite much effort, pancreatic cancer survival rates are still dismally low. Novel therapeutics may hold the key to improving survival. YM155 is a small molecule inhibitor that has shown antitumor activity in a number of cancers by reducing the expression of survivin. The aim of our study is to understand the mechanisms by which YM155 functions in pancreatic cancer cells. We established the antitumor effect of YM155 with in vitro studies in cultured cells, and in vivo studies using a mouse xenograft model. Our data demonstrated that YM155 reduced the expression of survivin; however, downregulation of survivin itself is insufficient to induce apoptosis in pancreatic cancer cells. We showed for the first time that treatment with YM155 increased death receptor 5 (DR5) expression in pancreatic cancer cells. We found that YM155 induced apoptosis by broad-spectrum inhibition of IAP family member proteins (e.g., CIAP1/2 and FLIP) and induced proapoptotic Bak protein upregulation and activation; the antitumor effect of YM155 treatment with either the DR5 agonist lexatumumab or gemcitabine on pancreatic cancer cells was synergistic. Our data also revealed that YM155 inhibits tumor growth in vivo, without apparent toxicity to the noncancerous human pancreatic ductal epithelial cell line. Together, these findings suggest that YM155 could be a novel therapeutic agent for pancreatic cancer. Mol Cancer Ther; 14(1); 80–89. ©2014 AACR.

Initiation of aberrant DNA methylation patterns and heterogeneity in precancerous lesions of human hepatocellular cancer

ABSTRACT While intratumor heterogeneity contributes to disease progression, metastasis, and resistance to chemotherapy, it also provides a route to understanding the evolution and drivers of disease. Defects in epigenetic landscapes are intimately linked to pathogenesis of a variety of human diseases, with epigenetic deregulation promoting tumorigenesis. Understanding epigenetic heterogeneity is crucial in hepatocellular carcinoma (HCC), where epigenetic alterations are frequent, early, and pathogenic events. We determined genome-wide DNA methylation and copy number variation leveraging the Infinium 450K in a series of regenerative nodules from within single patient livers. Bioinformatics strategies were used to ascertain within-patient heterogeneity, link epigenetic changes to clinical features, and determine their relevance to disease pathogenesis. Our data demonstrate that DNA methylation and copy number alterations evolve during the pre-neoplastic phase of HCC and independently segregate regenerative nodules into distinct clusters. Regenerative nodules with a high frequency of epigenetic changes have significantly lower copy number variation, suggesting that individual nodules have differential enrichment of epigenetic and genetic components, with both contributing to disease progression. Regenerative nodules were scored based on ‘epigenetic progression’ with higher scores associated with increased proliferation measured by Ki67 staining. Early events observed in epigenetically ‘aggressive’ nodules are enriched for genes involved in liver cancer. Our study demonstrates that marked epigenetic and genetic heterogeneity exists in early pre-neoplastic liver tissue within individual patients, emphasizing the potential contributions of each mechanism to driving liver disease progression, and it unveils strategies for identifying epigenetic drivers of hepatocellular carcinoma.

Abstract 418: Epigenetic regulation of glypican-3 in hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Glypican 3 (GPC3) is a cell surface heparan sulfate proteoglycan which plays critical roles in the control of cell division and growth regulation. Although repressed in most of adult tissues, GPC3 is found to be upregulated in different types of cancer, including hepatocellular carcinoma (HCC). To address whether the expression of GPC3 in HCC is regulated by epigenetic mechanisms, we studied the DNA methylation profiles of the two CpG islands in the GPC3 promoter by high resolution melting analysis (HRM) and by pyrosequencing. Histone modifications were also examined by ChIP assays to further explore how epigenetic changes contribute to GPC3 expression in HCC. Results: We observed hypomethylation in promoters of 5 different HCC cell lines (which express GPC3) but a partially hypermethylated pattern in HCO2, a HCC cell line in our panel which does not express GPC3. We measured average CpG methylation by pyrosequencing, with the average methylation values for GPC3 expressing cells at 7.74% vs. HCO2 at 43.25% (P<0.01). Similarly, the average methylation frequency in 9 different primary hepatocytes (also no GPC3 expression) is 31.85%, equals to that of HCO2 and much higher than GPC3 expressing cells (P<0.01). In tissues, the methylation profiles of GPC3 promoters are more complicated, where hypo- and hypermethylation could be found in both tumor and non-tumor adjacent tissues. In 34 pairs of tissues, 23 pairs (67.6%) have methylation patterns correlate with expression data. However, treatment of HCO2 with demethylating agent (5-aza-dC) resulted in little reactivation of GPC3 expression, suggesting that DNA methylation may not be the only factor modifying transcription of GPC3. Treatment of HCO2 with histone deacetylase inhibitor trichostatin-A (TSA) resulted in more than 10 folds of GPC3 expression, suggesting histone modification plays a critical role in regulation of GPC3. ChIP analysis in different HCC cell lines as well as primary hepatocytes revealed that the histone marker H3K9Ac (associated with transcription) is present at the GPC3 promoter region of Huh7 but absence in HCO2 and in primary hepatocytes, whereas occupancy of the promoter by repressive markers H3K9Me3 and H3K27Me3 is increased in HCO2 and primary hepatocytes but not Huh7. ChIP assay performed on two pairs of HCC tissues showed similar correlation data between GPC3 expression and histone modification. Conclusions: In this study, we showed that methylation is correlated with transcriptional repression of GPC3 in HCC cell lines, but demethylation of GPC3 promoter is not enough to reactivate the protein expression, suggesting DNA methylation is not the predominant regulatory mechanism for GPC3 gene. Histone modification, on the other hand correlates very tightly with GPC3 expression in HCC cell lines and tumors. Together, our data suggest that epigenetic alterations, including DNA methylation and histone modifications are critical for transcriptional regulation of GPC3 in HCC. Citation Format: Thu Le Trinh, William Puszyk, Chen Liu. Epigenetic regulation of glypican-3 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2014-418