Ming-Ta Sung

Mitochondrial dysfunction represses HIF-1α protein synthesis through AMPK activation in human hepatoma HepG2 cells

BACKGROUND Hypoxia-inducible factor-1α (HIF-1α) is an important transcription factor that modulates cellular responses to hypoxia and also plays critical roles in cancer progression. Recently, somatic mutations and decreased copy number of mitochondrial DNA (mtDNA) were detected in hepatocellular carcinoma (HCC). These mutations were shown to have the potential to cause mitochondrial dysfunction. However, the effects and mechanisms of mitochondrial dysfunction on HIF-1α function are not fully understood. This study aims to explore the underlying mechanism by which mitochondrial dysfunction regulates HIF-1α expression. METHODS Human hepatoma HepG2 cells were treated with various mitochondrial respiration inhibitors and an uncoupler, respectively, and the mRNA and protein expressions as well as transactivation activity of HIF-1α were determined. The role of AMP-activated protein kinase (AMPK) was further analyzed by compound C and AMPK knock-down. RESULTS Treatments of mitochondrial inhibitors and an uncoupler respectively reduced both the protein level and transactivation activity of HIF-1α in HepG2 cells under normoxia or hypoxia. The mitochondrial dysfunction-repressed HIF-1α protein synthesis was associated with decreased phosphorylations of p70(S6K) and 4E-BP-1. Moreover, mitochondrial dysfunction decreased intracellular ATP content and elevated the phosphorylation of AMPK. Treatments with compound C, an AMPK inhibitor, and knock-down of AMPK partially rescued the mitochondrial dysfunction-repressed HIF-1α expression. CONCLUSIONS Mitochondrial dysfunctions resulted in reduced HIF-1α protein synthesis through AMPK-dependent manner in HepG2 cells. GENERAL SIGNIFICANCE Our results provided a mechanism for communication from mitochondria to the nucleus through AMPK-HIF-1α. Mitochondrial function is important for HIF-1α expression in cancer progression.

Krüppel-like factor 4 modulates the migration and invasion of hepatoma cells by suppressing TIMP-1 and TIMP-2

Krüppel-like factor 4 (KLF4) plays important roles in development, stemness and tumorigenesis; however limited information is available on the detailed function of KLF4 in hepatocellular carcinoma (HCC). The objective of the present study was to examine the functional roles of KLF4 in the metastasis of HCC cells. KLF4 was overexpressed and knocked down by lentiviral transduction method in highly metastatic HCC cells. KLF4 overexpression in HCC cells led to inhibition of cell migration and invasion. These inhibitory effects were associated with the upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 by KLF4. Treatment with recombinant TIMP-1 decreased the migratory ability of HCC cells. Moreover, myeloperoxidase (MPO)-TIMP-1/TIMP-2 inactivator counteracted the KLF4-induced inhibition of cell migration/invasion. Consistently, KLF4 knockdown in HCC cells downregulated TIMP-1 and TIMP-2 expression, consequently promoting cell migration and invasion. Furthermore, we found that KLF4 regulated E-cadherin and epithelial-mesenchymal transition (EMT)-related proteins such as snail, vimentin and Bmi1 to modulate the cell migration ability. These results together demonstrated for the first time that KLF4 plays an important role in inhibiting the aggressiveness of HCC cells via upregulation of TIMP-1 and TIMP-2.

Peroxisome Proliferator-Activated Receptor γ Expression Is Inversely Associated with Macroscopic Vascular Invasion in Human Hepatocellular Carcinoma

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor that regulates cellular lipid and glucose metabolism and also plays an inhibitory role in various cancers. However, the role of PPARγ in hepatocellular carcinoma (HCC) remains controversial. This study aimed to investigate the prognostic value of PPARγ in HCC and its role in inhibiting tumor progression, namely, HCC cell growth, migration, and angiogenesis. Immunohistochemical PPARγ staining was examined in 83 HCC specimens to investigate the clinicopathological correlations between PPARγ expression and various parameters. The functional role of PPARγ was determined via PPARγ overexpression and knockdown in HCC cells. Patients with low HCC tissue PPARγ expression were significantly younger (p = 0.006), and exhibited more tumor numbers (p = 0.038), more macroscopic vascular invasion (MVI) (p = 0.008), and more advanced TNM (size of primary tumor, number of regional lymph nodes, and distant metastasis) stages at diagnosis (p = 0.013) than patients with high HCC tissue PPARγ expression. PPARγ knockdown increased HCC cell growth, migration, and angiogenesis, while PPARγ overexpression reduced HCC cell growth, migration, and angiogenesis. These results suggest that low PPARγ expression is an independent predictor of more MVI in HCC patients. PPARγ contributes to the suppression of HCC cell growth, migration, and angiogenesis. Therefore, PPARγ may be a therapeutic target in HCC patients.

The HGF/c-Met/COX-2 and Jagged1/Notch1/COX-2 Pathways as Molecular Targets for Gastric Cancer Treatment

In Taiwan, gastric cancer ranked fifth in total cancer mortality in 2012. Although the number of gastric cancer cases has declined in recent decades, it is still the second leading cause of cancer mortality worldwide. Therefore, more potential molecular targets and therapeutic options for gastric cancer treatment are urgently needed. The overexpression of COX-2 has been shown to be highly associated with metastasis, invasion and angiogenesis of gastric cancer. A positive correlation between COX-2 and c-Met (or Jagged1) expression also has been found in gastric cancer. Moreover, several studies indicated that hepatocyte growth factor (HGF)/c-Met and Jagged1/Notch1 pathways can up-regulate COX-2 expression, resulting in increased cell growth, migration and invasion in gastric cancer cells. Recently, various monoclonal antibodies or receptor tyrosine kinase inhibitors have been designed to target HGF/c-Met pathway and have shown potentials for gastric cancer therapy in clinical trials. In this article, we have summarized the molecular mechanisms of HGF/c-Met/COX-2 and Jagged1/ Notch1/COX-2 pathways, and the latest target therapies for gastric cancer.

Abstract 284: Down-regulated Kruppel-like factor 4 induces the migration and epithelial-mesenchymal transition in human hepatoma cells.

The transcriptional factor Kruppel-like factor 4 (KLF4) plays important roles in development, stemness, and tumorigenesis. Several studies have suggested that KLF4 is important in various tumors; however limited information is available on the detailed function of KLF4 in hepatocellular carcinoma. The objective of this study was to examine the functional roles of KLF4 in metastasis of hepatoma cells. In order to investigate the role of KLF4 in the progression of hepatocellular carcinoma, we used lentiviral transduction method to down-regulate KLF4 expression in Hep3B and HA22T/VGH hepatoma cell lines. The effects of KLF4 on cell migration, invasion and the expression of epithelial-mesenchymal transition (EMT) related proteins were evaluated in KLF4-knockdown hepatoma cells. Transwell migration assay and matrigel invasion assay revealed the migration and invasion abilities were significantly increased in KLF4-knockdown Hep3B or HA22T/VGH cells. Knockdown KLF4 in hepatoma cells resulted in decreased mRNA and protein expression of E-cadherin, and a concomitant increase of vimentin, MMP2 and MMP9. These results corresponded well with our finding that overexpression of KLF4 in Mahlavu hepatoma cells led to significant inhibition of cell migration and invasion through up-regulating E-cadherin and down-regulating vimentin, MMP2 and MMP9. Snail and Bmi1, repressors of E-cadherin gene expression, were also markedly decreased in KLF4-overexpressing Mahlavu cells. Therefore, these results together suggested that KLF4 is tightly involved in the epithelial-mesenchymal transition and metastasis in the tumor progression of hepatocellular carcinoma. Citation Format: Ming-Ta Sung, Hsin-Chen Lee, Cheng-Yuan Hsia, Chin-Wen Chi. Down-regulated Kruppel-like factor 4 induces the migration and epithelial-mesenchymal transition in human hepatoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 284. doi:10.1158/1538-7445.AM2013-284

Abstract 295: PPARγ-induced KLF4 in regulation of cell growth and migration in human hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recently, a zinc finger transcription factor Kruppel-like factor 4 (KLF4) was found to be reduced in tumors of HCC patients. Additionally, results from our laboratory showed that overexpressed KLF4 repressed epithelial-mesenchymal transition (EMT) process and cell motility by induction of E-cadherin expression and reduction of EMT markers in hepatoma cells. Interestingly, it has been found that PPARγ agonists upregulated KLF4 in colorectal cancer cells. However, the mechanism of PPARγ-induced KLF4 in the EMT process of HCC remains unclear. In this study, Hep3B, PLC/PRF/5, and Mahlavu human hepatoma cells were treated with PPARγ agonists rosiglitazone, troglitazone, and PPARγ antagonist GW9662, respectively. After rosiglitazone or troglitazone treatment for various time periods, growth inhibition was observed in both Hep3B and PLC/PRF/5 cells at 48 hours by surforhodamine B assay. Mahlavu cells showed less response to rosiglitazone and troglitazone treatment as compared to the other two cell lines. However, pretreatment of GW9662, a PPARγ antagonist, prior to PPARγ agonists treatment did not block the PPARγ agonists-induced cell growth inhibition. Moreover, cotreatment of PPARγ agonist and antagonist showed additive growth inhibition effect. In addition, rosiglitazone and troglitazone inhibited the migration of Hep3B, PLC/PRF/5 and Mahlavu cells differentially in transwell assay. Western blotting analysis showed upregulation of KLF4 and E-cadherin in Hep3B and PLC/PRF/5 cells after rosiglitazone and troglitazone treatment. Interestingly, decreased KLF4 and E-cadherin were observed in Hep3B, PLC/PRF/5, and Mahlavu cells after GW9662 treatment, indicating that the regulation of KLF4 and E-cadherin were PPARγ-dependent. Moreover, GW9662 also inhibited cell migration of Hep3B, PLC/PRF/5, and Mahlavu cells, with a concomitant decrease of vimentin. These results together suggested that PPARγ agonists and antagonist not only inhibited the growth but also decreased migration of hepatoma cells. Modulation of the PPARγ agonists induced expression of KLF4 and E-cadherin played important role in migration of hepatoma cells. Citation Format: Hui-Tzu Hsu, Ming-Ta Sung, Chin-Wen Chi. PPARγ-induced KLF4 in regulation of cell growth and migration in human hepatoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2013-295

Abstract 3442: Overexpression of Kruppel-like factor 4 inhibits the growth and migration in human hepatoma cells

The zinc-finger transcriptional factor Kruppel-like factor 4 (KLF4), can inhibit cell proliferation and maintain the differentiation of epithelial cells by regulating the expression of genes with GC-rich promoters, plays important roles in development and tumorigenesis. Recently, reduction of KLF4 expression has been reported in multiple gastrointestinal cancers such as gastric cancer, colorectal cancer and hepatocellular carcinoma (HCC), and this observation was associated well with tumor progression and poor clinical outcome. However, the exact role of KLF4 in the progression of HCC is still unclear. In this study, we used lentiviral transduction method to overexpress KLF4 in a highly metastatic HA22T/VGH hepatoma cell line. Using MTT and sulforhodamine B assay, we found that cell growth was decreased in KLF4-overexpressing HA22T/VGH cells as compared to parental cells. Moreover, wound healing assay revealed the migration ability was significantly suppressed in KLF4-overexpressing HA22T/VGH cells. Consistent with these findings, KLF4 overexpression also resulted in the up-regulation of E-cadherin expression and concomitant down-regulation of vimentin expression in HA22T/VGH cells. Moreover, our preliminary finding showed that KLF4 knockdown by transfection of KLF4 shRNA caused the decrease in E-cadherin expression in HEK293T cells. E-cadherin, a key adhesion molecule in the process of epithelial-mesenchymal transition (EMT) and tumor metastasis, was regulated by many transcription factors such as Snail. Interestingly, in this study we have found that KLF4 overexpression led to an obvious reduction in the Snail expression in HA22T/VGH cells. Therefore, these results together suggest that KLF4-induced E-cadherin expression may result from regulation at E-cadherin transcription level or inhibition of the expression of Snail. Furthermore, our findings also suggested that the role of KLF4 may be associated with the cell growth, EMT process and metastasis in the tumor progression of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3442. doi:1538-7445.AM2012-3442

Abstract 3423: The role of PGC1-α and KLF4 in inhibiting the progression of human hepatoma cells

Hepatocellular carcinoma (HCC) is one of the most common cancers with high mortality worldwide. Hypoxia is considered as a driving force to trigger the progression of malignancy, such as epithelial-mesenchymal transition (EMT) process, tumor metastasis, and recurrence. Because of the high recurrence rate caused by frequent metastasis and the lack of effective systemic treatment, development of new biomarkers and therapeutic targets for HCC treatment is urgently needed. Loss of E-cadherin is an early and critical step in EMT process and tumor metastasis. Consequently, modulation of the expression of E-cadherin may be a potential way to prevent tumor metastasis. Recently, we have demonstrated that overexpression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) not only down-regulated the cyclin D1 expression and inhibited growth of hepatoma HepG2 cells, but also up-regulated the E-cadherin expression and concomitantly down-regulated the mesenchymal marker, and decreased the vimentin expression as well as the migration of HepG2 cells. In this study, we observed a significant increase in the expression level of Kruppel-like factor 4 (KLF4) in HepG2 cells when PGC-1α was overexpressed by adenovirus mediated gene transfer. Using wound healing assay and Western blotting, we further observed increased cell migration and decreased expression of KLF4 in HepG2, Hep3B, and HA22T/VGH hepatoma cell lines under hypoxia condition. Moreover, the KLF4 expression level in HepG2 cells under hypoxia and combined with drug treatment was also examined. We found that treatment of sodium nitroprusside, a nitric oxide donor, can protect HepG2 cells from hypoxia- and glucose depletion-induced cytotoxicity through repressing the KLF4 and Bip/Grp78 expression. After treatment with a cytotoxic dose (1 μM) of doxorubicin, the KLF4 expression of HepG2 cells was decreased under normoxia while it was increased under hypoxia. These results together suggest that PGC-1α may regulate the expression of E-cadherin via KLF4, and the KLF4 expression correlated with the response of HepG2 cells to doxorubicin under hypoxia. PGC-1α and KLF4 may serve as potential targets to inhibit the growth and metastasis of hepatoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3423. doi:10.1158/1538-7445.AM2011-3423