Influence of hepatic artery occlusion on tumor growth and metastatic potential in a human orthotopic hepatoma nude mouse model: Relevance of epithelial–mesenchymal transition

Abstract

Hepatic artery ligation (HAL), transarterial embolization (TAE), and transarterial chemoembolization (TACE) have been treatment choices for unresectable hepatocellular carcinoma (HCC). Obstruction of tumor blood supply is one of the most important mechanisms of these therapeutics measures. Here we introduced HAL into a metastatic human HCC orthotopic nude mouse model (using MHCC97L and HepG2 cell lines) to examine the effects of hepatic blood flow obstruction on the metastatic potential of hepatic tumor cells, and to investigate the mechanisms underlying these effects. Our results indicated that HAL inhibited tumor growth but concomitantly elicited tumor adaptation and progression, with increased potential for invasion and distant metastases. The underlying proinvasive mechanism of HAL appeared to be associated with enhanced intratumoral hypoxia and epithelial-mesenchymal transition (EMT) due to hypoxia. This was in accord with the in vitro response of MHCC97L and HepG2 cells to hypoxia. The therapeutic effects of HAL could be enhanced by the phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002, through arrest of EMT in hepatic tumor cells. It could be useful in the development of mechanism-based combination therapies to enhance the initial antitumor response. (Cancer Sci 2010; 101: 120-128) R ooted in the belief that blocking vessel supply starves tumors to death,(1) multiple strategies for obstruction of hepatic arterial blood flow have achieved a pronounced therapeutic effect for hepatocellular carcinoma (HCC), especially unresectable HCC.(2,3) These measures include hepatic artery ligation (HAL), transarterial embolization (TAE), and transarterial chemoembolization (TACE). To some extent, recent rising antiangiogenic therapies such as sorafenib also develop conceptually from this theory.(2,4) Although overall survival of most patients was prolonged after treatment, this success was transient, without offering enduring cure. In some cases, a higher incidence of pulmonary metastasis has been reported following hepatic artery occlusion.(5-8) However, the precise mechanisms responsible for treatment failure are not yet clear.One plausible mechanism is hypoxia, one of the most prominent antitumor approaches, which works by blocking vessel supply. It has been noted that although hypoxia kills most tumor cells, it provides a strong selective pressure for the survival of the most aggressive and metastatic cells.(9) Such cells are proficient at escaping the noxious hypoxic microenvironment by activation of an invasive epithelial-mesenchymal transition (EMT) and other metastatic programs, ultimately leading to tumor recurrence or metastasis.(10) The majority of preclinical studies have focused on the significance of angiogenesis due to hypoxia on the enhanced malignant potential of HCC after blocking vessel supply, (11)(12)(13) but less attention has been paid to the tumor cells themselves. It is possible that the process of EMT could account for proinvasive consequences, since it is one of the central mechanis...

Activation of β-Catenin by Hypoxia in Hepatocellular Carcinoma Contributes to Enhanced Metastatic Potential and Poor Prognosis

“…Consistent with our previous report (17), it not only facilitated in vitro cell invasion in HCC but also resulted in peritoneal seeding and pulmonary metastasis in an in vivo HAL model. However, the growth of HCC cells and xenografts were suppressed by hypoxia.…”

“…Cell lysates were precleared by incubation with protein A agarose beads for 1 hour and divided into two: one for immunoprecipitation with the anti-HIF-1α antibody and the other with the anti-β-catenin antibody was used for immunoblotting. Immunofluorescence experiments were done according to previous methods (17).…”

“…Cytoplasmic and nuclear extracts from cultured cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). Immunoblotting was done as previously described (17) with the following antibodies: HIF-1α (1:1,000; Sigma), β-catenin (1:1,000; BD Biosciences), E-cadherin (1:500; Santa Cruz), plakoglobin (1:1,000; BD), vimentin (1:1,000; Sigma), N-cadherin (1:1,000; Santa Cruz), von Hippel-Lindau (1:1,000; Santa Cruz), GSK-3β (1:500; BD), Akt (1:1,000; Cell Signaling), and phosphorylated (p)-Akt (1:1,000; Cell Signaling). Coimmunoprecipitation was carried out in above buffer, followed by immunoblotting to identify precipitates and coprecipitates.…”

“…Hepatic artery ligation (HAL) was done in a spontaneous metastatic HCC model to induce intratumoral hypoxia (17). For the highly metastatic MHCC97-R cell line, 1 × 10 7 MHCC97-R, MHCC97-R-Mock (MHCC97-R-pLKO.1-scramble), and MHCC97-R-shRNA (MHCC97-R-pLKO.1-β-catenin) cells were orthotopically inoculated into the liver parenchyma of nude mice, according to the method previously described (17). After 14 days, HAL was performed by ligating the main branch of the hepatic artery under an operating microscope (n MHCC97-R = 12, n MHCC97-R-Mock = 6, and n MHCC97-R-shRNA = 6).…”

“…A parallel model using low metastatic potential Hep3B-R cell lines was also established and received similar treatment. Visceral organs, including the lungs and liver, were examined histopathologically by standard methods (17). Distant metastases were visualized using fluorescence stereomicroscopy (Leica Microsystems Imaging Solutions, Ltd.) and quantitatively analyzed by integrated absorbance measurements (12).…”