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ANTICANCER RESEARCH 34: 1709-1714 (2014)

Synergistic Inhibition of HCC and Cancer Stem Cell Proliferation by Targeting RAS/RAF/MAPK and WNT/β-Catenin Pathways

ROBERTO GALUPPO, ERIN MAYNARD, MALAY SHAH, MICHAEL F. DAILY, CHANGGUO CHEN, BRETT T. SPEAR and ROBERTO GEDALY

Department of Surgery, University of Kentucky, College of Medicine, Lexington, KY, U.S.A.

Abstract. Background/Aim: The aim of this study is to find countries, the incidence of HCC has been rising rapidly in synergistic effect using FH535 and sorafenib by targeting the recent decades due to infection with C virus (HCV) RAS/RAF/MAPK and WNT/β-catenin pathways. Materials and and alcohol use (2). Methods: 3H-Thymidine incorporation assays were performed Several signaling pathways have been found to be to address Huh7 and stem cell (LCSC) inhibition disregulated in HCC including the RAS/RAF/MAPK, using FH535 and sorafenib, alone and in combination. PI3K/AKT/mTOR, HGF/c-MET, IGF, VEGF, PDGF and Calcusyn analysis was used to calculate the combination index WNT/β-catenin pathways. Among them, de-regulation of the (CI). A western blot assay was performed to check for potential WNT/β-catenin pathway by far the most difficult to treat (3). targets. Results: FH535 and sorafenib caused inhibition of The WNT/β-catenin pathway involves three complexes: the Huh7 and LCSC. Combination therapy was significantly better ligand/receptor cell membrane complex, the cytosol β- than monotherapy in inhibition of HuH7. Combination with catenin destruction complex, and the nuclear β-catenin/ T sorafenib and FH535 was found to be synergistic in inhibition cell factor (TCF)/Lymphoid enhancer factor (LEF) of LCSC with a CI of less than 1. The western blot assay transcription complex (4, 5). Once the membrane complex is demonstrated enhanced cleaved poly (ADP-ribose) polymerase activated the cytosol destruction complex is de-activated, (PARP) and inhibition of cyclin D1, B-cell 2 (Bcl2), resulting in increased accumulation of β-catenin in the survivin and cellular myelocytomatosis oncogene (c-MYC). cytoplasm which enters the nucleus and interacts with the Conclusion: FH535 and sorafenib combination produced TCF/LEF family of transcripton factors, activating TCF/LEF synergistic effect on inhibition of HCC and LCSC. Our study target genes including cyclin D1, cellular myelocytomatosis demonstrated that FH535 can induce in these two oncogene (c-MYC) and survivin (6). different (HCC) cell lines. Aberrant activation of the WNT/β-catenin pathway has been observed in roughly 1/3 of HCCs. In HCC, nuclear and cellular Hepatocellular carcinoma (HCC) is the most common β-catenin accumulation, a hallmark of activated canonical primary malignancy of the liver and the third most common WNT/FZ signaling has been observed in 33-67% of tumors. cause of cancer-related death worldwide, representing a Roughly 20% of HCCs have mutations in the β-catenin gene significant health care problem (1). (7, 8). The chemical agents used to target WNT/β-catenin The prevalence of HCC differs greatly by geographical pathway are at the membrane, cytosol and nuclear complexes. location, reflecting variations in the main risk factors. Most Our group recently investigated the ability of FH-535, a dual cases of HCC (80%) arise in the Asia-Pacific and sub- inhibitor of the peroxisome proliferator-activated receptor Saharan African regions, where the dominant risk factor is (PPAR) and β-catenin/TCF/LEF to inhibit HCC and liver chronic infection with hepatitis B virus (HBV). In Western cancer stem cell growth in vitro. This drug was shown to inhibit growth of hepatoblastoma cell lines HepG2 and Huh7 (9). Sorafenib, a multi- inhibitor, has been found to be active against HCC in several pre-clinical and clinical studies. Correspondence to: Dr. Roberto Gedaly, 800 Rose Street, Room Sorafenib is considered the drug-of-choice to treat patients with C453, Lexington, Kentucky 40536-0293, U.S.A. Tel: +1 8593234661, advanced HCC that are deemed unsuitable for other types of Fax: +1 8592573644, e-mail: [email protected] surgical, ablative and embolization interventions (10). Key Words: Hepatocellular carcinoma, liver cancer stem cells, The aim of this study was to determine the sensitivity of epidermal , sorafenib, RAS/RAF/MAPK, PKI-587, PI- HCC and liver cancer stem cell (LCSC) lines using 103, rapamycin, mTOR complex 1, mTOR complex 2. sorafenib, FH-535 alone and in combination.

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Materials and Methods

Cell culture. The human HCC cell line Huh7 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS) at 37˚C with 5% CO2. LCSC (CelProgen, San Pedro, CA, USA) were cultured in CelProgen Liver Cancer Stem Cell Growth Media with 10% FBS.

Mouse tumorigenesis models. Female NOD/SCID mice (n=3) (NOD.CB17-prkdc^SCID/NCrSD, 4-5 weeks old) were purchased from Harlan Animal Research Laboratory (Indianapolis, IN, USA) and maintained in the Division of Laboratory Animal Resources facility at our Institution. Mice received filtered air, sterile water and irradiated food ad libitum. Tumors were generated by harvesting first passage LCSC that were trypsinized, washed and resuspended in a 50% mixture of Matrigel (BD Biosceince, San Diego, CA, USA) in CelProgen Liver Cancer Stem Cell Growth Media (serum-free) to a final cell count of 20,000 cells/ml. A volume of 0.1 ml of the cell suspension (2000 cells) was injected subcutaneously in the right flank of each mouse (10). The mice were weighed and checked for tumor growth every other week. Tumors were initially observed 28 days after inoculation, at which time the tumor was measured twice weekly using an optical caliper Figure 1. FH535 and sorafenib on inhibition of 3H-thymidine and tumor size was calculated using the following formula: length incorporation in Huh7 cells. ×(width)2 ×0.4 according to a published method (11). When the tumor size reached 940-1,020 mm3, the mice were euthanized with CO2. The tumors were isolated and fixed in 10% formalin for 48 h and then transferred to 70% ethanol. The tumors were then embedded in paraffin, cut to 5 μm sections and stained with Results hematoxylin and eosin for histological analysis. LCSC profile. Flow cytometric analysis indicated that the Chemicals and antibodies. FH535 and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methyl-3H- LCSC cells comprised 64.4% CD133-positive, 83.2% CD44- thymidine (two Ci/mM) was from MP Biomedicals (Costa Mesa, positive, 96.4% CD24-positive and 96.9% aldehyde A1- CA, USA). Antibodies from Cell Signaling(Boston, MA) were used positive cells. for Western Blot assay. Tumorigenesis of LCSC in animal model. Female NOD/SCID 3 H-Thymidine incorporation assay. Huh7 cells were plated in 96- mice (4-5 weeks old) were inoculated with 0.1 ml of the cell well plates at 2,500 cells/well in 0.2 ml DMEM with 10% FBS and suspension (2,000 cells) subcutaneously at the right flank of treated with different concentrations of FH535 and sorafenib, as single agents or in combination, and cultured for 72 h. 3H- each mouse. All mice developed tumors within 4-6 weeks 3 Thymidine incorporation assays were performed as described (13). after inoculation. Tumor size reached 940-1020 mm at day 60 and all mice were euthanized. Stained tumor sections were Western blot analysis. Huh7 cells were cultured in DMEM + analyzed by a trained liver pathologist at our Institution to 10%FBS in 100×20 mm tissue culture dishes until about 70% determine cancer development and characteristics. All mice confluence. The cells were treated with FH535 from 0 to 10 μM for developed poorly-differentiated lesions after inoculation of 38h. Dimethyl sulfoxide (DMSO) (<0.1%) were used as vehicle these liver cancer stem cells (CD133-, CD44-, and CD24- control. All the other procedures were performed as previously described (12). positive) seen on microscopic examination.

Statistical analysis. All analyses were performed using SPSS The combination of FH535 and sorafenib synergistically software version 19 (Chicago, IL, USA). Data are presented as inhibits LCSC proliferation. We found that FH535 in mean±SE. For nominal data, ANOVA followed by Tukey multiple combination with sorafenib was significantly better than range test was used; for two groups of continuous data, paired t- monotherapy on inhibition of LCSC proliferation (Figure 1). test was used. The level of statistical significance was set at CalcuSyn analysis demonstrated that combination therapy p<0.05. Analysis of synergism was performed using the software caused synergistic inhibition of LCSC proliferation. We also CalcuSyn version 2.0 from Biosoft® (Great Shelford, Cambrigde, found that FH535 and sorafenib in combination significantly UK). inhibited the Huh7 HCC cell line (Figure 2)

1710 Galuppo et al: Targeting the RAS/RAF/MAPK Signaling Pathway in HCC

Figure 2. FH535 and sorafenib on inhibition of 3H-thymidine incorporation in Liver Cancer Stem cells showing synergistic effects with combination therapy. Calculated combination index (CI) of less than 1.

Western blot assay after FH-535 treatment demonstrated cell signaling pathways involved in hepatic carcinogenesis. inhibition of survivin and BCL2 and an enhanced cleavage Our group and others have focused on inhibition of LCSCs of PARP. Western blot analysis demonstrated a dose- and differences in resistance patterns with non-liver CSC dependent inhibition of cyclin D1, survivin and BCL2 lines both in vitro and in vivo (15). LCSCs are thought to be expression at the protein level. These data also showed that responsible for tumor development, progression, recurrence levels of cleaved PARP are increased in Huh7 human HCC and metastasis, and targeting signaling pathways required for cells after FH-535 treatment (Figure 3). Expression of c- CSCs activation and proliferation should bring important and MYC, encoded by an oncogene that regulates numerous revolutionary advances in cancer therapeutics. genes important for cell proliferation, was inhibited by FH- Despite numerous efforts, the etiology of HCC 535 (Figure 4). tumorigenesis, whether originating from mature hepatocytes or stem/progenitor cells, remain unclear. Stem cells are Discussion defined by their potential for self-renewal and their ability to proliferate and differentiate into diverse cell types. This In recent years, numerous signaling pathways such as suggests that a tumor is comprised of a heterogeneous RAS/RAF/MAPK, WNT/β-catenin, EGFR, -like population of cells that form a distinct hierarchy. Although , AKT-mTOR (mammalian target of the existence of CSCs was first proposed over 40 years ago, rapamycin), notch and hedgehog have been implicated in it has only been slightly over a decade since Dick et al. first hepatic carcinogenesis (3). The WNT/β-catenin pathway has demonstrated a role for stem cells in hematological been associated with cellular processes such as development, malignancies (16). More recently, studies have provided growth, survival, regeneration, and self-renewal. Disruption convincing evidence that these cells do exist in solid tumors of this balance results from both genetic and epigenetic of many types including those of brain, breast, colorectal, changes found in many types of cancer, including colon liver, and prostate (17). The liver tumor cancer and HCC. Moreover, recent studies indicate that the microenvironment is a complex mixture of tumor cells within WNT/β-catenin signaling pathway has an important role in the extracellular matrix combined with an intricate mix of the maintenance of CSCs (14). Failure of cytotoxic stromal cells and the proteins they secrete. Together, these for advanced HCC and the development of elements contribute to the carcinogenic process (18) by sorafenib have significantly stimulated interest in different sustaining proliferation, evasion of growth suppressors,

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Figure 3. Western blot analysis showing enhanced levels of cleaved Figure 4. Western blot analysis showing enhanced levels of cyclin-D1, c- PARP, BCL2 and survivin. MYC and survivin.

resistance to cell death, induction of angiogenesis, activation protein and dickkopf family members, preventing activation of invasion and metastasis, reprogramming of energy of the pathway. The best-studied mutations in this pathway metabolism and evasion of immune destruction (19). We are the inherited and sporadic mutations in the tumor have recently been working on tumorigenesis and patterns of suppressor Adenomatous polyposis coli (APC), which reduce resistance to drugs in liver cancer. β-catenin degradation, causing increased β-catenin levels and In the present study, we initially focused on LCSC activation of target genes such as the oncogenes cyclin D1 profiling by measuring CSC markers and tumorigenesis and c-MYC (20). Mutations of β-catenin in HCC are often experiments using low cell densities. We then examined located in the CTNNB1 gene exon 3, which encodes the responses to therapy with single agents and drug phosphorylation site for glycogen synthase kinase 3 (GSK-3) combination in two different HCC cell lines including (21). Numerous other signaling pathways have been involved LCSCs. Lastly, we performed a western blot assay to analyze in HCC carcinogenesis. The WNT/β-catenin pathway plays for significant potential targets such as cyclin D1, survivin, an important role in stem cell self-renewal and BCL2 and oncogene c-MYC. Our main focus was to differentiation. Pro-angiogenic factors such as VEGF, determine patterns of response to therapy and the possibility , EGF, PDGF, (HGF) of finding a synergistic effect using combination drugs induce angiogenic signaling via RAS/RAF/MEK/ERK, targeting RAS/RAF/MAPK and WNT/β-catenin pathways. mTOR and WNT signal transduction pathways can We have confirmed by flow cytometry that these cells contribute to HCC progression. The WNT/β-catenin pathway were positive for CD133 (64.4%), CD44 (83.2%), CD24 has been described as one of most difficult pathways to (96.4%) and aldehyde A1 (96.9%). All SCID mice inoculated target in HCC. We studied Huh7 proliferation and the subcutaneously with a low dose of LCSCs (2,000 cells) response to sorafenib and FH-535 alone and in combination. developed tumors, whereas our previous experience and We demonstrated that the sorafenib-and-FH-535 combination published data from other groups indicated that 100,000 is significantly better than monotherapy in inhibition of HCC HCC cells were needed to develop tumors in SCID mice. proliferation as shown in 3H-thymidine incorporation assay. Tumors from these LCSCs demonstrated features of poorl- We previously demonstrated an additive effect of targeting differentiated cancer with a significant proportion of PI3K/mTOR and RAS/RAF/MAPK pathways with several pleomorphism and mitosis. different compounds in vitro and in vivo (12). Other The WNT/β-catenin pathway has been extensively studied investigators have tried combinations targeting different and known to be important in HCC proliferation. In normal pathways to induce HCC inhibition in vitro and in vivo. cells, extracellular WNT ligands can interact with a host of In the present study, using the CalcuSyn software, we secreted antagonists, including secreted frizzled-related found that FH-535 and sorafenib synergistically inhibit

1712 Galuppo et al: Targeting the RAS/RAF/MAPK Signaling Pathway in HCC

LCSC with a combination index (CI) of less than 1. To the 6 Takebe N, Harris PJ, Warren RQ and Ivy SP: Targeting cancer best of our knowledge, this is the first report of synergistic stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. inhibition of HCC and LCSCs targeting RAS/RAF/MAPK Nat Rev Clin Oncol 8: 97-106, 2011. 7 Lee HC, Kim M and Wands JR: Wnt/Frizzled signaling in and WNT/β-catenin pathway in combination. hepatocellular carcinoma. Front Biosci J Virtual Libr 11: 1901- We also analyzed levels of the apoptosis inhibitors 1915, 2006. survivin and BCL2. Survivin inhibits caspase activation, 8 Giles RH, van Es JH and Clevers H: Caught up in a Wnt storm: leading to inhibition of apoptosis. Survivin, whose Wnt signaling in cancer. Biochim Biophys Acta 1653: 1-24, expression is highly regulated by the cell cycle and is only 2003. 9 Handeli S and Simon JA: A small-molecule inhibitor of TCF/B- found in the G2-M phase of the cell cycle is regulated by β- catenin. Cleaved PARP, another marker of apoptosis, was catenin signaling down-regulates PPAR; and PPARD activities. Mol Cancer Ther 7(3): 521-529, 2008. enhanced in Huh7 after FH-535 treatment. We also 10 Llovet JM et al: Sorafenib in advanced hepatocellular demonstrated that FH-535 inhibits cyclin D1 in Huh7 cells. carcinoma. N Engl J Med 359: 378-390, 2008. In conclusion, HCC has proven to be a very heterogeneous 11 Liu L et al: Sorafenib blocks the RAF/MEK/ERK pathway, disease. Regardless of the recent advances in the inhibits tumor angiogenesis, and induces tumor cell apoptosis in understanding HCC pathophysiology, it remains a complex hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66: and poorly-understood disease. Numerous signaling 11851-11858, 2006. pathways such as RAS/RAF/MAPK, WNT/β-catenin, EGFR, 12 Newell P et al: Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and insulin-like growth factor receptor, AKT-mTOR, notch, rapamycin in vivo. J Hepatol 51: 725-733, 2009. hedgehog have been implicated in hepatic carcinogenesis and 13 Gedaly R et al: PI-103 and sorafenib inhibit hepatocellular their components represent molecular targets for therapy in carcinoma cell proliferation by blocking RAS/RAF/MAPK and HCC. Interest in the CSC hypothesis is increasing, and PI3K/AKT/mTOR pathways. Anticancer Res 30: 4951-4958, according to it, cancer initiation, progression, recurrence, 2010. metastasis and therapy resistance are unique properties 14 Takahashi-Yanaga F and Kahn M: Targeting Wnt signaling: Can implicitly dependent on CSC subsets. Our LCSC (positive we safely eradicate cancer stem cells? Clin Cancer Res 16(12): 3153-3162, 2010. for CD133, CD44 and CD24) were able to develop HCC 15 Gedaly R et al: PKI-587 and sorafenib alone and in combination with very low cell dose. LCSC-derived tumors demonstrated on inhibition of liver cancer stem cell proliferation. J Surg Res, typical characteristics of poorly differentiated HCC. FH-535 2013. and sorafenib combined synergistically inhibit LCSC 16 Lapidot T et al: A cell initiating human acute myeloid leukaemia proliferation. Apoptosis was enhanced in Huh7 cells after after transplantation into SCID mice. Nature 367: 645-648, 1994. inhibiting the WNT/β-catenin pathway with FH-535. 17 Visvader JE and Lindeman GJ: Cancer stem cells in solid However, understanding the role of hepatic CSCs remains tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8: 755-768, 2008. limited and its role in tumorigenesis, tumor progression and 18 Hernandez–Gea V, Toffanin S, Friedman SL and Llovet JM: resistance to treatment deserves further investigation. Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma. Gastroenterology References 19 Hanahan D and Coussens LM: Accessories to the crime: functions of cells recruited to the . 1 Llovet JM, Burroughs A and Bruix J: Hepatocellular carcinoma. Cancer Cell 21: 309-322, 2012. Lancet 362: 1907-1917, 2003. 20 Van de Wetering M et al: The beta-catenin/TCF-4 complex 2 El-Serag HB: Hepatocellular carcinoma. N Engl J Med 365: imposes a crypt progenitor phenotype on colorectal cancer cells. 1118-1127, 2011. Cell 111: 241-250, 2002. 3 Llovet JM and Bruix J: Molecular targeted therapies in 21 Zucman-Rossi J et al: Genotype-phenotype correlation in hepatocellular carcinoma. Hepatol Baltim Md 48: 1312-1327, hepatocellular adenoma: new classification and relationship with 2008. HCC. Hepatol Baltim Md 43: 515-524, 2006. 4 Reya T and Clevers H: Wnt signalling in stem cells and cancer. Nature 434: 843-850, 2005. 5 Cavard C et al: Wnt/β-catenin pathway in hepatocellular carcinoma pathogenesis and liver physiology. (2008). at Received December 4, 2013 Accepted December 17, 2013

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