Mcl-1 as a potential therapeutic target for human hepatocelluar carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.

Keywords

Mcl-1 / potential target / hepatocellular carcinoma

References

[1] EI-SeragHB. Hepatocellular carcinoma. N Engl J Med, 2011, 365(12): 1118-1127

[2] ChenJG, ZhangSW. Liver cancer epidemic in China: past, present and future. Semin Cancer Biol, 2011, 21(1): 59-69 PMID: 21144900

[3] BruixJ, LlovetJM. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepotology, 2002, 35(3): 519-524

[4] ChungV. Systemic therapy for hepatocellular carcinoma and cholangiocarcinoma. Surg Oncol Clin N Am, 2015, 24(1): 187-198 PMID: 25444475

[5] KangMH, ReynoldsCP. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res, 2009, 15(4): 1126-1132 PMID: 19228717 PMCID: 3182268

[6] CraigRW, JabsEW, ZhouP, et al. . Human and mouse chromosomal mapping of the myeloid cell leukemia-1 gene: MCL1 maps to human chromosome 1q21, a region that is frequently altered in preneoplastic and neoplastic disease. Genomics, 1994, 23(2): 457-463 PMID: 7835896

[7] MojsaB, LassotI, DesagherS. Mcl-1 ubiquitination: unique regulation of an essential survival protein. Cells, 2014, 3(2): 418-437 PMID: 24814761 PMCID: 4092850

[8] GeserickP, WangJ, FeoktistovaM, et al. . The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin. Cell Death Dis, 2014, 5: e1412 PMID: 25210795 PMCID: 4540197

[9] De VeirmanK, Van GinderachterJA, LubS, et al. . Multiple myeloma induces Mcl-1 expression and survival of myeloid-derived suppressor cells. Oncotarget, 2015, 6(12): 10532-10547 PMID: 25871384 PMCID: 4496373

[10] YanJ, ZhongN, LiuG, et al. . Usp9x and Noxa-mediated Mcl-1 downregulation contributes to pemetrexed-induced apoptosis in human non-small-cell lung cancer cells. Cell Death Dis, 2014, 5: e1316 PMID: 24991768 PMCID: 4123075

[11] JacqueminG, GranciV, GallouetAS, et al. . Quercetin-mediated Mcl-1 and surviving downregulation restores TRAIL-induced apoptosis in non-Hodgkin lymphoma B cells. Haematologica, 2012, 97(1): 38-46 PMID: 21933852 PMCID: 3248929

[12] ZhangT, ZhaoC, LuoL, et al. . The expression of Mcl-1 in human cervical cancer and its clinical significance. Med Oncol, 2012, 29(3): 1985-1991 PMID: 21674276

[13] MajiS, SamalSK, PattanaikL, et al. . Mcl-1 is an important therapeutic target for oral squamous cell carcinomas. Oncotarget, 2015, 6(18): 16623-16637 PMID: 26009874 PMCID: 4599294

[14] OpfermanJT, LetaiA, BeardC, et al. . Development and maintenance of B and T lymphocytes requires antiapoptotic Mcl-1. Nature, 2003, 426(6967): 671-676 PMID: 14668867

[15] OpfermanJT, IwasakiH, OngCC, et al. . Obligate role of antiapoptotic Mcl-1 in the survival of hematopoietic stem cells. Science, 2005, 307(5712): 1101-1104 PMID: 15718471

[16] VickB, WeberA, UrbanikT, et al. . Knockout of myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes. Hepatology, 2009, 49(2): 627-636 PMID: 19127517 PMCID: 2753874

[17] PanR, RuvoloVR, WeiJ, et al. . Inhibition of Mcl-1 with the pan-Bcl-2 family inhibitor (-)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia. Blood, 2015, 126(3): 363-372 PMID: 26045609 PMCID: 4504949

[18] MartinAP, MitchellC, RahmaniM, et al. . Inhibition of Mcl-1 enhances lapatinib toxicity and overcomes lapatinib resistance via BAK-dependent autophagy. Cancer Biol Ther, 2009, 8(21): 2084-2096 PMID: 19823038 PMCID: 3887451

[19] SieghartW, LosertD, SrommerS, et al. . Mcl-1 overexpression in hepatocelluar carcinoma: a potential target for antisense therapy. J Hepatology, 2006, 44(1): 151-157

[20] NijhawanD, FangM, TraerE, et al. . Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev, 2003, 17(12): 1475-1486 PMID: 12783855 PMCID: 196078

[21] CraigRW. Mcl-1 provides a window on the role of the Bcl-2 family in cell proliferation, differentiation and tumorigenesis. Leukemia, 2002, 16(4): 444-454 PMID: 11960321

[22] VranaJA, BieszczadCK, CleavelandES, et al. . A Mcl-1 overexpression Burkitt lymphoma subline exhibits enhanced survival on exposure to serum deprivation, topoisomerase inhibitors, or staurosporine but remains sensitive to 1-β-D-arabinofuranosylcytosine. Cancer Res, 2002, 62(3): 892-900 PMID: 11830549

[23] CadwellC, ZambettiGP. The effects of wild-type p53 tumor suppressor activity and mutant p53 gain-of-function on cell growth. Gene, 2001, 277(1-2): 15-30 PMID: 11602342

[24] FritschRM, SchneiderG, SaurD, et al. . Translational repression of Mcl-1 couples stress-induced eIF2 alpha phosphorylation to mitochondrial apoptosis initiation. J Biol Chem, 2007, 282(31): 22551-22562 PMID: 17553788

[25] FleischerB, Schulze-BergkamanH, SchuchmannM, et al. . Mcl-1 is an anti-apoptotic factor for human hepatocellular carcinoma. Int J Oncol, 2006, 28(1): 25-32 PMID: 16327976

[26] SuLY, ShiYX, YanMR, et al. . Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway. Acta Pharmacologica Sinica, 2015, 36(12): 1514-1519 PMID: 26592508 PMCID: 4816232

[27] Schulze-BergkamanH, KrammerPH. Apoptosis in cancer-implication for therapy. Semin Oncol, 2004, 31(1): 90-119

[28] GarridoC, KroemerG. Life’s smile, death’s grin: vital functions of apoptosis-executing proteins. Curr Opin Cell Biol, 2004, 16(6): 639-646 PMID: 15530775

[29] VelaL, GonzaloO, NavalJ, et al. . Direct interaction of Bax and Bak proteins with Bcl-2 homology domain 3 (BH3)-only proteins in living cells revealed by fluorescence complementation. J Biol Chem, 2013, 288(7): 4935-4946 PMID: 23283967 PMCID: 3576097

[30] ZhangCZ, ChenGG, MerchantJL, et al. . Interaction between ZBP-89 and p53 mutants and its contribution to effects of HDACi on hepatocellular carcinoma. Cell Cycle, 2012, 11(2): 322-334 PMID: 22214764 PMCID: 3293381

[31] LeuJI, DumontP, HafeyM, et al. . Mitochondrial p53 activate Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol, 2004, 6(5): 443-450 PMID: 15077116

[32] PerciavalleRM, OpfermanJT. Delving deeper: MCL-1's contributions to normal and cancer biology. Trends Cell Biol, 2013, 23(1): 22-29 PMID: 23026029

[33] RahmaniM, AustMM, BensonEC, et al. . PI3K/mTOR inhibition markedly potentiates HDAC inhibitor activity in NHL cells through BIM-and MCL-1-dependent mechanisms in vitro and in vivo. Clin Cancer Res, 2014, 20(18): 4849-4860 PMID: 25070836 PMCID: 4166554

[34] AkgulC. Mcl-1 is a potential therapeutic target in multiple types of cancer. Cell Mol Life Sci, 2009, 66(8): 1326-1336 PMID: 19099185