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The over-expression of survivin enhances the chemotherapeutic efficacy of YM155 in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. The inability of chemotherapeutic drugs to selectively target HCC tumor cells because of their predominant resistant phenotype to most conventional anticancer agents bestows a major obstacle for the clinical management of HCC. In this report, we have examined and demonstrated the remarkable heterogeneity of expression of survivin and its phosphorylated active form (p-survivin) in HCC patients' tissues and cell lines. Furthermore, the expression of survivin and p-survivin in HCC cell lines was found to be associated with response to the small-molecule survivin suppressant YM155. Therefore, in the HCC cell lines that express elevated level of survivin and p-survivin, YM155 efficiently inhibited their proliferation, induced cell cycle arrest and apoptosis resulting in DNA damage through the dysregulation of cell-cycle checkpoint-related regulatory genes. Importantly, YM155 yielded significantly better therapeutic effect than sorafenib when tested in an orthotopic mouse model using patient-derived HCC xenografts with elevated survivin and p-survivin expression. Our results clearly demonstrated that the level of survivin and p-survivin expression could serve as molecular predictive biomarkers to select potential YM155-responsive patients, in a move towards delivering precision medicine for HCC patients.

The over-expression of survivin enhances the chemotherapeutic efficacy of YM155 in human hepatocellular carcinoma

Tissue specimens, cell lines and reagents
The collection of tumor and adjacent normal liver tissues from HCC patients were approved by our Institutional Review Board (IRB) and all tissues studied were provided by the Tissue Repository of the National Cancer Centre Singapore (NCCS) and Cancer Center, Sun Yat-Sen University. Written informed consent was obtained from all participating patients and all clinical and histopathological data provided to the researchers were rendered anonymous. Liver cancer cells were cultured in Dulbecco's modified Eagle's medium (HepG2, HuH7, PLC/PRF/5, HCCLM3, HLE, Mahlavu, SNU-449, SK-HEP-1), with 10% FBS and 100 units/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen, Carlsbad, CA). All cell lines were maintained at 37°C in the presence of 5% CO 2 . Sorafenib was from Bayer HealthCare Pharmaceuticals, Inc. and YM155 was synthesised by MedChemexpress Co. Ltd and Biochempartner Co. Ltd (Shanghai, China).

RNA extraction, microarray and Real-time quantitative RT-PCR (qRT-PCR) analysis
Total RNA from the tissue samples or cell lines was extracted using TRIzol reagent (Invitrogen). The quality and quantity of isolated total RNA were assessed using the Agilent 2100 Bioanalyzer and NanoDrop ND-1000 Spectrophotometer (Agilent, Santa Clara, CA, USA). Microarray analysis was performed as described [22]  and are accessible through ArrayExpress public database with accession numbers E-MEXP-84 and E-TABM-292. The microarray data were further analysed using Ingenuity Pathway Analysis (IPA) Software. For mRNA detection by qRT-PCR, the total RNA was reversely transcribed by using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, CA). qPCR was performed using SsoFast™ EvaGreen® Supermix (Bio-Rad) with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as an internal control as described previously, and fold changes were calculated via relative quantification (2 -ΔCt ).

Immunofluorescence analysis
HCC cells were seeded in the BD Falcon™ 8-well CultureSlide and treated with YM155 (10 ng/ml) for 24 hours. The treated cells were fixed and incubated with primary antibodies against p-H2AX and then incubated with Alexa Fluor® 488 goat anti-rabbit IgG (Invitrogen). Slides were counterstained with Hoechst 33342 and imaged using a confocal laser-scanning microscope (Carl Zeiss).

Animal studies
All experiments were approved by the Institutional Animal Care and Use Committee of SingHealth. All mice studies were done in the Animal Unit with AAALAC accreditation in National Cancer Center Singapore. Mahlavu-and HuH7-luciferase expressing cells were firstly subcutaneously injected into 5-to 6-week-old athymic mice (2 mice per group, BioLASCO Taiwan Co, Ltd) to generate tumor. The tumor-bearing mice were anesthetized with Hypnorm/Midazolam and a small piece of tumor was harvested and orthotopically transplanted to the liver of the recipient mice as described previously [18] to establish the orthotopic mouse liver tumor xenograft model. One week after implantation, the mice were imaged and grouped. Mice with similar signals were grouped (10 mice/group) and treated either with YM155, sorafenib or saline for 7 weeks.
Sorafenib was administered at the effective dose of 30 mg/kg/dose, p.o. Mice were treated with YM155 for 7 days with a continuous intraperitoneal injection of 10 mg/kg, followed by a 7-day rest period. Each cycle was repeated every 14 days for a total of 4 cycles of treatment. Tumor growth was monitored every week by bioluminescence imaging using the Xenogen IVIS Lumina system (Xenogen Corporation, Hopkinton, MA).