Cyclin E1 Inhibition Can Overcome Sorafenib Resistance In

Published OnlineFirst November 24, 2015; DOI: 10.1158/1078-0432.CCR-15-0499

Cancer Therapy: Preclinical Clinical Cancer Research Cyclin E1 Inhibition can Overcome Sorafenib Resistance in Hepatocellular Carcinoma Cells Through Mcl-1 Suppression Chiun Hsu1,2,3, Liang-In Lin4, Yu-Che Cheng4, Zi-Rui Feng2,3, Yu-Yun Shao1,2, Ann-Lii Cheng1,2,3,5,6, and Da-Liang Ou1,3

Abstract

Purpose: To clarify the effects of cyclin E1 suppression on Results: Cyclin E1 mRNA and protein expressions were sup- antitumor efficacy of sorafenib in hepatocellular carcinoma cells pressed after sorafenib treatment in sorafenib-sensitive but not in and to explore the potential of combining sorafenib with cyclin- sorafenib-resistant hepatocellular carcinoma cells. Changes in dependent kinase (CDK) inhibition in therapy. cyclin E2 or D1 were not correlated with sorafenib sensitivity. Experimental Design: The effects of cyclin E1 suppression on The knockdown of cyclin E1 expression reversed the resistance of sorafenib-induced apoptosis were tested in both sorafenib- hepatocellular carcinoma cells to sorafenib in terms of cell growth sensitive (Huh-7 and HepG2, IC50 5–6 mmol/L) and sorafe- and apoptosis induction, whereas the overexpression of cyclin E1 nib-resistant (Huh-7R and HepG2R, IC50 14–15 mmol/L) increased the resistance to sorafenib. The growth-inhibitory and hepatocellular carcinoma cells. The activity of pertinent signal- apoptosis-inducing effects of sorafenib were enhanced by flavo- ing pathways and the expression of cell cycle and apoptosis- piridol, and Mcl-1 suppression was determined to play a critical related proteins were measured using Western blotting. Efficacy role in mediating this enhancing effect. of sorafenib combined with the pan-CDK inhibitor flavopiridol Conclusions: The cyclin E1 suppression in hepatocellular was tested both in vitro and in xenograft experiments. The carcinoma cells may serve as a pharmacodynamic biomarker for pertinent downstream mediators of antitumor efficacy were predicting sorafenib efficacy. The combination of sorafenib and tested in transient transfection and RNA interference CDK inhibitors may improve the efficacy of sorafenib in hepa- experiments. tocellular carcinoma. Clin Cancer Res; 1–10. 2015 AACR.

Introduction contribute to design strategies for overcoming sorafenib resistance (8). The multikinase inhibitor sorafenib is currently the standard The dysregulation of cell-cycle control is a hallmark of systemic treatment for patients with advanced hepatocellular cancer. The overexpression of cyclins is generally associated carcinoma (1, 2). Sorafenib has been found to exert many "off- with advanced-stage disease and poor prognosis in many types target effects" that may contribute to its antitumor efficacy, in of cancers, including hepatocellular carcinoma (9–13). addition to the inhibition of Raf kinase signaling and tumor Although molecular agents targeting cyclin-dependent kinases angiogenesis (3, 4). Multiple mediators in the apoptosis (CDK) and other cell-cycle regulators have been extensively regulatory pathways, including Mcl-1 and survivin, may studied, their roles in cancer therapy were not established until regulate the antitumor efficacy of sorafenib in cancer cells recently, when the CDK inhibitor palbociclib was demonstrat- (5–7). Identifying these pathways and molecules will greatly ed to prolong the progression-free survival of breast cancer patients who received hormonal therapy (14–16). Preclinical studies have indicated that cell-cycle inhibitors can reverse the 1Graduate Institute of Oncology, College of Medicine, National Taiwan resistance of cancer cells to hormonal, cytotoxic, and molecular University, Taipei, Taiwan. 2Department of Oncology, National Taiwan targeted therapies (17–19). These preclinical and clinical data University Hospital, Taipei, Taiwan. 3National Center of Excellence for suggest that cell-cycle inhibitors may play a crucial role in Clinical Trial and Research, National Taiwan University Hospital,Taipei, combination anticancer therapy. Taiwan. 4Graduate Institute of Clinical Laboratory Sciences and Med- ical Biotechnology, College of Medicine, National Taiwan University, Sorafenib can suppress several key cell-cycle regulators, Taipei, Taiwan. 5Department of Internal Medicine, National Taiwan including E2F1, cyclin D, cyclin E1, and CDKs, which may 6 University Hospital, Taipei, Taiwan. Graduate Institute of Toxicology, contribute to its antitumor efficacy (5, 6, 20–22). We deter- College of Medicine, National Taiwan University, Taiwan. mined cyclin E1 expression in hepatocellular carcinoma cells Note: Supplementary data for this article are available at Clinical Cancer was associated with sensitivity to sorafenib (20). In current Research Online (http://clincancerres.aacrjournals.org/). study, we sought to clarify the mechanisms of sorafenib- Corresponding Author: Da-Liang Ou, College of Medicine, National Taiwan induced cyclin E1 suppression in hepatocellular carcinoma University, No. 1 Jen Ai Road, Section 1, Taipei 100, Taiwan. Phone: 8862-3366- cells, especially the regulatory roles of the E2F1–Rb–cyclin 8226; Fax: 8862-3366-8234; E-mail: [email protected] E1 complex (23, 24). The potential for improving the efficacy doi: 10.1158/1078-0432.CCR-15-0499 of sorafenib in hepatocellular carcinoma by combination with 2015 American Association for Cancer Research. CDK inhibition was also explored.

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of the luciferase gene in the pGL4.17-base luciferase expression Translational Relevance plasmid (Promega; Supplementary Fig. S1; ref. 24). Three differ- In this study, we demonstrated that the suppression of ent deletion fragments of the cyclin E1 promoter (389 to þ747, cyclin E1 by sorafenib in hepatocellular carcinoma cells is 192 to þ747, and þ515 to þ747) were generated. Huh-7 cells correlated with its therapeutic efficacy. Adding of the pan-CDK were transfected with individual cyclin E1 reporter constructs and inhibitor flavopiridol can significantly improve the efficacy of cotransfected with pGL4.73 [hRluc/SV40], which constitutively sorafenib both in vitro and in vivo. The suppression of Mcl-1 expresses Renilla luciferase, to normalize transfection efficiency. was found to be a critical mediator for improved therapeutic The promoter activities with or without sorafenib treatment were efficacy. The results suggest the potential of using cell-cycle determined using a Dual-Luciferase Assay Kit (Promega). The inhibitors in combination therapy for hepatocellular binding activity of E2F1 to cyclin E1 promoter with or without carcinoma. sorafenib treatment was analyzed by ChIP assay.

Tumor xenograft experiments The protocol for the xenograft experiments was approved by the Materials and Methods Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University (Taipei, Taiwan). All the Cell culture animal studies were performed according to the criteria outlined The hepatocellular carcinoma cell lines tested in this study were in the Guide for the Care and Use of Laboratory Animals prepared HepG2, Hep3B, PLC-5, SK-Hep1, SNU398 (from the ATCC), and by the National Academy of Sciences and published by the NIH þ þ Huh-7 (from the Health Science Research Resources Bank). Sor- (Bethesda, MD). Male BALB/c athymic (nu /nu ) mice were afenib-resistant cell lines, Huh-7R and HepG2R, were generated inoculated subcutaneously with Huh-7 or Huh-7R cells. Sorafe- by continuous treatment of Huh-7 and HepG2 cells combined nib was administered daily by gavage and flavopiridol 3 times per m with sorafenib up to 10 mol/L to mimic the clinical phenom- week through intraperitoneal injection. Tumor volume and body enon of acquired resistance. No further authentication was con- weight were recorded every 7 days. At the end of drug treatment, ducted in our laboratory. tumor samples were collected for Western blotting, immunohistochemical analysis, and a terminal deoxynucleotidyl transferase– Chemicals and other reagents mediated dUTP nick end labeling (TUNEL) assay (25). The compounds used in this study were sorafenib (Bayer- Schering Pharma), U0126 (MEK kinase inhibitor, Calbiochem), Statistical analysis fl ZM336372 (Raf kinase inhibitor, Merck KGaA), and avopiridol All data were representative of at least three independent experi- (Selleck Chemicals). The antibodies used for Western blotting, ments. Quantitative data were expressed as mean SD. Compar- immunohistochemical staining, and chromatin immunoprecip- isons were analyzed using Student t test and ANOVA. Significance itation (ChIP) assays were cyclin E1 (BD Biosciences), Bcl-XL was defined as P < 0.05. Animal survival time was calculated using (R&D Systems), ERK 2, phospho-ERK 1/2, Rb, CDK2, CDK4, Bcl- the Kaplan–Meier method and compared using a log-rank test. 2, GAPDH, actin (Santa Cruz Biotechnology), E2F1, cyclin E2, phospho-cyclin E1, cyclin D1, phospho-cyclin D1, phospho-Rb, Results Mcl-1, Bcl-XL (Cell Signaling Technology), and CD31 (Abcam). Suppression of cyclin E1 expression in hepatocellular carcinoma cells by sorafenib correlated with sorafenib efficacy Cell viability, cell cycle, and apoptosis assays The extent of cyclin E1 suppression after sorafenib treatment Cell viability was assessed using an MTT [3-(4, 5-dimethylthia- correlated favorably with the sensitivity to sorafenib (Fig. 1A and zol-2-yl)-2, 5- diphenyltetrazolium bromide] assay The cell-cycle Supplementary Fig. S2A). However, the baseline levels of cyclin E1 distribution and fraction of apoptotic cells after drug treatment were not correlated with sorafenib sensitivity (Supplementary Fig. was assessed using flow cytometry, as previously described (7). S2B). The data suggested that the changes in cyclin E1 levels after The expression of apoptosis-related proteins after drug treatment sorafenib treatment may serve as a pharmacodynamic marker in was measured using Western blotting and the Human Apoptosis hepatocellular carcinoma. Sorafenib suppressed cyclin E1 mRNA Array kit (Proteome Profiler, R&D Systems). expression in sorafenib-sensitive hepatocellular carcinoma cells (Huh-7 and HepG2, IC50 5–6 mmol/L) but not in sorafenib- Modulation of cyclin E, Mcl-1, and E2F1 in vitro resistant cells (Huh-7R and HepG2R, IC50 14–15 mmol/L). The The overexpression or knockdown of cyclin E1, Mcl-1, or E2F1 effects on cyclins E2 and D1 mRNA expression did not correlate was performed through transient transfection or siRNA, respec- favorably with the sensitivity to sorafenib (Fig. 1B). Cyclin E1 tively. The primer/plasmid sequences are summarized in Supple- expression was not suppressed by U0126 or the Raf kinase fi mentary Table S1. The ef cacy of overexpression/knockdown was inhibitor ZM336372 in the sensitive hepatocellular carcinoma fi con rmed by performing qRT-PCR and Western blotting. The cells, supporting a Raf kinase–independent mechanism (Fig. 1B effects of cyclin E1 modulation on the sensitivity of hepatocellular and Supplementary Figs. S2A and S3). carcinoma cells to drug treatment were measured using the MTT To confirm the effects of cyclin E1 modulation on sorafenib assay and flow cytometry. sensitivity, the IC50 of sorafenib was measured after the knockdown or overexpression of cyclin E1. The knockdown of cyclin E1, Reporter assay of the cyclin E1 promoter activity but not cyclin E2, significantly increased the sensitivity to sor- Proximal promoter fragments of cyclin E1, spanning 389 to afenib in both sensitive and resistant hepatocellular carcinoma þ747 and containing 6 E2F-binding sites, were cloned upstream cells (Fig. 2A and Supplementary Fig. S4). Cyclin E1 knockdown

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Figure 1. Suppression of cyclin E1 by sorafenib correlated with sorafenib sensitivity in hepatocellular carcinoma cells. A, correlation between the IC50 of sorafenib and the expression of total and phosphorylated cyclin E1. IC50 was measured using the MTT assay after 72-hour sorafenib treatment. Cell lysates were collected after 24-hour treatment with 10 mmol/L sorafenib. Quantification of protein expression, using the ImageJ software (NIH, Bethesda, MD), was expressed as the ratio of sorafenib-treated/control cells. B, cyclin E1, E2, and D1 mRNA expression were assessed by performing real-time quantitative reverse transcription- polymerase chain reaction. The sorafenib-sensitive (Huh-7 and HepG2) and sorafenib-resistant (Huh-7R and HepG2R) hepatocellular carcinoma cells were treated with sorafenib or U0126 (selective MEK inhibitor) for 24 hours. enhanced the apoptosis-inducing effects of sorafenib in hepato- suppression of Mcl-1 expression (Fig. 2E). These data indicate that cellular carcinoma cells (Fig. 2B and Supplementary Fig. S5), cyclin E1 expression plays a crucial role in mediating antitumor and cyclin E1 overexpression was associated with reversal of efficacy of sorafenib in hepatocellular carcinoma cells. sorafenib-induced apoptosis in sorafenib-sensitive hepatocellular carcinoma cells (Fig. 2C). Cyclin E1 overexpression significantly Sorafenib inhibited cyclin E1 expression through increased viability in the sorafenib-sensitive hepatocellular carci- downregulation of E2F1 noma cells after sorafenib treatment (Fig. 2D). The apoptosis- To clarify the regulatory mechanisms of cyclin E1 expression by enhancing effects of cyclin E1 knockdown were associated with sorafenib, the expression of E2F1 and Rb proteins was measured

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Figure 2. Effects of cyclin E1 modulation on sorafenib sensitivity. A, knockdown of cyclin E1 (top), but not cyclin E2, increased the sensitivity of hepatocellular carcinoma cells to sorafenib, as indicated by the decrease in IC50 assessed using the MTT assay (bottom). Hepatocellular carcinoma cells were transfected with siRNA directed against cyclin E1 (si-CCNE1), cyclin E2 (si-CCNE2), or a negative control (si-NC) siRNA. The efﬁcacy of cyclin E1/E2 knockdown was measured by Western blotting. , P < 0.05 compared with si-NC. B, cyclin E1 knockdown enhanced the apoptosis-inducing effects of sorafenib in hepatocellular carcinoma cells. Hepatocellular carcinoma cells were transfected with si-CCNE1 or a negative-control siRNA and treated with sorafenib (10 mmol/L, S10) for 72 hours. , P < 0.05 compared with si-NC. C, cyclin E1 overexpression partially reversed the apoptosis-inducing effects of sorafenib in Huh-7 and HepG2 cells but had negligible effects on Huh-7R and HepG2R cells. Columns in B and C indicate the means of three independent experiments. Bars, SD. , P < 0.05; , P < 0.01, compared with Empty-S10. D, cyclin E1 overexpression increased sorafenib resistance in hepatocellular carcinoma cells (top). Huh-7 and Huh-7R cells were transfected with cyclin E1 or empty vectors and then treated with sorafenib 10 mmol/L or control. The IC50 of sorafenib in hepatocellular carcinoma cells after cyclin E1 overexpression were assessed using the MTT assay (bottom). , P < 0.05, compared with Empty. E, effects of cyclin E1 knockdown on apoptosis-related proteins in hepatocellular carcinoma cells. Hepatocellular carcinoma cells were transfected with si-CCNE1 or a negative control siRNA and treated with sorafenib (10 mmol/L, S10) for 48 hours and whole-cell lysates were subjected to Western blotting.

in sorafenib-sensitive and sorafenib-resistant hepatocellular car- efficacy of sorafenib can be improved by cell-cycle modulation. cinoma cells. Sorafenib suppressed E2F1 and Rb expression only Flavopiridol could suppress cyclin E1 and Rb protein levels in in sorafenib-sensitive hepatocellular carcinoma cells (Fig. 3A). hepatocellular carcinoma cells in a dose-dependent manner The changes in the levels of CDK2, the primary CDK activated by (Supplementary Fig. S7). Sorafenib-induced apoptosis could be cyclin E1, was not consistent with the sensitivity of hepatocellular significantly enhanced by the addition of flavopiridol in both carcinoma cells to sorafenib (Fig. 3A). Sorafenib inhibited the sorafenib-sensitive and sorafenib-resistant hepatocellular carci- luciferase activity of all the cyclin E1 promoter fragments, which noma cells (Fig. 4A and Supplementary Fig. S8). However, the contained the 6 E2F-binding sites previously reported (24), change in cell-cycle distribution after addition of flavopiridol did suggesting a transcriptional regulation on cyclin E1 expression not correlate with the antitumor synergy between the two agents (Fig. 3B). The association of E2F1 with the cyclin E1 promoter- (Supplementary Fig. S9). The apoptosis-enhancing effects of enhancer in hepatocellular carcinoma cells was confirmed using a flavopiridol were associated with the suppression of Mcl-1 expres- ChIP assay (Fig. 3C), and E2F1 knockdown could suppress the sion (Fig. 4B), consistent with findings derived using cyclin E1 expression of total and phospho-cyclin E1 in hepatocellular knockdown (Fig. 2E). Screening using an apoptosis array did not carcinoma cells (Supplementary Fig. S6). detect changes in other apoptosis-related proteins consistent with the antitumor enhancement (Supplementary Fig. S10). Mcl-1 Enhancement of the antitumor activity of sorafenib by overexpression reversed the apoptosis induced by sorafenib by flavopiridol flavopiridol (Fig. 4C), whereas Mcl-1 knockdown further The combination of sorafenib and the CDK inhibitor flavopir- enhanced the apoptosis induction (Fig. 4D and Supplementary idol was tested in vitro and in vivo to explore whether the antitumor Fig. S5). These data indicate that flavopiridol can enhance the

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Figure 3. Sorafenib inhibited cyclin E1 expression through downregulation of E2F1. A, sorafenib suppressed the expression of E2F1 and Rb proteins in sorafenib- sensitive (Huh-7 and HepG2) but not in sorafenib-resistant (Huh-7R and HepG2R) cells. Whole-cell lysate was collected after 24-hour treatment of the cells with sorafenib 10 mmol/L and was subjected to Western blot analysis. B, sorafenib- induced E2F1 suppression reduced cyclin E1 transcription. The 50-deletion constructs of the cyclin E1 promoter (389 to þ747 bp, containing six E2F- binding sites) were transfected into Huh-7 cells, and the relative luciferase activity of each promoter fragment after sorafenib treatment is shown on the right. , P < 0.01, compared with the control group. C, ChIP assay of E2F1 association with the cyclin E1 promoter-enhancer in Huh-7 and HepG2 cells. Negative control (Neg Con) was chromatin immunoprecipitated with a normal mouse IgG. Positive control (Pos Con) was chromatin immunoprecipitated with an anti-RNA polymerase II antibody. Input was 0.1% of the sonicated chromatin before immunoprecipitation.

antitumor efficacy of sorafenib and that cyclin E1 and Mcl-1 play tumor angiogenesis, however, were not obvious (Fig. 5C). The critical roles in mediating this antitumor enhancement. suppression of cyclin E1 and Mcl-1 was observed in tumor lysates, In vivo experiments also supported the enhancement of anti- consistent with in vitro study results (Fig. 5D and Supplementary tumor efficacy by flavopiridol, particularly in the sorafenib-resis- Fig. S12). Figure 6 depicts the potential effects of cell-cycle tant xenografts. The combination of sorafenib and flavopiridol regulation on sorafenib-induced apoptosis in hepatocellular significantly delayed tumor growth (Fig. 5A) and improved ani- carcinoma cells. mal survival (Fig. 5B), without significant increase in animal toxicity (Supplementary Fig. S11). Flavopiridol increased tumor cell apoptosis and decreased tumor cell proliferation induced by Discussion sorafenib treatment, which were also more prominent in the In this study, we demonstrated that cyclin E1 suppression by sorafenib-resistant xenografts. The effects of flavopiridol on sorafenib in hepatocellular carcinoma cells correlated with

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Figure 4. Synergistic antitumor activity between sorafenib and the CDK inhibitor flavopiridol. A, induction of apoptosis by sorafenib and/or flavopiridol, measured using flow cytometry (sub-G1 fraction analysis). B, effects of sorafenib and flavopiridol on apoptosis-related proteins in hepatocellular carcinoma cells. Hepatocellular carcinoma cells were treated with the drugs for 48 hours and whole-cell lysates were subjected to Western blotting. (Continued on the following page.)

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AB 1.0 1.0 Huh-7 xenograft model Huh-7R xenograft model )

3 4,000 Vehicle

) 1,400 Fla-3 3 Vehicle 0.8 3,000 S10 1,200 Fla-3 0.8 Fla-3 + S10 1,000 S10 Fla-3 + S10 2,000 800 Huh-7 Huh-7R 0.6 0.6 600 1,000 400 Vehicle Vehicle

Tumor volume (mm volume Tumor Flavopiridol Flavopiridol 200 Survival fraction Survival Sorafenib Sorafenib Tumor volume (mm volume Tumor 0.4 0.4 0 0 Sorafenib + Sorafenib + 0 5 10 15 20 25 30 35 40 0 510152025 30 35 40 flavopiridol flavopiridol Days of treatment Days of treatment Tx start Tx stop Tx start Tx stop 0.2 0.2 Huh-7 Huh-7R 200 40 60 80 200 40 60 80 Vehicle Fla-3 Vehicle Fla-3 Days after tumor cell implantation Days after tumor cell implantation Huh-7 Huh-7R

Group Vehicle Fla-3 S10 S10+Fla-3 S10 S10+Fla-3 S10 S10+Fla-3

CDHuh-7 Huh-7R Huh-7 Huh-7R 1.4 1.2 1.0 Mcl-1 0.8 14 14 0.6 12 12 0.4 10 10 0.2 8 8 ratio Mcl-1/actin 0.0 CD31 6 6 4 4

MVD (/HPF) 1.4

2 MVD (/HPF) 2 0 0 1.2 1.0 5 Bcl-XL 0.8 4 5 0.6 0.4 3 4 0.2 3 ratio Bcl-XL/actin 0.0 2 TUNEL 2 1.6 1.4 1 1 1.2 E2F1 % TUNEL (+) cells % 1.0 0 0 0.8 % TUNEL (+) cells % 0.6 40 0.4

E2F1/actin ratio 0.2 0.0 40 30 1.8 30 1.6 20 1.4 20 T-cyclin E1 Ki67 1.2 10 1.0 10 0.8

% Ki67 (+) cells 0.6 % Ki67 (+) cells 0 0 0.4 0.2 S10 S10 0.0

Fla-3 Fla-3 E1/actin ratio T-cyclin Vehicle Vehicle S10 S10 S10 + Fla-3 S10 + Fla-3 Fla-3 Fla-3 Vehicle Vehicle S10 + Fla-3 S10 + Fla-3

Figure 5. Flavopiridol enhanced the antitumor efficacy of sorafenib in vivo. Huh-7 or Huh-7R cells were injected subcutaneously into male BALB/c athymic nude mice. Mice were treated as indicated (V, vehicle; S10, sorafenib 10 mg/kg/d, Fla-3, flavopiridol 3 mg/kg/d three times a week). A, difference in tumor growth (n ¼ 5 in each group). B, difference in animal survival (n ¼ 10 in each group). C, changes in tumor microvessel density (CD31 staining; MVD), tumor cell apoptosis (TUNEL assay), and tumor cell proliferation (Ki67 staining) after drug treatment. , P < 0.05; , P < 0.01 derived using Student t test. Columns, means (n ¼ 5); bars, SD. D, changes in cyclin E1, E2F1, and apoptosis-related proteins in tumor lysates. Western blotting results were scanned and quantified using the ImageJ software (NIH, Bethesda, MD) and expressed as target/actin ratios. Tx, treatment. HPF, high power field; NC, negative control. therapeutic efficacy of sorafenib. Adding the pan-CDK inhibitor a critical mediator of the improved therapeutic efficacy. These flavopiridol can significantly improve the efficacy of sorafenib findings provide the rationale of design for combination therapy both in vitro and in vivo. The suppression of Mcl-1 was found to be for hepatocellular carcinoma.

(Continued.) C, Mcl-1 overexpression reversed apoptosis induced by sorafenib plus flavopiridol. Huh-7 and Huh-7R cells were transfected with pCMV6-Mcl-1-Myc- DDK (V-Mcl-1) or control vectors and then treated with control or sorafenib (10 mmol/L; Sor10) plus flavopiridol (0.1 mmol/L; Fla0.1) for 48 hours. Whole-cell lysates were collected for Western blotting. Apoptosis was measured using flow sub-G1 fraction analysis. D, Mcl-1 knockdown enhanced apoptosis induced by sorafenib plus flavopiridol. Huh-7 and Huh-7R cells were transfected with si-Mcl-1 or scrambled siRNA for 12 hours and then treated with sorafenib plus flavopiridol for 72 hours. Columns, means of three independent experiments; bars, SD. , P < 0.05; , P < 0.01.

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Figure 6. Schematic of the cell cycle–related proteins regulated by sorafenib in hepatocellular carcinoma cells. indicated activation, indicated inhibition, and indicated decrease.

Although the dysregulated cell-cycle control in cancer cells has pan-CDK inhibitors is the off-target adverse events. However, our been extensively studied, most clinical trials on single-agent data indicate that flavopiridol can enhance the antitumor efficacy treatment with CDK inhibitors or other cell-cycle regulators have of sorafenib in doses without significant single-agent effects on reported significant antitumor efficacy. One reason may be the Mcl-1 expression and tumor control. This finding raises the issue functional redundancy of cell-cycle control mechanisms, which of whether the single-agent antitumor activity of CDK inhibitors occur at the genetic, biologic, and biochemical levels (26). How- should be demonstrated to justify their use in combination ever, our finding that only cyclin E1 but not E2 contributes to treatment. Future clinical trials should clarify whether specificor sorafenib resistance suggests that individual cell-cycle regulators multitargeted CDK inhibitors can yield a more favorable thera- may still play specific functional roles. The suppression of cyclin peutic index in clinics. E1 may serve as a pharmacodynamic marker of sorafenib efficacy There are several challenges to incorporating cell-cycle regula- in hepatocellular carcinoma, and further study on cyclin E1 tors in systemic therapy for hepatocellular carcinoma. The first is expression in hepatocellular carcinoma tumor tissues prospec- to identify biomarkers to predict treatment response. It was tively collected from patients before and after sorafenib treatment hypothesized that CDK inhibitors and other cell-cycle regulators would help validate our findings. may be of greatest benefit in cancer patients with known aberra- Results from our study and other investigators support Mcl-1 as a tions in cell-cycle control, such as cyclin D amplification or p16 key mediator of cell survival and drug resistance in hepatocellular loss (34). However, results from the palbociclib trial for breast carcinoma (5, 6, 27, 28). Mcl-1 may be suppressed by sorafenib cancer did not support this hypothesis (15). Therefore, although through downregulation of E2F1, which also suppress cyclin E1 aberrations in cell-cycle control in hepatocellular carcinoma have expression in hepatocellular carcinoma cells. Our data indicate that been repeatedly reported, whether these aberrations can serve as regulation of the E2F1–Rb–cyclin E1 complex may play crucial markers for patient enrichment remains to be tested (35–38). The roles in mediating sorafenib resistance in hepatocellular carcinoma second challenge involves watching for treatment-induced mye- cells. Mcl-1 expression is also regulated by multiple signaling losuppression. Although the myelosuppression reported in recent pathways, including MAPK, JAK/STAT, and PI3K/AKT, in cancer clinical trials on CDK inhibitors has generally been well tolerated, cells (29, 30). Therefore, Mcl-1 expression can serve as a pharma- it may pose an increased risk of complications in hepatocellular codynamic marker of different types of combination therapy. carcinoma patients because of the patients' underlying cirrhosis Recent developments of CDK inhibitors for cancer treatment and hypersplenism. Therefore, identifying the biologic effective have focused on selective CDK4/6 inhibitors including palboci- dose in future clinical trials on hepatocellular carcinoma is critical, clib, abemaciclib, and ribociclib. Although single-agent activity especially in trials on combination therapy. has been revealed in various cancer types, their major values In conclusion, cyclin E1 suppression contributes to sorafenib- appear to be in combination therapy to enhance efficacy of other induced apoptosis in hepatocellular carcinoma cells. Future stud- systemic therapies (31). Our data support the use of CDK inhi- ies are necessary to validate its value in predicting sorafenib bitors in combination therapy for hepatocellular carcinoma. Pan- efficacy. CDK inhibitors such as flavopiridol or dinaciclib may have better antitumor activity through both cell-cycle regulation and the Disclosure of Potential Conflicts of Interest suppression of the expression of antiapoptotic proteins, including C. Hsu reports receiving speakers bureau honoraria from Bayer-Schering Mcl-1, Bcl-XL, and XIAP (32, 33). A potential concern about using Pharma. A.-L. Cheng is consultant/advisory board member for and reports

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receiving speakers bureau honoraria from Bayer-Schering Pharma. No potential The authors thank the Liver Disease Prevention and Treatment Foundation, conﬂicts of interest were disclosed by the other authors. Taiwan, for logistical support.

Authors' Contributions Grant Support This work is supported by grants NSC 101-2325-B-002-039, NSC 102- Conception and design: C. Hsu, D.-L. Ou 2325-B-002-038, NSC 100-2314-B-002-058-MY3, NSC 102-2314-B-002- Development of methodology: C. Hsu, L.-I. Lin, Y.-C. Cheng, Z.R. Feng, Y.-Y. 142-MY3, and NSC 101-2321-B-002-014 from the National Science Council; Shao, D.-L. Ou MOST 103-2314-B-002-112-MY3 from the Ministry of Science and Tech- Acquisition of data (provided animals, acquired and managed patients, nology; and NHRI-EX102-9911BC from the National Health Research provided facilities, etc.): C. Hsu, Y.-C. Cheng, Z.-R. Feng Institutes. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the computational analysis): C. Hsu, L.-I. Lin, Y.-C. Cheng, Y.-Y. Shao, D.-L. Ou payment of page charges. This article must therefore be hereby marked Writing, review, and/or revision of the manuscript: C. Hsu, D.-L. Ou advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Administrative, technical, or material support (i.e., reporting or organizing this fact. data, constructing databases): C. Hsu, L.-I. Lin, A.-L. Cheng Study supervision: C. Hsu, D.-L. Ou, A.-L. Cheng

Received March 3, 2015; revised October 2, 2015; accepted October 27, 2015; Acknowledgments published OnlineFirst November 24, 2015.

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OF10 Clin Cancer Res; 2015 Clinical Cancer Research

Cyclin E1 Inhibition can Overcome Sorafenib Resistance in Hepatocellular Carcinoma Cells Through Mcl-1 Suppression

Chiun Hsu, Liang-In Lin, Yu-Che Cheng, et al.

Clin Cancer Res Published OnlineFirst November 24, 2015.

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