Original Article Cabazitaxel, a Novel Chemotherapeutic Alternative for Drug-Resistant Hepatocellular Carcinoma

Am J Cancer Res 2018;8(7):1297-1306 www.ajcr.us /ISSN:2156-6976/ajcr0077675

Original Article Cabazitaxel, a novel chemotherapeutic alternative for drug-resistant hepatocellular carcinoma

Ronggao Chen1,2, Qiyang Cheng1,2, Kwabena Gyabaah Owusu-Ansah1,2, Jun Chen1,2, Guangyuan Song1,2, Haiyang Xie1,2,3,4,5, Lin Zhou1,2,3,4,5, Xiao Xu1,2,3,4,5, Donghai Jiang1,2,3,4,5, Shusen Zheng1,2,3,4,5

1Department of Surgery, Division of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310000, Zhejiang Province, China; 2NHFPC Key Laboratory of Combined Multi-Organ Transplantation, Hangzhou 310000, Zhejiang Province, China; 3Key Laboratory of The Diagnosis and Treatment of Organ Transplantation, CAMS; 4Key Laboratory of Organ Transplantation, Hangzhou 310003, Zhejiang Province, China; 5Collaborative Innovation Center for Diagnosis Treatment of Infectious Diseases, Hangzhou 310000, Zhejiang Province, China Received April 10, 2018; Accepted June 29, 2018; Epub July 1, 2018; Published July 15, 2018

Abstract: The prognosis of advanced hepatocellular carcinoma (HCC) patients remains extremely poor, partially due to the development of acquired resistance to sorafenib and chemotherapy. Cabazitaxel, a semisynthetic taxane, has been approved for the therapy of docetaxel-resistant prostate cancer. However, no studies have been performed on the effect of cabazitaxel on HCC, and whether cabazitaxel remains sensitive in chemotherapy-resistant and sorafenib-resistant HCC cells is not clear. Our results demonstrate that cabazitaxel is highly toxic to HCC cell lines in a time- and dose-dependent manner by inducing G2/M phase arrest and apoptosis in vitro. Cabazitaxel also significantly suppresses HCC tumor growth in vivo. In chemotherapy-resistant HCC cell Huh-TS-48 with P-gp- overexpression, cabazitaxel shows less cross-resistant to other chemotherapeutic agents. The resistance fold of cabazitaxel, doxorubicin, paclitaxel, docetaxel and vinorelbine is 1.53, 8.60, 38.58, 15.53 and 18.06 respectively. Furthermore, sorafenib-resistant HCC cell SK-sora-5 is still sensitive to cabazitaxel. The IC50 values of cabazitaxel after 72 h exposure for parental cell SK-hep-1 and resistant cell SK-sora-5 are 0.84 and 0.73 nM. The results indicate that cabazitaxel is a potential agent to treat HCC after developing chemotherapy resistance caused by overexpression of P-gp and acquired resistance to sorafenib, and might improve prognosis in advanced HCC patients.

Keywords: Hepatocellular carcinoma, cabazitaxel, acquired resistance, sorafenib, P-gp

Introduction the response rate is quite low, and the mean prolongation of survival is modest at about 2-3 Globally, hepatocellular carcinoma (HCC), which months. Another limit to the efficacy of this accounts for 90% of primary liver cancers, is medication is the acquired resistance. Although the fifth most common malignancy and the almost 40% of HCC tumors are initially con- third leading cause of cancer-related mortality trolled by sorafenib treatment, these patients [1]. Curative treatments, such as surgical re- acquire resistance over time and experience section and liver transplantation, are suitable disease progression. Currently, there is no for early HCC. Nevertheless, the majority of existing second-line therapy for patients who HCC patients present with advanced stage at relapse following sorafenib therapy in clinic. diagnosis, making chemotherapy and targeted Systematic chemotherapy has been applied for agents the best option for controlling tumor over 30 years, but still lacks definite evidence progression in such patients [2-5]. of improving prognosis [6]. Traditional therapeutic agents including doxorubicin, cisplatin Sorafenib, a multi-kinase inhibitor, is the only and 5-FU have been limited by resistance, sys- approved systemic treatment that prolongs temic toxicity and poor efficacy [7-9]. MDR1 overall survival in select advanced HCC pati- (encoding P-gp) is one of the most vital reasons ents [4, 5]. Despite an evident survival benefit, which cause multidrug resistance and leads to Novel chemotherapy for hepatocellular carcinoma failure in HCC chemotherapy [10-13]. Partially and acquired resistance to sorafenib, and mig- due to the development of resistance to ht improve prognosis in advanced HCC patients. sorafenib and chemotherapy, the prognosis of advanced HCC patients remains extremely Materials and methods poor. This creates an unmet medical need for Cell culture and mice alternative therapies for advanced HCC. Novel therapeutic agents are urgently required to All cells were purchased from China Center for improve the situation. Type Culture Collection (CCTCC). Human hepatocellular carcinoma cell lines SK-hep-1, SM- Cabazitaxel, a semisynthetic taxane, has been MC7721, Huh-7, HCC-LM3, Huh-TS-48 and SK- approved by the American Food and Drug sora-5 were maintained in minimum essential Agency (FDA) and European Medicines Agency medium (Gibco-Invitrogen, USA) supplemented for therapy of castration-resistant prostate can- with 10% fetal bovine serum at 37°C in a hu- cer with disease progression after docetaxel midified incubator with 5%2 CO . Female aged treatment [14, 15]. The TROPIC trial demon- 4-5 weeks of athymic nude (nu/nu) mice were strated that cabazitaxel offers an overall sur- purchased from Shanghai SLAC animal facility. vival benefit of 2.4 months over mitoxantrone All animals care and experiments were con- in such patients. Besides prostate cancer, clini- ducted according to the Guidelines of Zhejiang cal efficacy has been reported in metastatic University Animal Care Committee. breast cancer patients resistant to former tax- anes [16, 17]. The antitumor activity spectrum Antitumor activity in tumor-bearing mice of cabazitaxel in mice bearing human xenografts is broad [18, 19]. Same with other tax- HCC-LM3 cells (4 × 106 cells in 0.1 mL PBS) anes, cabazitaxel exerts cytotoxic effect throu- were implanted into the right flanks of the gh mitotic arrest by stabilizing microtubules, homozygous nude athymic mice (female, 5-6- which leads to cell death [20]. Compared with weeks old). Three days after implantation, xe- paclitaxel and docetaxel, cabazitaxel possess- nografts were randomly divided into 2 groups es a relatively low affinity for P-gp, which may and treated with different regimens: (i) vehicle; offer potential for a wider antitumor spectrum (ii) cabazitaxel at 10 mg/kg, i.v. The same treat- and more durable response. Although cabazi- ment regimens were repeated every 6 days for taxel has been used clinically in different can- a total of 4 cycles. Two perpendicular diameters cers, its effect on HCC has not been researched (width and length) of the tumors were mea- before. Whether cabazitaxel remains sensitive sured every 3 days until the animals were termi- in chemotherapy-resistant and sorafenib-resis- nated. After the animals were killed, the tumor tant HCC cells is still unclear. tissues were removed and weighted. Caba- zitaxel solution (1 mg/ml) was prepared by mix- Herein, we demonstrate that cabazitaxel is ing 1 volume of DMSO, 1 volume of polysorbate highly toxic to HCC cell lines in a time- and 80, and 38 volumes of sterile water. dose-dependent manner by inducing G2/M Establishment of drug resistant cell lines phase arrest and apoptosis in vitro. Cabazita- xel substantially inhibits HCC tumor growth Huh-TS-48 cells were produced based on a in vivo as well. To investigate whether chemo- strategy of pulsed exposure to paclitaxel with therapy-resistant and sorafenib-resistant HCC time-stepwise increments. Briefly, Huh-7 cells cells are sensitive to cabazitaxel, chemothera- were exposed to 50 nM paclitaxel in time incre- py-resistant HCC cell line Huh-TS-48 with P-gp ments ranging from 0.5 h to 24 h (0.5, 1, 2, 4, overexpression and sorafenib-resistant HCC 12, 24 h) in the adaptation stage. The surviving cell line SK-sora-5 were established. Cabazi- cells were amplified in paclitaxel free medium. taxel is much less cross-resistant in Huh-TS- Each pulse treatment was repeated 3 times, 48 than doxorubicin, paclitaxel, docetaxel and followed by exposure to the next longer time- vinorelbine. Furthermore, SK-sora-5 is still sen- course. In the consolidation stage, previously sitive to cabazitaxel. These results indicate th- selected cells were exposed to ten pulses of at cabazitaxel offers a potential approach for treatment with 50 nM paclitaxel for 48 h. The treating HCC after experiencing chemotherapy resulting cell line was named Huh-TS-48 and resistance caused by overexpression of P-gp was maintained in paclitaxel-free medium.

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SK-sora-5 cells were selected based on con- and DNA content were determined using a tinuous exposure to sorafenib using a dose- Coulter Epics V instrument (Beckman Coulter, stepwise incremental strategy from SK-hep-1. Inc., Fullerton, CA). In the adaptation stage, SK-hep-1 cells were exposed to sorafenib for 72 h in stepwise in- Analysis of apoptosis crements of concentrations ranging from 1 μM Annexin V/PI apoptosis detection kit (Beyotime, to 2 μM (1, 2 μM). Following each dose-induced Haimen, China) was used to detect cell apopto- step, surviving cells were amplified in sorafenib- sis according to the manufacturer’s instruc- free medium. After repeating 3 times (for each tions. Briefly, cells were harvested and washed dose), cells were exposed to the next higher twice with PBS after treatment for 48 h. Then concentration of sorafenib. In the consolidation the cells were suspended with 400 μL of bind- stage, previously selected cells were continu- ing buffer, 5 μL of Annexin V antibody conjugat- ously cultured in medium containing 5 μM ed with fluorescein isothiocyanate (FITC) and 5 sorafenib, until they multiplied normally in me- μL of PI solution. After being incubated in the dium containing 5 μM sorafenib. The resulting mixture for 15 min at room temperature in the resistant cell line (SK-sora-5) was maintained dark, the percentage of apoptotic cells was de- in medium containing 5 μM sorafenib. termined by flow cytometry.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra- Western blotting zolium bromide assay (MTT assay) Equal amounts (30 μg/lane) of proteins were Cells were harvested and resuspended to a fractionated on 12% SDS-PAGE gels and trans- final concentration of 10^4 cells/ml. Aliquots of ferred to polyvinylidene difluoride membranes. the cell suspension were evenly distributed into The membranes were incubated with anti-Cdc- 96-well tissue culture plates. After one night of 25c, anti-Cdc2, anti-pCdc2, anti-cyclin B1, anti- incubation, the designated columns were treat- Bcl2, anti-PARP and anti-β-actin primary anti- ed with drug regimes. Four hours prior to the bodies (Cell Signaling Technology, Danvers, end of time point, MTT solution was added. The MA, USA), respectively. After washing with TBS medium containing MTT was replaced with 150 containing 0.1% (v/v) Tween 20, the membr- μL of DMSO in each well to dissolve the forma- anes were incubated with peroxidase-conjugat- zan crystals after 4 h-incubation. The absor- ed goat anti-mouse or rabbit secondary anti- bance in individual wells was determined at bodies. The western blot bands were visualized 570 nM using a microplate reader (Bio-Rad, using ECL kits in accordance with the manufac- Sunnyvale, CA). turer’s instructions (Abcam, Cambridge, MA, USA). β-actin was used for normalization of pro- Colony-forming assay tein loading. SK-hep-1, Huh-7 cells were seeded into 6-well Statistical analysis plates in triplicates at a density of 500 cells/ well and were maintained with or without caba- Data are presented as mean ± standard error zitaxel for 10 days. SK-hep-1 and SK-sora-5 of three independent experiments. Two-sided cells were seeded into 6-well plates in tripli- Student’s t-test was used to determine the sta- cates at a density of 500 cells/well, and were tistical difference between various experimen- maintained with or without sorafenib for 12 tal and control groups. Differences were con- days. The cell clones were stained with Giemsa. sidered statistically significant at a level of P < 0.05. Statistical analysis of the data presented Analysis of cell cycle was performed by SPSS version 22.0 (SPSS, Cell cycle distribution was determined by flow Chicago, IL, USA). cytometric analysis. Briefly, after drug exposure Results for 48 h, both detached and attached cells were harvested and washed twice with PBS, Cabazitaxel shows high cytotoxic effects on followed by fixation in 75% ethanol diluted in HCC cells PBS. Cells were then incubated in PBS containing 100 μL/mL RNase and 40 μL/mL propidium To test the cytotoxic effects of cabazitaxel on iodide (PI) at room temperature for 0.5 h before HCC cells, we examined the sensitivities of four flow cytometric analysis. Cell cycle distribution HCC cell lines (SK-hep-1, SMMC7721, Huh-7,

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ited HCC cell proliferation in vitro. Those findings had raised a clinically relevant concern about whether this phenomenon would also oc- cur in vivo. As shown in Figure 2A-C, after four treatment cycles, the tumors of cabazitaxel treated mice were significantly smaller than those of the control group. The mean tumor volumes of control and cabazitaxel group were 539.9 ± 85.2, 121.7 ± 29.3 mm3 (n = 6, p < 0.01), and mean tumor weights were 0.29 ± 0.06, 0.06 ± 0.03 g (n = 6, p < 0.01). Furthermore, removed HCC-LM3 xenograft tumors were tested by immu- nohistochemistry staining with Ki-67. As shown in Figure 2D, Ki-67-positive cells significantly reduced in the cabazitaxel group, compared with the control group. Taken together, these results indi- Figure 1. Effect of cabazitaxel on HCC cell lines in vitro. A. HCC cells were cated that cabazitaxel treat- treated with increasing doses of cabazitaxel (0-50 nM) for 12 h, 24 h, 48 h, ment could significantly inhib- 72 h. Then, HCC cells proliferation was assessed by the MTT assay. B. SK- hep-1 and Huh-7 cells’ colony formation with or without 5 nM cabazitaxel it HCC tumor proliferation in treatment. C. The IC50s of cabazitaxel after 24 h, 48 h and 72 h treatment vivo. were determined for each cell line; Data are shown as mean ± SD. CTL, control; CBT, cabazitaxel. Cabazitaxel triggers G2/M cell cycle arrest in HCC cells

HCC-LM3) to cabazitaxel using MTT assay. As The effect of cabazitaxel on cell cycle arrest shown in Figure 1A, cabazitaxel treatment sub- in SK-hep-1 and Huh-7 cells was studied. As stantially inhibited HCC cell proliferation in a shown in Figure 3A, when treated with 5 nM time- and dose-dependent manner. Colony for- cabazitaxel for 48 hours, the proportion of mation assays demonstrated that when treat- cells at G2-M phase significantly increased ed with 5 nM cabazitaxel, SK-hep-1 and Huh-7 both in SK-hep-1 and Huh-7 from 5.14% to cells both showed fewer and smaller colonies 57.77% and from 7.71% to 55.54%, respective- than the control group (Figure 1B). The IC50s of ly. Furthermore, we conducted a detailed ana- cabazitaxel after 24 h, 48 h and 72 h treatment lysis of several regulatory proteins associated were determined for each cell line (Figure 1C). with cabazitaxel-induced mitotic arrest. As de- IC50 values of 72 h cabazitaxel exposure for picted in Figure 3B, treatment of cabazitaxel four HCC cell lines were all below 5 nM, ranging resulted in a decrease in Cdc25c, Cdc2, pCdc2 from 0.84 to 4.52 nM. Taken together, we dem- and cyclin B1 protein levels. In a word, our data onstrate that cabazitaxel is effective in inhibit- support that cabazitaxel induces G2/M phase ing cell viability of various HCC cell lines at arrest in HCC cells via Cdc25C/Cdc2/cyclin B1 nanomolar concentrations. pathway.

Cabazitaxel suppresses HCC tumor growth in Cabazitaxel induces apoptosis in HCC cells mouse xenograft models Annexin V and propidium iodide staining were The results presented above demonstrated used to assess apoptosis. The results indicat- that cabazitaxel treatment substantially inhib- ed that in comparison with the control group,

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the ratio of apoptotic cells statistically increased when exposed to 5 nM cabazitaxel for 48 h in SK-hep-1 and Huh- 7 (Figure 4A), increasing from 3.6% to 21% and from 4.7% to 20.3%, respectively. Then, we analyzed the molecules likely to be involved in cabazitaxel-induced apoptosis. As shown in Figure 4B, exposure to cabazitaxel led to a decli- ne in anti-apoptotic protein Bcl2 and a stronger cleavage of poly ADP-ribose polymera- Figure 2. Effect of cabazitaxel on HCC tumor growth in vivo. The tumor volume (A) and tumor weight at the end of experiments (B) were compared in se (PARP). Taken together, ca- control and cabazitaxel group. (C) The tumor mass with or without cabazi- bazitaxel promotes apopto- taxel treatment. Data are shown as mean ± SD. **P < 0.01 vs. control group. sis in HCC cells through the Ki-67 immunohistochemistric staining (D, × 200) were performed. CTL, con- Bcl2/PARP pathway. trol; CBT, cabazitaxel. Cabazitaxel is much less cross-resistant in P-gp- overexpressed HCC cell

Elevated levels of P-gp plays a major role in acquired resistance to paclitaxel in HCC [21]. To explore whether cabazitaxel remains sensitive in P-gp-overexpressed HCC cell, we selected Huh-TS-48 based on pulsed exposure to paclitaxel with a time-stepwise increments strategy to build a P-gp-overexpressed HCC cell model. Compared to parent cell Huh-7, overexpression of P-gp was confirmed in Huh- TS-48 by western blot (Figure 5A). MTT assay showed cross-resistant of Huh-TS-48 to doxorubicin, paclitaxel, docetaxel and vinorelbine, all known to have a high affinity for P-gp (Table 1). The resistance fold was 8.60, 38.58, 15.53 and 18.06 respectively. Next, we tested the effect of cabazitaxel on Huh-7 and Huh- TS-48 (Figure 5B, 5C). The re- Figure 3. Cell cycle analysis of cabazitaxel-treated cells. A. Cabazitaxel treat- sults showed that the resis- ment caused G2/M arrest. SK-hep-1 and Huh-7 were treated with or without tance fold of cabazitaxel is 5 nM cabazitaxel for 48 h. B. Western blot analysis of Cdc25c, Cdc2, pCdc2 and Cyclin B1 proteins after cabazitaxel treatment. Data are shown as mean 1.53 (Table 1), which is sig- ± SD. *P < 0.05 vs. control group; **P < 0.01 vs. control group. CTL, control; nificantly smaller than the for- CBT, cabazitaxel. mer four drugs (Figure 5D).

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sorafenib using MTT assays. As shown in Figure 6B, the IC50 values of 72 h sorafenib exposure for SK-hep-1 and SK-sora-5 were 3.86 and 8.12 μM (P < 0.01). These two experiments validated that SK-sora-5 is resistant to sorafenib, compared with SK- hep-1. Next, we examined the effect of cabazitaxel on SK-hep-1 and SK-sora-5. As depicted in Figure 6C, the IC50 values of 72 h cabazitaxel exposure for SK-hep-1 and SK-sora-5 were 0.84 and 0.73 nM. Taken together, these results demonstrate that cabazitaxel is still sensitive in HCC cell resistant to sorafenib.

Discussion

HCC is a major global heal- Figure 4. Effect of cabazitaxel on HCC cells apoptosis. A. Cabazitaxel pro- th issue, especially in China, motes apoptosis in HCC cells. SK-hep-1 and Huh-7 were treated with or with little improvement in pro- without 5 nM cabazitaxel for 48 h. FITC, Annexin V/PI staining was used to gnosis during the past few identify apoptosis. The data are shown as mean ± S.D. from three experi- decades. Hence, novel thera- ments. *P < 0.05 vs. control group; **P < 0.01 vs. control group. B. Bcl2, peutic agents are urgently re- PARP and c-PARP proteins were measured by Western blot. CTL, control; CBT, quired. Herein, we offer the cabazitaxel. most comprehensive preclinical assessment of a semisyn- Taken together, we demonstrated that cabazi- thetic taxane, cabazitaxel, in HCC. Cabazitaxel taxel is much less cross-resistant in P-gp-over- shows high toxicity in HCC cell lines at nano- expressed HCC cell compared to doxorubicin, molar concentrations by inducing G2/M phase paclitaxel, docetaxel and vinorelbine. arrest and apoptosis in vitro, and significantly suppresses HCC tumor growth in vivo. More- Cabazitaxel is sensitive in sorafenib-resistant over, cabazitaxel is much less cross-resistant HCC cell in chemotherapy-resistant HCC cell, and rema- Acquired resistance limits the efficacy of sora- ins sensitive to sorafenib-resistant HCC cell. fenib treatment in clinic. To test whether cabazitaxel is still sensitive in sorafenib-resistant Cabazitaxel is a second-generation semisynth- HCC cell, we selected SK-sora-5 based on con- etic taxane approved by American FDA for the tinuous exposure to sorafenib using a dose- therapy of patients with metastatic castration- stepwise incremental strategy to establish a resistant prostate cancer with disease progres- sorafenib-resistant HCC cell model. Colony for- sion after docetaxel-containing treatment [14]. mation assays demonstrated that while the In addition, clinical trials of cabazitaxel conta- number and size of colonies are comparable ined phase I/II studies in patients with various between SK-hep-1 and SK-sora-5 in the control advanced solid tumors, including metastatic group, SK-hep-1 showed much fewer and small- breast cancer, lung cancer and colorectal can- er colonies than SK-sora-5 when treated with 5 cer, in which partial and complete responses μM sorafenib (Figure 6A). We then examined were observed [16, 22-24]. Besides, cabazitax- the sensitivities of SK-hep-1 and SK-sora-5 to el showed antitumor activity in animals bear-

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peutic agents in SK-hep-1 and Huh-7 cell, thus may avoid certain dose-related toxic effects and improve patient tol- erance in clinic. Besides, we further confirmed that cabazitaxel treatment could gener- ate anti-cancer effects on HCC in vivo. Mechanistically, cabazitaxel induced HCC cell G2/M phase arrest via Cdc- 25C/Cdc2/cyclin B1 pathway and apoptosis through the bcl2/PARP pathway, consis- tent with its effect on other cancer cells [20].

Previously, chemotherapy was not routinely used in terminal stage HCC, as it was consid- Figure 5. Effect of cabazitaxel on P-gp-overexpressed HCC cell. A. P-gp was ered chemo-refractory and measured by Western blot in Huh-7 and Huh-TS-48. B. Huh-7 and Huh-TS-48 were treated with increasing doses of cabazitaxel (0-5 nM) for 72 h. Then, because of adverse events cell proliferation was assessed by the MTT assay. C. Resistance fold was (AEs) [26]. Numerous studies determined by dividing IC50 values of Huh-TS-48 by that of Huh-7 cells. D. have reported low response Resistance fold of cabazitaxel, doxorubicin, paclitaxel, docetaxel and vinorel- rates of single or combined bine was calculated. The data are shown as mean ± S.D. from three experi- chemotherapeutic agents in ments, *P < 0.05 vs. cabazitaxel, **P < 0.01 vs. cabazitaxel. CBT, cabazitaxel; DOX, doxorubicin; DTX, docetaxel; VB, vinorelbine; PTX, paclitaxel. HCC. Though when compared with best supportive care, doxorubicin was initially shown Table 1. Drug sensitivity of Huh-7 and Huh-TS-48 to improve survival [27], two Huh-TS-48 subsequent clinic trials show- Drug (72 h) Huh-7 IC50 (nM) IC50 (nM) RF ed a low response rate when Doxorubicin 299.67 ± 44.81 2578.33 ± 301.22 8.60 used as monotherapy or combination regimens [7]. The Paclitaxel 11.70 ± 3.04 451.33 ± 36.47 38.58 combination regimens PIAF Docetaxel 5.67 ± 0.96 88.05 ± 14.71 15.53 (cisplatin, interferon, doxoru- Vinorelbine 8.92 ± 2.22 161.09 ± 16.36 18.06 bicin and 5-fluorouracil) sh- Cabazitaxel 2.69 ± 0.38 4.11 ± 0.53 1.53 owed positive outcomes with Note: The IC50s of five chemotherapeutic agents after 72 h treatment were deter- a median overall survival of mined for Huh-7 and Huh-TS-48. Resistance fold (RF) was determined by dividing IC50 values of Huh-TS-48 by that of Huh-7 cells. 8.9 month [28]. However, a subsequent study comparing PIAF with doxorubicin failed ing glioblastoma xenografts [25]. However, the to meet the primary endpoint (OS: 8.6 mo vs. effect of cabazitaxel on HCC has not been pre- 6.8 mo, P = 0.83), with no survival benefit [29]. viously researched. To our best knowledge, this To date, chemotherapy (monotherapy or combi- is the only study to demonstrate the cytotoxic nation regimens) has been researched in abun- effect of cabazitaxel on HCC in vitro and in vivo. dant clinical trials in HCC, but no significant Our results revealed that cabazitaxel treatment persuasive efficacy in prolonging survival has inhibits HCC cell proliferation in a time- and been shown. Resistance is one of the major dose-dependent manner. Cabazitaxel at nano- reasons leading to chemotherapy failure in molar concentrations was sufficiently enough HCC. The mechanisms of resistance include to affect HCC cells in MTT and colony formation drug transport and metabolism, alterations in assays. As shown in Table 1, cabazitaxel had drug targets, DNA damage repair, activation of dose advantage over the other chemothera- prosurvival pathways, ineffective induction of

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Sorafenib was the first approved targeted agent which demonstrated survival bene- fits in advanced HCC patients. Two landmark phase III trials (SHARP and the Asia- Pacific studies) showed sorafenib as a prominent progre- ss in the treatment of terminal HCC with prolonged overall survival (10.7 mo vs. 7.9 mo, 6.5 mo vs. 4.2 mo) [4, 5]. Despite initial response to sorafenib, acquired resistance has been shown to limit the efficacy. Currently, no alternative second-line drugs are available after the failure of sorafenib therapy. There- fore, seeking for substitute therapy after sorafenib resistance is promising and worth- Figure 6. Effect of cabazitaxel on sorafenib-resistant HCC cell. A. SK-hep-1 while. In our study, sorafenib- and SK-sora-5 cells’ colony formation with or without 5μM sorafenib treat- resistant HCC cell SK-sora-5 ment. B. SK-hep-1 and SK-sora-5 were treated with increasing doses of was successfully established sorafenib (0-10 μM) for 72 h. Then, cell proliferation was assessed by the to imitate the setbacks in MTT assay. The IC50s of sorafenib after 72 h treatment were determined, Data are shown as mean ± S.D, *P < 0.05 vs. SK-hep-1, **P < 0.01 vs. targeted therapies in HCC. SK-hep-1. C. SK-hep-1 and SK-sora-5 were treated with increasing doses of SK-sora-5 became resistant cabazitaxel (0-50 nM) for 72 h. Then, cell proliferation was assessed by the to sorafenib as constantly MTT assay. The IC50s of cabazitaxel after 72 h treatment were determined, cultured in drug-contained Data are shown as mean ± S.D. **P < 0.01 vs. SK-hep-1, N.S vs. SK-hep-1. media, mimicking initial res- CTL, control; sora, sorafenib. ponse and subsequent resistance to sorafenib therapy of cell death and etc. MDR1 (encoding P-gp) is HCC in clinic. MTT and colony formation assays one of the most vital reasons which cause mul- show that SK-sora-5 remains sensitive to ca- tidrug resistance and lead to chemotherapy bazitaxel. Our data demonstrated that cabazi- failure in HCC [9, 10]. The expression of P-gp is taxel offers a potential approach for HCC with found in 80-90% of HCC patients and has been acquired resistance to sorafenib. Hence, com- shown to negatively correlate with response to bination or sequential therapy of cabazitaxel chemotherapy in one study [11]. In another and sorafenib may be a novel effective regi- study, P-gp expression is significantly higher men to treat advanced HCC. The mechanistic both in HCC neoplastic and perineoplastic tis- basis of acquired sorafenib resistance will be sues, compared with cirrhotic and cholestatic explored in further study. liver tissues [12]. In this study, chemotherapy- resistant HCC cell line Huh-TS-48 with P-gp Our study has two important implications. First, overexpression was successfully established to as far as we know, it is the only study demon- mimic the obstacles in chemotherapy of HCC strating the effect of cabazitaxel on HCC both in clinic. Huh-TS-48 exhibited a phenotype of in vitro and in vivo. Second, we tested the effe- MDR after continuous exposure to paclitaxel, ct of cabazitaxel on two novel drug-resistant as HCC is mostly chemotherapy-resistant and cell models, which were built to imitate the set- known to have innate and acquired MDR [30]. backs in the systematic therapy of advanced MTT assays show that cabazitaxel is much less HCC in clinic. The results indicated that cabazi- cross-resistant in Huh-TS-48, indicating cabazitaxel is a potential agent to treat HCC after taxel as an effective agent for HCC with multi- experiencing chemotherapy resistance caused drug resistance. by overexpression of P-gp and acquired resis-

1304 Am J Cancer Res 2018;8(7):1297-1306 Novel chemotherapy for hepatocellular carcinoma tance to sorafenib, and might improve progno- Jean-François S, Ivan B, Dieter H, Tom G, Ming- sis in advanced HCC patients. Nevertheless, hua S, Marius M, Dimitris V, and Jordi B. Sora- additional studies involving HCC patients and fenib in advanced hepatocellular carcinoma. N patient-derived xenograft models will be need- Engl J Med 2008; 359: 378-90. ed to refine response prediction of cabazitaxel [5] Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, and optimize selection in clinic. Further studies Kim JS, Luo R, Feng J, Ye S, Yang TS, Xu J, Sun Y, Liang H, Liu J, Wang J, Tak WY, Pan H, Burock are needed to identify the molecular basis of K, Zou J, Voliotis D, Guan Z. Efficacy and safety cabazitaxel resistance which may promote the of sorafenib in patients in the Asia-Pacific re- development of effective drug combination gion with advanced hepatocellular carcinoma: strategies and maximize the anti-tumor effects a phaseIII randomised, double-blind, placebo- of cabazitaxel. In conclusion, our findings pro- controlled trial. Lancet Oncol 2009; 10: 25-34. vide evidence for future development of ca- [6] Asghar U, Meyer T. Are there opportunities for bazitaxel as a novel chemotherapeutic agent chemotherapy in the treatment of hepatocel- against HCC in clinical settings. lular cancer? J Hepatol 2012; 56: 686-95. [7] Burroughs A, Hochhauser D, Meyer T. Systemic Acknowledgements treatment and liver transplantation for hepatocellular carcinoma: two ends of the therapeu- This research was supported by Innovative tic spectrum. Lancet Oncol 2004; 5: 409-18. Research Groups of National Natural Science [8] Ganne-Carrie N, Trinchet JC. Systemic treat- Foundation of China (No. 81721091); National ment of hepatocellular carcinoma. Eur J Gas- S&T Major Project (No. 2017ZX10203205); troenterol Hepatol 2004; 16: 275-81. [9] Szakacs G, Paterson JK, Ludwig JA, Booth- Zhejiang International Science and Techno- Genthe C, Gottesman MM. Targeting multidrug logy Cooperation Project (NO. 2016C04003); resistance in cancer. Nat Rev Drug Discov Zhejiang Provincial Natural Science Found- 2006; 5: 219-34. ation (LY18H160017). [10] Ng IO, Liu CL, Fan ST, Ng M. Expression of P-glycoprotein in hepatocellular carcinoma. A Disclosure of conflict of interest determinant of chemotherapy response. Am J Clin Pathol 2000; 113: 355-63. None. [11] Serena B, Lorella P, Lory SC, Giorgio S, Claudio T. Gene expression of ABC proteins in hepato- Address correspondence to: Shusen Zheng and cellular carcinoma, perineoplastic tissue, and Donghai Jiang, Department of Surgery, Division of liver diseases. Mol Med 2002; 8: 318-25. Hepatobiliary and Pancreatic Surgery, First Affiliated [12] Cox J, Weinman S. Mechanisms of doxorubicin Hospital, School of Medicine, Zhejiang University, resistance in hepatocellular carcinoma. Hepat Hangzhou 310000, Zhejiang Province, China. E-mail: Oncol 2016; 3: 57-9. [email protected] (SSZ); [email protected]. [13] Park JG, Lee SK, Hong IG, Kim HS, Lim KH, cn (DHJ) Choe KJ, Kim WH, Kim YI, Tsuruo T, Gottesman MM. MDR1 gene expression: its effect on drug References resistance to doxorubicin in human hepatocellular carcinoma cell lines. J Natl Cancer Inst [1] Lindsey AT, Freddie B, Rebecca LS, Jacques F, 1994; 86: 700-5. Joannie LT, Ahmedin J. Global cancer statis- [14] de Bono JS, Oudard S, Ozguroglu M, Hansen S, tics, 2012. Ca A Cancer Journal for Clinicians Machiels, JP, Kocak I, Gravis G, Bodrogi I, 2015; 61: 69-90. Mackenzie MJ, Shen L. Prednisone plus caba- [2] Schwartz M, Roayaie S, Konstadoulakis M. zitaxel or mitoxantrone for metastatic castra- Strategies for the management of hepatocel- tion-resistant prostate cancer progressing af- lular carcinoma. Nat Clin Pract Oncol 2007; 4: ter docetaxel treatment: a randomised open- 424-32. label trial. Lancet 2010; 376: 1147-54. [3] Forner A, Reig ME, de Lope CR, Bruix J. Current [15] Pean E, Demolis P, Moreau A, Hemmings RJ, strategy for staging and treatment: the BCLC O’Connor D, Brown D, Shepard T, Abadie E, update and future prospects. Semin Liver Dis Pignatti F. The European medicines agency re- 2010; 30: 61-74. view of cabazitaxel (Jevtana?) for the treat- [4] Josep ML, Sergio R, Vincenzo M, Philip H, Ed- ment of hormone-refractory metastatic pros- ward G, Jean-Frédéric B, Andre Cosme de O, tate cancer: summary of the scientific assess- Armando S, Jean-Luc R, Alejandro F, Myron S, ment of the committee for medicinal products Camillo P, Stefan Z, Luigi B, Tim FG, Peter RG, for human use. Oncologist 2012; 17: 543-9.

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