Cancer Chemother Pharmacol (2015) 76:257–267 DOI 10.1007/s00280-015-2787-7

ORIGINAL ARTICLE

Efficacy of perifosine alone and in combination with sorafenib in an HrasG12V plus shp53 transgenic mouse model of hepatocellular carcinoma Mi Na Kim1 · Simon Weonsang Ro2 · Do Young Kim1,2 · Da Young Kim2 · Kyung‑Ju Cho2 · Jeon Han Park3 · Ho Yeong Lim4 · Kwang‑Hyub Han1,2 

Received: 16 April 2015 / Accepted: 21 May 2015 / Published online: 3 June 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Purpose  Perifosine has shown antitumor activity via inhibition of Akt phosphorylation in many advanced solid tumors. This study investigated the efficacy of perifosine alone and in combination with sorafenib in a transgenic mouse model of HCC. Methods  The mouse model of HCC was generated by hydrodynamic injection of transposons encoding HrasG12V and short-hairpin RNA downregulating p53. The transgenic mice were treated with perifosine alone and in combination with sorafenib to evaluate efficacy of drugs on tumor growth and survival. Results  Treatment with perifosine for 5 weeks, alone and in combination with sorafenib, strongly inhibited tumor growth and increased survival. Perifosine inhibited HCC cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Furthermore, its combination with sorafenib enhanced these effects. In addition, Akt phosphorylation was decreased by perifosine and further decreased by combination treatment. Although perifosine alone did not

* Do Young Kim [email protected] * Kwang‑Hyub Han [email protected] 1

Department of Internal Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun‑gu, Seoul 120‑752, Korea

2

Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea





3



4



Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

appear to activate the caspase pathway, combination treatment increased the cleavage of caspase-3, caspase-9, and poly (ADP-ribose) polymerase. Conclusions  The preclinical effect that current study showed represents a strong rationale for clinical trials using perifosine alone and in combination with sorafenib in the treatment of HCC patients. Keywords  Hepatocellular carcinoma · Perifosine · Sorafenib · PI3K/Akt pathway · Ras/Raf/MAPK pathway Abbreviations ALT Alanine aminotransferase AST Aspartate aminotransferase ALP Alkaline phosphatase ERK Extracellular signal-regulated kinase HCC Hepatocellular carcinoma GAPDH Glyceraldehyde 3-phosphate dehydrogenase GEM Genetically engineered mouse MAPK Mitogen-activated protein kinase MEK MAPK kinase PARP Poly (ADP-ribose) polymerase PI3K Phosphatidylinositol 3-kinase SD Standard deviation Shp53 Short-hairpin RNA downregulating p53 TUNEL Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling VEGF Vascular endothelial growth factor

Introduction Hepatocellular carcinoma (HCC) is the sixth most common malignancy and the third leading cause of cancerrelated mortality worldwide [1]. Although the management

13

258

of HCC has improved significantly, curative treatments such as surgical resection, liver transplantation, and locoablative therapy provide benefits for only a small subset of patients [2, 3]. Therefore, novel therapeutic strategies are needed in the management of HCC. Sorafenib, an oral multikinase inhibitor, blocks tumor cell proliferation by targeting the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathway and inhibits angiogenesis by targeting vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, and platelet-derived growth factor receptor-β [4, 5]. Sorafenib is the only guideline-recommended systemic drug for treating advanced HCC [6]. Despite the benefit provided by sorafenib, the response rate of sorafenib is relatively low [4, 5]. Thus, additional treatment options are needed to improve survival in patients with advanced HCC. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays an important role in the development and progression of HCC [7]. Aberrations in the PI3K/Akt pathway have been detected in 40–50 % of HCC tumors [8]. For this reason, the PI3K/Akt axis is a promising drug target for HCC therapy. Perifosine, a synthetic alkylphosphocholine antitumor agent, is an oral Akt inhibitor that targets the pleckstrin homology domain of Akt to prevent its translocation to the plasma membrane [8]. Perifosine has shown antitumor activity in patients with advanced solid tumors [9–12] and exerts substantial antitumor activity in human HCC cell lines [13]. However, an antitumor effect in HCC has not been demonstrated in vivo. Perifosine and sorafenib target separate stages along two different signaling pathways, providing a strong rationale for the use of this drug combination [14]. Furthermore, compared with single-drug treatment, combination treatment targeting these two different signaling pathways may be a better treatment option by overcoming mechanisms of drug resistance [15]. Recent studies reported that activation of the PI3K/Akt pathway mediated the acquired resistance to sorafenib in HCC, and this resistance was overcome by Akt inhibition [16, 17]. However, whether the combined use of perifosine and sorafenib has therapeutic potential in HCC has not yet been determined. This study aimed to investigate the efficacy of perifosine in transgenic mouse model of HCC. Furthermore, it was investigated whether the effects of perifosine and sorafenib are synergistic when administered in combination.

Cancer Chemother Pharmacol (2015) 76:257–267

using animals were performed in strict accordance with the Guidelines and Regulations for the Care and Use of Laboratory Animals in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities. The care and use of laboratory animals were based on the Guidelines and Regulations for the Use and Care of Animals at Yonsei University College of Medicine. Drugs Perifosine was provided by Aeterna Zentaris GmbH (Frankfurt, Germany). Sorafenib was purchased from Bayer AG (Leverkusen, Germany). Transgenic mouse model of HCC A transgenic mouse model of HCC has been established by a hydrodynamic injection method, coupled with the Sleeping Beauty transposon system [18]. The transposon-encoding plasmids pT2/HrasG12V and pT2/shp53 and the transposase-encoding plasmid pT2/C-Luc//PGK-SB13 were prepared with endotoxin-free Plasmid Maxi Kits (Qiagen, Hilden, Germany). To generate the double-transgenic mice, 12.5 µg of pT2/HrasG12V and 14.0 µg of pT2/shp53 were mixed with 9.0 µg of pT2/C-Luc//PGK-SB13. The transposon plasmids pT2/HrasG12V, pT2/shp53, and pT2/C-Luc// PGK-SB13 were described previously [18]. For hydrodynamic injection, 12.5 µg of pT2/HrasG12V and 14.0 µg of pT2/shp53 were mixed with 9.0 µg of pT2/C-Luc//PGKSB13 and then suspended in 2 ml of Ringer’s lactated solution. The DNA solution was injected into the lateral tail veins of mice (0.1 ml/g body weight) in <7 s. Animal treatment protocol After hydrodynamic injection, 20 mice were randomly assigned to the following four experimental groups (each group, n  = 5): control group, perifosine group, sorafenib group, and combination group. Drugs were administered five times per week by oral gavage beginning at day 2 after hydrodynamic injection. Doses of drugs administered were 30 mg/kg/day for perifosine and 60 mg/kg/day for sorafenib. The mice of combination group were administered using the same dose and schedule for perifosine and sorafenib as described for the single-drug treatment. All mice in control group received an equal volume of normal saline by oral gavage using the same treatment schedule.

Materials and methods Liver tissue processing and biochemical tests Animals Male 5- to 6-week-old C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea). All experiments

13

The drug-treated mice were euthanized 5 weeks after hydrodynamic injection. At necropsy, body weight and liver weight were measured, and the ratio of liver weight

Cancer Chemother Pharmacol (2015) 76:257–267

to body weight was determined. The livers were fixed overnight in freshly prepared neutral buffered formalin and then embedded in paraffin. Blood was obtained via cardiac puncture at the time of killing. Serum was obtained by centrifugation, and the extent of hepatic injury was assessed by measuring serum alanine aminotransferase (ALT; reference range 31–46 U/l), aspartate aminotransferase (AST; reference range 70–115 U/l), albumin (reference range 2.7–3.5 g/dl), and alkaline phosphatase (ALP; reference range 118–207 U/l). Animal survival test In a series of experiments designed to measure survival, 40 mice were randomly assigned to the four groups (each group, n = 10): control, perifosine, sorafenib, and combination group. Mice were administered with each drug following the treatment protocol described above. Survival was calculated as the time from the date of hydrodynamic injection to the time of death. If a mouse shows signs indicating distress (deterioration of the general state of the mice, severe piloerection, abnormal posture, or behavior), euthanasia was performed. Western blot analysis Western blot analysis was performed using standard methods. Equal amounts of protein extract of each group were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a membrane, and probed with primary antibodies. The following primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA): anti-Akt, anti-phospho-Akt, antiphospho-S6, anti-MAPK kinase (MEK), anti-phosphoMEK, anti-extracellular signal-regulated kinase (ERK)1/2, anti-phospho-ERK1/2, anti-caspase-3, anti-cleaved caspase-3, anti-caspase-9, anti-cleaved caspase-9, anti-poly (ADP-ribose) polymerase (PARP), anti-cleaved PARP. The primary antibody for VEGF was purchased from Abcam (Cambridge, TX, USA). As an internal control, an antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technology) was used. The secondary antibody was goat anti-rabbit IgG polyclonal antibody (Dako, Carpinteria, CA, USA). Detection of the target proteins on the membranes was performed using the ECL western blotting detection reagents. Immunohistochemical staining Liver tissue sections were deparaffinized in xylene and rehydrated through a gradual decrease in ethanol concentration. Antigens were then unmasked using sodium citrate

259

buffer. The sections were incubated overnight at 4 °C with primary antibodies against Ki67 (Abcam), CD31 (Abbiotec; San Diego, CA, USA), and VEGF (Abcam). The sections were then incubated with biotinylated secondary antibody. The color reaction was visualized with diaminobenzidine, and tissues were counterstained with hematoxylin. The proliferation index (PI) was determined by counting the number of Ki67-positive cells among at least 1000 cells in five randomly selected fields (magnification 200×) and expressed as percentage. The number of CD31-positive blood vessels and VEGF-positive cells was determined by counting at least 1000 cells in five randomly selected fields (magnification 200×) and expressed as percentage values. Apoptosis detection Apoptosis was detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Liver tissues were embedded in paraffin blocks, and 4-µm sections were cut and stained with the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Cell Signaling Technology). The stained sections of tumors of each group were reviewed, and the apoptosis index, determined by TUNEL staining, was determined by counting at least 1000 cells in five randomly selected fields (magnification 200×) and expressed as percentage values. Statistical analyses Data are expressed as mean ± SD. Differences between groups were analyzed using Student’s t test. The Kaplan– Meier method was used to determine the probability of survival as a function of time, and the differences between groups were compared by two-sided log-rank test. P values <0.05 were regarded to indicate statistical significance. Statistical analyses were performed using the SPSS software (version 18.0; SPSS Inc., Chicago, IL, USA).

Results Effect of perifosine alone and in combination with sorafenib on tumor growth in an HrasG12V plus shp53 transgenic mouse model of HCC To investigate the antitumor effect of perifosine alone and in combination with sorafenib, the livers of the transgenic mice were examined after 5 weeks of drug administration. Gross morphology of the harvested livers showed that perifosine alone and in combination with sorafenib strongly inhibited tumor growth. Tumor nodules decreased in size and number in all drug-treated groups compared to the

13

260

control group (Fig. 1a). In addition, the ratio of liver weight to body weight decreased in mice treated with perifosine (0.085  ± 0.010, P  = 0.024) or sorafenib (0.114 ± 0.025, P  = 0.043) compared with that of the control group (0.172  ± 0.045). In mice of the combination group, the ratio of liver weight to body weight was lower than that of the control group (0.083 ± 0.021, P = 0.006), but did not differ significantly from either single-drug treatment group

Fig. 1  Effect of perifosine alone and in combination with sorafenib on tumor growth and survival in an HrasG12V plus shp53 transgenic mouse model of hepatocellular carcinoma. a Harvested livers of mice from each group 5 weeks after hydrodynamic injection. b Average ratio of liver weight to body weight of each group 5 weeks after hydrodynamic injection. *P < 0.05 compared to the control group; #P > 0.05 compared to the perifosine group or the sorafenib group. c Kaplan–Meier survival curves of HrasG12V plus shp53 transgenic mice treated with perifosine alone, sorafenib alone, or perifosine plus sorafenib. All drug-treated groups showed a significant increase in survival compared to the control group (all P < 0.05, log-rank test)

13

Cancer Chemother Pharmacol (2015) 76:257–267

(P  = 0.927 compared to the perifosine group; P  = 0.072 compared to the sorafenib group) (Fig. 1b). Total body weight did not change significantly after 5 weeks of drug administration in any of the groups (control group, from 18.6 ± 0.4 to 20 ± 1.2; perifosine group, from 19.7 ± 0.2 to 21.2 ± 1.3; sorafenib group, from 20.2 ± 0.4 to 21.4 ± 1.9; combination group, from 20.1 ± 1.3 to 20.2 ± 0.7; all P > 0.05).

Cancer Chemother Pharmacol (2015) 76:257–267 Table 1  Values of serum biochemical parameters after 5 weeks of treatment

261

Group

AST (U/l)a

ALT (U/l)

Albumin (g/dl)

ALP (U/I)

Control Perifosine Sorafenib

179.7 ± 74.3 77.7 ± 12.5 256.5 ± 154.9

74.7 ± 34.9 51.0 ± 21.8 206.5 ± 150.6

2.5 ± 0.1 2.8 ± 0.3 3.1 ± 0.4

428.3 ± 193.9 325.0 ± 53.6 682.0 ± 438.4

Combination

411.5 ± 19.1

611.5 ± 253.9

2.8 ± 0.8

611.5 ± 253.9

a

  Control group versus combination group, P < 0.05

Biochemical parameters To assess the extent of hepatic injury, biochemical parameters were checked and compared between groups. Levels of AST, ALT, and ALP appeared to be decreased in the perifosine group and increased in the sorafenib and combination treatment groups. However, these differences were not significant (all P > 0.05) except for increased AST levels in the combination group (P < 0.05). Serum albumin appeared to be increased among all drug-treated groups compared to the control group, but these differences were not significant (all P > 0.05) (Table 1). Effect of perifosine alone and in combination with sorafenib on survival in an HrasG12V plus shp53 transgenic mouse model of HCC The median survival was 46.5 days for mice in the control group, 73 days for mice in the perifosine group, 58.5 days for mice in the sorafenib group, and 59.5 days for mice in the combination group. Results of Kaplan–Meier analysis showed a significant survival advantage for all drug-treated groups compared to the control group (P < 0.05, log-rank test). However, survival did not differ significantly between mice treated with perifosine alone or sorafenib alone (P > 0.05, log-rank test), and combination treatment did not increase survival compared with either single-drug treatment (P > 0.05, log-rank test) (Fig. 1c). Effect of perifosine alone and in combination with sorafenib on PI3K/Akt and Ras/Raf/MAPK pathways Changes in the phosphorylation levels of key proteins in PI3K/Akt and Ras/Raf/MAPK pathways were determined by western blot analysis. Perifosine alone and in combination with sorafenib significantly decreased levels of phosphorylated Akt by 29 and 35.3 %, respectively, compared to the control group (all P < 0.05), but sorafenib alone did not decrease Akt phosphorylation. The effect of combination treatment on phosphorylation of Akt was not significantly greater than the effect of perifosine alone (P > 0.05) (Fig. 2a, b). Levels of

phosphorylated S6, a downstream target of Akt, were also decreased by 19.5 and 26.6 % in the perifosine and combination groups, respectively (all P < 0.005 compared to the control group), and the effect of combination treatment was significantly greater than that of perifosine alone (P < 0.05) (Fig. 2a, c). In contrast, the level of phosphorylated S6 was not decreased by sorafenib alone. Total Akt levels did not differ significantly between groups (Fig. 2a). The level of phosphorylated MEK was decreased in the sorafenib group compared to the control group by 13.9 %, but this difference was not significant (P > 0.05). Combination treatment significantly downregulated p-MEK compared to the control group by 28 % (P < 0.05) (Fig. 3a, b). Similarly, the level of phosphorylated ERK1/2 was decreased by 48.9 % and 55 % in mice treated with sorafenib alone and combination treatment, respectively (all P < 0.05 compared to the control group) (Fig. 3a, c). However, the effect of combination treatment on phosphorylation of MEK and ERK1/2 was not significantly greater than the effect of sorafenib alone (all P > 0.05). Interestingly, mice treated with perifosine showed higher levels of MEK and ERK1/2 phosphorylation compared to the control group (Fig. 3b, c). Total MEK and ERK1/2 levels did not differ significantly between the groups (Fig. 3a). Effect of perifosine alone and in combination with sorafenib on tumor cell proliferation in an HrasG12V plus shp53 transgenic mouse model of HCC The effect of perifosine alone and in combination with sorafenib on tumor cell proliferation was investigated as measured by Ki67 staining. Mice in all drug-treated groups showed decreased Ki67-positive cells compared to the control group (Fig. 4a). As shown in Fig. 4b, the mean PI based on Ki67 staining of liver tissue sections was 47 ± 9.1 for the control group and was significantly decreased in mice treated with perifosine (25 ± 6.1; P < 0.05, compared to the control group) or sorafenib (19 ± 2.6; P < 0.001, compared to the control group). The effect of combination treatment on tumor cell proliferation (PI, 14.6 ± 2.7) was significantly greater than that of perifosine alone or sorafenib alone (all P < 0.05).

13

262 Fig. 2  Inhibition of Akt and S6 phosphorylation by perifosine alone and in combination with sorafenib in HrasG12V plus shp53 transgenic mice liver tissues. a Evaluation of Akt, phospho-Akt, and phospho-S6 in tumor lysates by western blot analysis. GAPDH was used as a loading control. b Plot based on GAPDH-normalized phosphoAkt band densities shown in a. c Plot based on GAPDH-normalized phospho-S6 band densities shown in a. Data are expressed as mean ± SD of three experiments. All statistical tests were two-sided. *P < 0.05, compared to the control group; #P > 0.05, compared to the perifosine group; $P < 0.05, compared to the perifosine group

Fig. 3  Inhibition of MEK and ERK 1/2 phosphorylation by perifosine alone and in combination with sorafenib in HrasG12V plus shp53 transgenic mice liver tissues. a Evaluation of MEK, phospho-MEK, ERK 1/2, and phospho-EKR 1/2 in tumor lysates by western blot analysis. GAPDH was used as a loading control. b Plot based on GAPDH-normalized phosphoMEK band densities shown in a. c Plot based on GAPDHnormalized phospho-ERK 1/2 band densities shown in a. Data are expressed as mean ± SD of three experiments. All statistical tests were two-sided. *P > 0.05, compared to the control group; **P < 0.05, compared to the control group

13

Cancer Chemother Pharmacol (2015) 76:257–267

Cancer Chemother Pharmacol (2015) 76:257–267

263

Fig. 4  Effect of perifosine alone and in combination with sorafenib on tumor cell proliferation and tumor cell apoptosis in an HrasG12V plus shp53 transgenic mouse model of hepatocellular carcinoma. a Immunohistochemical staining for Ki67 (magnification ×200). Mice in all drug-treated groups showed decreased Ki67-positive cells compared to the control group. b Proliferation index based on the quantitation of Ki67 staining. Data are expressed as mean ± SD. *P < 0.05, compared to the control group; **P < 0.001, compared to control

group; #P < 0.05. c Histological examination of TUNEL-stained liver tissues (magnification ×200). Mice treated in all drug-treated groups showed increased TUNEL-stained cells compared to the control group. d Apoptosis index based on the quantitation of TUNEL staining. Data are expressed as mean ± SD of three experiments. All statistical tests were two-sided. *P < 0.001 compared to control group; # P < 0.05

Effect of perifosine alone and in combination with sorafenib on tumor cell apoptosis in an HrasG12V plus shp53 transgenic mouse model of HCC

are critical components of caspase-mediated apoptosis [13]. Although the level of cleaved caspase-3 was significantly increased by sorafenib alone, single-drug treatments had a little effect on the cleavage of other apoptotic markers. However, combination treatment significantly increased cleavage of caspase-3, caspase-9, and PARP. Levels of uncleaved caspase-3, caspase-9, and PARP did not differ significantly between groups (Fig. 5).

The effect of perifosine alone and in combination with sorafenib on tumor cell apoptosis was examined by TUNEL assay. Mice treated in all drug-treated groups showed increased TUNEL-stained cells compared to the control group (Fig. 4c). The mean apoptosis index on percentage of TUNEL-stained cells was 1.8 ± 0.8 for the control group and was significantly increased in mice treated with perifosine (5.4 ± 1.1; P < 0.001, compared to the control group) or sorafenib (7.0 ± 1.6; P < 0.001, compared to the control group). The effect of combination treatment on apoptosis (apoptosis index, 9.6 ± 2.4) was significantly greater than that of perifosine alone or sorafenib alone (all P < 0.05) (Fig. 4d). Tumor cell apoptosis was also evaluated by western blot analysis of caspase-3, caspase-9, and PARP, which

Effect of perifosine alone and in combination with sorafenib on tumor angiogenesis in an HrasG12V plus shp53 transgenic mouse model of HCC To determine the effect of perifosine alone and in combination with sorafenib on tumor angiogenesis, tumor sections were stained with antibody against CD31. Mice in all drug-treated groups showed less CD31-positive vessels compared to the control group (Fig. 6a). The mean

13

264

Cancer Chemother Pharmacol (2015) 76:257–267

Fig. 5  Effect of perifosine alone and in combination with sorafenib on apoptosis in an HrasG12V plus shp53 transgenic mouse model of hepatocellular carcinoma. a Evaluation of caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, PARP, and cleaved PARP in tumor lysates by western blot analysis. GAPDH was used as a loading control. b Plot based on GAPDH-normalized cleaved caspase-3 band densities shown in a. c Plot based on GAPDHnormalized cleaved caspase-9 band densities shown in a. Data are expressed as mean ± SD of three experiments. All statistical tests were two-sided. *P > 0.05 compared to the control group; **P < 0.01 compared to the control group

Fig. 6  Effect of perifosine alone and in combination with sorafenib on angiogenesis in an HrasG12V plus shp53 transgenic mouse model of HCC. a Histological examination of CD31 staining (magnification ×200). Mice in all drug-treated groups showed less CD31-positive

vessels compared to the control group. b Quantitation of CD31positive blood vessels. Data are expressed as mean ± SD. All statistical tests were two-sided. *P < 0.005 compared to control group; # P < 0.005

number of CD31-positive vessels in the control group was 598 ± 44.4, which was significantly decreased by treatment with perifosine (502 ± 31.1) or sorafenib (360  ± 15.8) (all P < 0.005) (Fig. 6b). The combination group showed significantly less CD31-positive vessels (320 ± 15.8) than the group treated with perifosine alone or the sorafenib group (all P < 0.005).

Discussion

13

This is the first preclinical study showing that perifosine as a single-drug treatment and in combination with sorafenib strongly inhibits tumor growth and improves survival in a mouse model of HCC generated by hydrodynamic injection of the transgenes HrasG12V and shp53.

Cancer Chemother Pharmacol (2015) 76:257–267

Results of a previous study showed that phosphorylation of Akt, ERK, and MEK was elevated in transgenic mice expressing HrasG12V [18]. Although induction of HCC was low in mice expressing HrasG12V alone, coexpression of HrasG12V plus shp53 improved the success rate [18]. Therefore, to evaluate inhibitors of the PI3K/Akt and Ras/ Raf/MAPK pathways, double-transgenic mouse model of HCC expressing HrasG12V and shp53 was used in this study. Recent studies showed that activation of the PI3K/Akt pathway is frequently observed and associated with aggressive tumor phenotype in human HCC [19–21]. The PI3K/ Akt pathway has been demonstrated to have the significant role in cell growth, survival, differentiation, and metabolism, and its inhibition causes cell death associated with apoptosis [22]. Thus, the PI3K/Akt pathway is a promising target for the treatment of HCC. In addition, overexpression of Akt alone has an important role in hepatocarcinogenesis, and Akt is almost ubiquitously upregulated in human HCC [23]. Perifosine is an orally active synthetic alkylphosphocholine Akt inhibitor, and the clinical efficacy of this drug has been reported in various solid tumors in which Akt activation is associated with poor prognosis [8, 24–26]. Perifosine also showed substantial antitumor activity in human HCC cell lines by inhibiting Akt phosphorylation [13]. The present study confirmed this finding in vivo by demonstrating antitumor effects and inhibition of Akt phosphorylation in transgenic mice treated with perifosine. Significant decrease in the ratio of liver weight to body weight and in the level of phosphorylated Akt was shown in the perifosine group (Figs. 1, 3). Previous studies have reported that simultaneous targeting of PI3K/Akt and Ras/Raf/MAPK pathways strongly induces cell death in transformed cells [27], suggesting that concomitant inhibition of these two pathways may be an effective treatment option for a variety of diseases characterized by the activation of these pathways [28]. The PI3K/ Akt and Ras/Raf/MAPK pathways are the main activated pathways in HCC cells [29]. Moreover, many studies have shown that sorafenib activates the PI3K/Akt pathway [30– 33], and its inhibition enhances the efficacy of sorafenib [29, 34]. For that reason, the hypothesis was that combining the Akt inhibitor perifosine with sorafenib could increase its antitumor effect. The enhanced decrease in the phosphorylation of Akt, S6, MEK, and ERK in the combination group (Figs. 3, 4) supports this hypothesis. Furthermore, results of the present study suggest that perifosine triggers the phosphorylation of MEK and ERK, which is blocked by sorafenib. The simultaneous blockade of Akt and MEK/ ERK signaling cascades may prevent clinical resistance to these drugs. The antitumor effects of perifosine alone and in combination with sorafenib may be explained by three potential

265

mechanisms. First, perifosine may directly suppress tumor cell proliferation by the inhibition of phosphorylation of Akt and its downstream effector S6, which regulate cell proliferation. Sorafenib also inhibits tumor cell proliferation by inhibiting the Ras/Raf/MAPK pathway [35]. This hypothesis is supported by the results of this study showing attenuated tumor cell proliferation after single-drug treatment with perifosine or sorafenib. The increased inhibition of cell proliferation with combination treatment, which was demonstrated by Ki67 immunohistochemistry (Fig. 5), is consistent with the important roles of PI3K/Akt and Ras/ Raf/MAPK signaling pathways in HCC cell proliferation. Because multiple proliferation and survival pathways are involved in driving the proliferation of HCC cells, the simultaneous inhibition of several relevant pathways may produce a synergistic effect. Second, the observed antitumor effect of perifosine alone and in combination with sorafenib may be a result of drug-induced apoptosis, as indicated by the results of the TUNEL assay (Fig. 6). Perifosine has been shown to induce apoptosis in hepatoma cells via activation of caspase-3 and caspase-9 [13]. In addition, sorafenib was recently found to induce apoptosis in several human cancer lines, including the HCC cell line HepG2, by downregulating the expression of the levels of the anti-apoptotic protein [36]. Thus, the role of apoptosis was further investigated by western blot analysis, which revealed a significant increase in the cleavage of caspase-3 and caspase-9 with combination treatment. In addition, the cleavage of PARP, a downstream substrate of the caspase cascade and marker of apoptosis [37], was detected only in mice receiving combination treatment. However, the effect of perifosine alone on caspase-dependent apoptosis, which was observed in vitro [13], was not detected in this in vivo study. Finally, perifosine may inhibit angiogenesis, which is required for HCC growth. The hypothesis that perifosine affects tumor growth primarily by suppressing angiogenesis is supported by data of present study showing less CD31-positive blood vessels in HCC tumors of perifosinetreated mice compared to untreated controls. This effect was enhanced by combination treatment. Further study is needed to better understand the mechanisms underlying the anti-angiogenic activity of perifosine. There are some unresolved issues in this study. First, despite enhanced inhibition of tumor cell proliferation and induction of apoptosis, combination treatment did not significantly improve survival compared with perifosine or sorafenib alone. Results of this study showed that serum levels of liver injury-related bioparameters (AST, ALT, and ALP) were substantially elevated in the combination group, which may have affected survival. However, in normal mice treated with combination treatment, serum levels of liver injuryrelated bioparameters were not elevated (data not shown).

13

266

Recent study also showed that perifosine and sorafenib combination treatment was feasible with manageable toxicity in patients with Hodgkin lymphoma [38]. Second, although this double-transgenic mouse model (HrasG12V plus shp53) of HCC has a strong advantage for testing perifosine and sorafenib, the results should be confirmed in a xenograft model established using a human HCC cell line. In conclusion, this is the first in vivo study demonstrating that perifosine can inhibit HCC tumor growth. Furthermore, its combination with sorafenib enhances the antitumor effect of perifosine through the simultaneous inhibition of PI3K/Akt and Ras/Raf/MAPK signaling pathways. Owing to the limitations of currently available therapies, there is an urgent need for new therapeutic options for advanced HCC patients. The preclinical effect that current study showed represents a strong rationale for clinical trials using perifosine alone and in combination with sorafenib in the treatment of HCC patients. Acknowledgments  This research was supported by grants from the Korean Association for the Study of Liver and was supported by a faculty research grant of Yonsei University College of Medicine for 2013 (6-2013-0004). The authors are grateful to Dong-Su Jang (Medical Illustrator, Medical Research Support Section, Yonsei University College of Medicine, Seoul, Korea) for his help with the figure. Conflict of interest None.

References 1. Forner A, Llovet JM, Bruix J (2012) Hepatocellular carcinoma. Lancet 379:1245–1255. doi:10.1016/s0140-6736(11)61347-0 2. Bruix J, Sherman M (2005) Management of hepatocellular carcinoma. Hepatology 42:1208–1236. doi:10.1002/hep.20933 3. Chan SL, Mo FK, Johnson PJ, Liem GS, Chan TC, Poon MC, Ma BB, Leung TW, Lai PB, Chan AT, Mok TS, Yeo W (2011) Prospective validation of the Chinese University prognostic index and comparison with other staging systems for hepatocellular carcinoma in an Asian population. J Gastroenterol Hepatol 26:340–347. doi:10.1111/j.1440-1746.2010.06329.x 4. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Haussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378–390. doi:10.1056/ NEJMoa0708857 5. Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, 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 K, Zou J, Voliotis D, Guan Z (2009) Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 10:25–34. doi:10.1016/s1470-2045(08)70285-7 6. Bruix J, Sherman M (2011) Management of hepatocellular carcinoma: an update. Hepatology 53:1020–1022. doi:10.1002/hep.24199 7. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2:489–501. doi:10.1038/nrc839

13

Cancer Chemother Pharmacol (2015) 76:257–267 8. Patel V, Lahusen T, Sy T, Sausville EA, Gutkind JS, Senderowicz AM (2002) Perifosine, a novel alkylphospholipid, induces p21(WAF1) expression in squamous carcinoma cells through a p53-independent pathway, leading to loss in cyclin-dependent kinase activity and cell cycle arrest. Cancer Res 62:1401–1409 9. Chiarini F, Del Sole M, Mongiorgi S, Gaboardi GC, Cappellini A, Mantovani I, Follo MY, McCubrey JA, Martelli AM (2008) The novel Akt inhibitor, perifosine, induces caspase-dependent apoptosis and downregulates P-glycoprotein expression in multidrug-resistant human T-acute leukemia cells by a JNK-dependent mechanism. Leukemia 22:1106–1116. doi:10.1038/leu.2008.79 10. Leighl NB, Dent S, Clemons M, Vandenberg TA, Tozer R, Warr DG, Crump RM, Hedley D, Pond GR, Dancey JE, Moore MJ (2008) A Phase 2 study of perifosine in advanced or metastatic breast cancer. Breast Cancer Res Treat 108:87–92. doi:10.1007/ s10549-007-9584-x 11. Knowling M, Blackstein M, Tozer R, Bramwell V, Dancey J, Dore N, Matthews S, Eisenhauer E (2006) A phase II study of perifosine (D-21226) in patients with previously untreated metastatic or locally advanced soft tissue sarcoma: A National Cancer Institute of Canada Clinical Trials Group trial. Invest New Drugs 24:435–439. doi:10.1007/s10637-006-6406-7 12. Crul M, Rosing H, de Klerk GJ, Dubbelman R, Traiser M, Reichert S, Knebel NG, Schellens JH, Beijnen JH, ten Bokkel Huinink WW (2002) Phase I and pharmacological study of daily oral administration of perifosine (D-21266) in patients with advanced solid tumours. Eur J Cancer 38:1615–1621 13. Fei HR, Chen G, Wang JM, Wang FZ (2010) Perifosine induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cell lines by blockade of Akt phosphorylation. Cytotechnology 62:449–460. doi:10.1007/s10616-010-9299-4 14. Teachey DT, Grupp SA, Brown VI (2009) Mammalian target of rapamycin inhibitors and their potential role in therapy in leukaemia and other haematological malignancies. Br J Haematol 145:569–580. doi:10.1111/j.1365-2141.2009.07657.x 15. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA (2008) Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 11:32–50. doi:10.1016/j.drup.2007.11.003 16. Chen KF, Chen HL, Tai WT, Feng WC, Hsu CH, Chen PJ, Cheng AL (2011) Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells. J Pharmacol Exp Ther 337:155–161. doi:10.1124/jpet.110.175786 17. Zhai B, Hu F, Jiang X, Xu J, Zhao D, Liu B, Pan S, Dong X, Tan G, Wei Z, Qiao H, Jiang H, Sun X (2014) Inhibition of Akt reverses the acquired resistance to sorafenib by switching protective autophagy to autophagic cell death in hepatocellular carcinoma. Mol Cancer Ther 13:1589–1598. doi:10.1158/1535-7163. mct-13-1043 18. Ju HL, Ahn SH, Kim DY, Baek S, Chung SI, Seong J, Han KH, Ro SW (2013) Investigation of oncogenic cooperation in simple liver-specific transgenic mouse models using noninvasive in vivo imaging. PLoS One 8:e59869. doi:10.1371/journal. pone.0059869 19. Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, Factor VM, Thorgeirsson SS (2006) Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 130:1117–1128. doi:10.1053/j.gastro.2006.01.006 20. Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, Tovar V, Roayaie S, Minguez B, Sole M, Battiston C, Van Laarhoven S, Fiel MI, Di Feo A, Hoshida Y, Yea S, Toffanin S, Ramos A, Martignetti JA, Mazzaferro V, Bruix J, Waxman S, Schwartz M, Meyerson M, Friedman SL, Llovet JM (2008) Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology 135:1972–1983. doi:10.1053/j.gastro.2008.08.008

Cancer Chemother Pharmacol (2015) 76:257–267 21. Wang C, Cigliano A, Delogu S, Armbruster J, Dombrowski F, Evert M, Chen X, Calvisi DF (2013) Functional crosstalk between AKT/mTOR and Ras/MAPK pathways in hepatocarcinogenesis: implications for the treatment of human liver cancer. Cell Cycle 12:1999–2010. doi:10.4161/cc.25099 22. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB (2005) Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4:988–1004. doi:10.1038/nrd1902 23. Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, Destefanis G, Delogu S, Zimmermann A, Ericsson J, Brozzetti S, Staniscia T, Chen X, Dombrowski F, Evert M (2011) Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology 140:1071–1083. doi:10.1053/j.gastro.2010.12.006 24. Chee KG, Longmate J, Quinn DI, Chatta G, Pinski J, Twardowski P, Pan CX, Cambio A, Evans CP, Gandara DR, Lara PN Jr (2007) The AKT inhibitor perifosine in biochemically recurrent prostate cancer: a phase II California/Pittsburgh cancer consortium trial. Clin Genitourin Cancer 5:433–437. doi:10.3816/ CGC.2007.n.031 25. Posadas EM, Gulley J, Arlen PM, Trout A, Parnes HL, Wright J, Lee MJ, Chung EJ, Trepel JB, Sparreboom A, Chen C, Jones E, Steinberg SM, Daniels A, Figg WD, Dahut WL (2005) A phase II study of perifosine in androgen independent prostate cancer. Cancer Biol Ther 4:1133–1137 26. Dasmahapatra GP, Didolkar P, Alley MC, Ghosh S, Sausville EA, Roy KK (2004) In vitro combination treatment with perifosine and UCN-01 demonstrates synergism against prostate (PC-3) and lung (A549) epithelial adenocarcinoma cell lines. Clin Cancer Res 10:5242–5252. doi:10.1158/1078-0432.ccr-03-0534 27. Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH, Wiedemeyer R, Stegh AH, Bradner JE, Ligon KL, Brennan C, Chin L, DePinho RA (2007) Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 318:287–290. doi:10.1126/science.1142946 28. De J, Brown RE (2010) Tissue-microarray based immunohistochemical analysis of survival pathways in nodular sclerosing classical Hodgkin lymphoma as compared with non-Hodgkin’s lymphoma. Int J Clin Exp Med 3:55–68 29. Llovet JM, Bruix J (2008) Molecular targeted therapies in hepatocellular carcinoma. Hepatology 48:1312–1327. doi:10.1002/ hep.22506 30. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, Evers BM (2010) PI-103 and sorafenib inhibit hepatocellular

267 carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res 30:4951–4958 31. Huynh H, Ngo VC, Koong HN, Poon D, Choo SP, Thng CH, Chow P, Ong HS, Chung A, Soo KC (2009) Sorafenib and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma. J Cell Mol Med 13:2673–2683. doi:10.1111/j.1582-4934.2009.00692.x 32. Manov I, Pollak Y, Broneshter R, Iancu TC (2011) Inhibi tion of doxorubicin-induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib—the role of extracellular signal-regulated kinase counteraction. FEBS J 278:3494–3507. doi:10.1111/j.1742-4658.2011.08271.x 33. Piguet AC, Saar B, Hlushchuk R, St-Pierre MV, McSheehy PM, Radojevic V, Afthinos M, Terracciano L, Djonov V, Dufour JF (2011) Everolimus augments the effects of sorafenib in a syngeneic orthotopic model of hepatocellular carcinoma. Mol Cancer Ther 10:1007–1017. doi:10.1158/1535-7163.mct-10-0666 34. Strowski MZ, Cramer T, Schafer G, Juttner S, Walduck A, Schipani E, Kemmner W, Wessler S, Wunder C, Weber M, Meyer TF, Wiedenmann B, Jons T, Naumann M, Hocker M (2004) Helicobacter pylori stimulates host vascular endothelial growth factorA (vegf-A) gene expression via MEK/ERK-dependent activation of Sp1 and Sp3. FASEB J 18:218–220. doi:10.1096/fj.03-0055fje 35. Chaparro M, Gonzalez Moreno L, Trapero-Marugan M, Medina J, Moreno-Otero R (2008) Review article: pharmacological therapy for hepatocellular carcinoma with sorafenib and other oral agents. Aliment Pharmacol Ther 28:1269–1277. doi:10.1111/j.1365-2036.2008.03857.x 36. Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, Schwartz B, Simantov R, Kelley S (2006) Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 5:835–844. doi:10.1038/nrd2130 37. Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG (1993) Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 53:3976–3985 38. Guidetti A, Carlo-Stella C, Locatelli SL, Malorni W, Mortarini R, Viviani S, Russo D, Marchiano A, Sorasio R, Dodero A, Farina L, Giordano L, Di Nicola M, Anichini A, Corradini P, Gianni AM (2014) Phase II study of perifosine and sorafenib dual-targeted therapy in patients with relapsed or refractory lymphoproliferative diseases. Clin Cancer Res 20:5641–5651. doi:10.1158/1078-0432.ccr-14-0770

13