Proteasome inhibitor interacts synergistically with autophagy inhibitor to suppress proliferation and induce apoptosis in hepatocellular carcinoma

BACKGROUND:

The ubiquitin‐proteasome system and autophagy‐lysosome system are 2 major protein degradation pathways in eukaryotic cells, which are tightly linked to cancer. Proteasome inhibitors have been approved in clinical use against hematologic malignancies, but their application in solid tumors is uncertain. Moreover, the role of autophagy after proteasome inhibition is controversial.

METHODS:

Two proteasome inhibitors, 2 autophagy inhibitors, and 3 hepatocellular carcinoma (HCC) cell lines were investigated in the current study. In vitro, cell proliferation was evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, cell apoptosis was evaluated by flow cytometry analysis of annexin‐V/propidium iodide staining, and autophagy was evaluated by green fluorescent protein‐light chain 3 (GFP‐LC3) redistribution and LC3 Western blot analysis. In vivo, Ki‐67 staining was used to detect cell proliferation, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL) staining was used to detect apoptosis, and electron microscopy and p62 immunohistochemical staining were used to detect autophagy.

RESULTS:

Proteasome inhibitors suppressed proliferation, induced apoptosis, and activated autophagy in HCC cell lines in vitro, and autophagy exerted a protective role after proteasome inhibition. In vivo, anticancer effects of bortezomib on the MHCC‐97H orthotopic model (human HCC cells) were different from the effects observed on the Huh‐7 subcutaneous model (human HCC cells). The autophagy inhibitor chloroquine interacted synergistically with bortezomib to suppress proliferation and induce apoptosis in both tumor models.

CONCLUSIONS:

The current results indicated that simultaneous targeting of the proteasome and autophagy pathways may represent a promising method for HCC treatment. Cancer 2012. © 2012 American Cancer Society.     

HGF/c-Met在肝细胞癌中作用的研究进展

肝细胞癌是世界上最具破坏力的癌症之一,其主要治疗方式为手术治疗,但大部分患者确诊时已失去手术机会,仅部分患者可行手术治疗,且术后的转移率及复发率较高,近期相关研究发现,肝细胞生长因子(HGF)/间充质-上皮转换受体(c-Met)轴的异常激活与HCC的转移潜力及预后不良相关,为肝细胞癌的靶向治疗提供新方向。目前针对HGF/c-Met信号传导途径的有效治疗药物仍处于研究重点,现就HGF/c-Met通路在肝细胞癌中的研究进展进行综述。     

索拉非尼治疗中晚期肝细胞癌的不良反应及其处理

背景与目的：SHARP（sorafenib hcc assessment randomized protocol）和ORIENTAL（sorafenib in patients in asia-pacific region with hepatocellular carcinoma）两个Ⅲ期临床试验证实,多激酶抑制剂索拉非尼在一定程度上能延长晚期肝癌患者的总生存期（overall survival,OS）,并显著改善无进展生存期（progress free survival,PFS）和延长疾病进展时间（time to progression,TTP）,但治疗过程中出现的各种不良反应在一定程度上会影响其临床治疗。本研究使用索拉非尼治疗25例中晚期肝细胞癌患者,并对其不良反应情况及临床处理进行总结。方法：选取2008年1月-2009年10月期间符合原发性肝癌临床诊断标准的晚期肝癌患者25例,给予索拉非尼治疗所有患者均满足以下标准：⑴化疗栓塞术后病情进展;⑵广泛门脉癌栓无法栓塞;⑶肝门部、腹膜后淋巴结或肺、骨等多发转移者;⑷弥漫乏血供肿瘤;⑸签署知情同意书。在开始服用索拉非尼治疗后的前12周,观察患者不同级别不良反应发生情况,并给予相应临床处理。结果：共25例患者接受索拉非尼治疗,其中男性17例,女性8例,年龄范围30～72岁,平均51.44岁。期间9例患者死亡,其中3例在服药开始的12周内死亡。3例患者中断索拉非尼治疗,其中2例为治疗不满12周停药,1例为服药5个月后停药。共20例患者符合观察标准。不良反应发生情况如下：手足皮肤反应（hand-foot-skin reaction,HFSR）4例（4/20）,腹泻4例（4/20）,脱发5例（5/20）,皮疹4例（4/20）,乏力8例（8/20）,白细胞和血小板减少4例（4/20）,高血压1例（1/20）,腹痛1例（1/20）。这些不良反应经临床处理后,20例患者均能继续接受索拉非尼治疗。结论：索拉非尼治疗中晚期肝细胞癌具有良好的安全性和良好的耐受性。     

Pilot study of combination chemotherapy of S-1, a novel oral DPD inhibitor, and interferon-alpha for advanced hepatocellular carcinoma with extrahepatic metastasis

BACKGROUND: To the authors' knowledge, there is no effective therapy for extrahepatic metastasis of hepatocellular carcinoma (HCC). In a pilot study, the results of combination therapy of S-1, a novel oral dehydropyrimidine dehydrogenase (DPD) inhibitor, and interferon-alpha (IFN-alpha) are reported for HCC patients with extrahepatic metastasis. METHODS: Twelve patients with extrahepatic metastasis of HCC were enrolled in the pilot study. S-1 was administered orally at a dose based on body surface area, twice daily after a meal, for 4 weeks. IFN-alpha was injected subcutaneously on Days 1, 3, and 5 of each week. One course consisted of consecutive administration for 28 days followed by 14 days rest. RESULTS: An objective response was observed in 3 (25%) of 12 patients. The overall 1-year survival rate was 61.7%. Grade 3 leukocytopenia was observed in 1 patient (8.3%). No severe toxicity or treatment-related deaths were observed. CONCLUSIONS: The combination therapy of S-1 and IFN-alpha appears to be highly efficacious, with low toxicity in patients with extrahepatic metastases of HCC. The combination chemotherapy of oral S-1 and subcutaneous IFN-alpha is a potentially promising treatment strategy for advanced HCC with extrahepatic metastasis.     

Sorafenib continuation or discontinuation in patients with unresectable hepatocellular carcinoma after a complete response

Aims

To assess the efficacy of continued administration of sorafenib for patients with unresectable hepatocellular carcinoma (HCC) treated with local regional therapy (LRT) after a complete response (CR), also, the adverse events of sorafenib after discontinuation of administration were observed.

Methods

Between April 2008 and May 2012, 956 consecutive patients with unresectable HCC treated with LRT (transarterial chemoembolization, radiofrequency ablation) combined with sorafenib were retrospectively investigated. Of these, 157 patients with a CR were enrolled: 102 of them continued to receive sorafenib (test group) and the other 55 stopped receiving sorafenib (control group).

Results

The median recurrence-free survival (RFS), post-complete response overall survival (pOS) and overall survival (OS) in the test and control groups were 11 months (95% CI: 6.1, 15.9), 25 months (95% CI: 20.7, 29.3) and 33 months (95% CI: 29.2, 36.8) and 12 months (95% CI: 10.4, 13.6), 28 months (95% CI 24.2, 31.8) and 34 months (95% CI: 30.8, 37.2) respectively. The differences in RFS, pOS and OS between the groups were not significant (P = 0.768, 0.797 and 0.730, respectively). The adverse events related to sorafenib resolved after discontinuation of administration and the quality of life (QoL) scores improved.

Conclusions

Patients with unresectable HCC who achieved a CR did not benefit from continued sorafenib in terms of RFS, pOS or OS. The adverse events of sorafenib were reversible, and discontinuation of sorafenib may improve the QoL of patients who have achieved a CR.     

Sorafenib therapy in advanced hepatocellular carcinoma: the SHARP trial

Until now, no effective systemic treatment options have been available for patients with unresectable advanced hepatocellular carcinoma (HCC). In the Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP), patients with unresectable advanced HCC with Child–Pugh liver function class A and who had not received prior systemic therapy, received either oral sorafenib (400 mg twice daily) or placebo until radiological and symptomatic progression. The two groups of patients were well balanced with respect to baseline characteristics. The study was stopped at the second planned interim analysis because of an advantage in the median overall survival (10.7 vs 7.9 months; hazard ratio: 0.69; 95% CI: 0.55–0.87; p < 0.001) and the median time to radiological progression (5.5 vs 2.8 months; p < 0.001) in the sorafenib arm. However, sorafenib was not able to increase the time to symptomatic progression. In terms of toxicity, there were more cases of diarrhea, weight loss, hand–foot skin reaction and hypophosphatemia among the patients receiving sorafenib, the majority of which were of grade 1 or 2 severity. The SHARP trial has demonstrated that sorafenib is effective in prolonging median survival and time-to-progression in patients with advanced HCC and that it is generally well tolerated with a manageable adverse events profile.     

索拉非尼治疗肝细胞癌的疗效评价及不良反应

目的：观察索拉非尼治疗肝细胞癌的临床疗效及不良反应。方法：回顾性分析我院2008年7月至2009年12月间应用索拉非尼400mg每日两次治疗21例肝细胞癌患者的临床疗效与不良反应。结果：2例由于在服药后1月内出现肝功能异常而停药,未进行肿瘤评价。19例接受过1次以上实体瘤治疗反应评价标准（RECIST Response Evaluation Criteria in Solid Tumours 1.0版）的评价,其中无客观缓解者,14例（73.7%）疾病稳定且稳定时间大于3月。5例（26.3%）持续肿瘤进展。药物不良反应最常见的为手足皮肤反应、高血压、腹泻,其次有脱发、乏力、纳差、口腔溃疡、肝功能异常、发热等,不良反应一般在服药后1-2周内出现,其中有2例不能耐受不良反应而减少药物剂量,2例由于肝功能异常而停药。结论：索拉非尼治疗肝细胞癌患者以疾病稳定者多见,应严密监测并及时处理不良反应,以保证持续用药。     

索拉非尼治疗肝细胞癌的疗效评价及不良反应

目的:观察索拉非尼治疗肝细胞癌的临床疗效及不良反应。方法:回顾性分析我院2008年7月至2009年12月间应用索拉非尼400mg每日两次治疗21例肝细胞癌患者的临床疗效与不良反应。结果:2例由于在服药后1月内出现肝功能异常而停药,未进行肿瘤评价。19例接受过1次以上实体瘤治疗反应评价标准(RECIST Response Evaluation Criteria in Solid Tumours 1.0版)的评价,其中无客观缓解者,14例(73.7%)疾病稳定且稳定时间大于3月。5例(26.3%)持续肿瘤进展。药物不良反应最常见的为手足皮肤反应、高血压、腹泻,其次有脱发、乏力、纳差、口腔溃疡、肝功能异常、发热等,不良反应一般在服药后1-2周内出现,其中有2例不能耐受不良反应而减少药物剂量,2例由于肝功能异常而停药。结论:索拉非尼治疗肝细胞癌患者以疾病稳定者多见,应严密监测并及时处理不良反应,以保证持续用药。     

Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma

Background:

Sorafenib is the only drug approved for the treatment of hepatocellular carcinoma (HCC). The bioenergetic propensity of cancer cells has been correlated to anticancer drug resistance, but such correlation is unclear in sorafenib resistance of HCC.

Methods:

Six sorafenib-naive HCC cell lines and one sorafenib-resistant HCC cell line (Huh-7R; derived from sorafenib-sensitive Huh-7) were used. The bioenergetic propensity was calculated by measurement of lactate in the presence or absence of oligomycin. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, and siRNA of hexokinase 2 (HK2) were used to target relevant pathways of cancer metabolism. Cell viability, mitochondrial membrane potential, and sub-G1 fraction were measured for in vitro efficacy. Reactive oxygen species (ROS), adenosine triphosphate (ATP) and glucose uptake were also measured. A subcutaneous xenograft mouse model was used for in vivo efficacy.

Results:

The bioenergetic propensity for using glycolysis correlated with decreased sorafenib sensitivity (R²=0.9067, among sorafenib-naive cell lines; P=0.003, compared between Huh-7 and Huh-7 R). DCA reduced lactate production and increased ROS and ATP, indicating activation of oxidative phosphorylation (OXPHOS). DCA markedly sensitised sorafenib-resistant HCC cells to sorafenib-induced apoptosis (sub-G1 (combination vs sorafenib): Hep3B, 65.4±8.4% vs 13±2.9% Huh-7 R, 25.3± 5.7% vs 4.3±1.5% each P<0.0001), whereas siRNA of HK2 did not. Sorafenib (10 mg kg⁻¹ per day) plus DCA (100 mg kg⁻¹ per day) also resulted in superior tumour regression than sorafenib alone in mice (tumour size: −87% vs −36%, P<0.001).

Conclusion:

The bioenergetic propensity is a potentially useful predictive biomarker of sorafenib sensitivity, and activation of OXPHOS by PDK inhibitors may overcome sorafenib resistance of HCC.     

MicroRNA-93 activates c-Met/PI3K/Akt pathway activity in hepatocellular carcinoma by directly inhibiting PTEN and CDKN1A

To assess the role of microRNAs (miR) in hepatocellular carcinoma (HCC), we performed comprehensive microRNA expression profiling using HCC cell lines and identified miR-93 as a novel target associated with HCC. We further verified miR-93 expression levels in advanced HCC tumors (n=47) by a direct PCR assay and found that elevated miR-93 expression level is significantly correlated with poor prognosis. Elevated miR-93 expression significantly stimulated in vitro cell proliferation, migration and invasion, and additionally inhibited apoptosis. We confirmed that miR-93 directly bound with the 3′ untranslated regions of the tumor-suppressor genes PTEN and CDKN1A, respectively,and inhibited their expression. As a result of this inhibition, the c-Met/PI3K/Akt pathway activity was enhanced. IHC analysis of HCC tumors showed significant correlation between c-Met protein expression levels and miR-93 expression levels. Knockdown of c-Met inhibited the activation of the c-Met/PI3K/Akt pathway regardless of hepatocyte growth factor (HGF) treatment, and furthermore reduced the expression of miR-93 in these HCC cells. miR-93 also rendered cells to be more sensitive to sorafenib and tivantinib treatment. We concluded that miR-93 stimulated cell proliferation, migration, and invasion through the oncogenic c-Met/PI3K/Akt pathway and also inhibited apoptosis by directly inhibiting PTEN and CDKN1A expression in human HCC.     

Sorafenib in combination with transarterial chemoembolization and bronchial arterial chemoinfusion in the treatment of hepatocellular carcinoma with pulmonary metastasis

Aim: Hepatocelluar carcinoma (HCC) with pulmonary metastasis is considered incurable. This study addresses the efficacy of the combination of systemic therapy using sorafenib and local treatment using transarterial chemoembolization (TACE) for intrahepatic and bronchial transarterial chemoinfusion (TAI) for pulmonary lesions for this condition. Methods: In all, 52 HCC patients with pulmonary metastasis were treated with sorafenib and TACE/TAI for intrahepatic and intrapulmonary lesions. Response to treatment, progression‐free survival (PFS), overall survival (OS) and treatment‐induced adverse effects were analyzed. Results: With a median follow‐up time of 11.4 months, radiologically confirmed complete response (CR), partial response (PR), stable disease and disease progression for intrahepatic disease were observed in 0, 22, 23 and seven patients, respectively; radiologically confirmed CR, PR, stable disease and disease progression observed for intrapulmonary lesions were in 1, 8, 25 and 18 patients, respectively. Median OS and PFS was 12.0 and 10.0 months, respectively. Median OS of patients who achieved response (i.e., CR + PR + stable disease) in their gross lesion(s) was 14.0 and 13.0 months, respectively, as compared to 4.0 and 3.0 months for patients who progressed (P < 0.003). Significant prognosticators for OS and PFS included performance status, Barcelona Clinic Liver Cancer stage and response to treatment. The combined treatment strategy was well tolerated. Conclusion: The combination of sorafenib, TACE and TAI produced median OS and PFS of 12 and 10 months, respectively, in HCC patients with lung metastasis. The outcomes of patients who achieved a response to their gross lesions were significantly better than those who had disease progression. Further investigation is warranted to test the efficacy of this treatment combination.     

Sorafenib for advanced hepatocellular carcinoma (HCC): impact of rationing in the United Kingdom

Background:

The prognosis for hepatocellular carcinoma (HCC) is dependent upon tumour stage, performance status (PS), severity of underlying liver disease, and the availability of appropriate therapies. The unavailability of sorafenib may have a significantly adverse effect on the prognosis of UK patients with advanced HCC. During the study period, access to sorafenib was at the discretion of local health funding bodies, a process that may delay or deny access to the drug and that remains in place for Wales, Scotland, and Northern Ireland. Here, we attempt to address the impact of this system on patients with advanced HCC in the United Kingdom.

Methods:

This is a retrospective study performed in the two largest specialist hepatobiliary oncology units in the United Kingdom. Funding applications were made to local funding bodies for patients with advanced HCC for whom sorafenib was considered appropriate (advanced HCC not suitable for loco-regional therapies, compensated chronic liver disease, PS 0–2).

Results:

A total of 133 applications were made, of which 57 (43%) were approved and 76 (57%) declined. Demographics and prognostic factors were balanced between the two groups. This cohort had a number of adverse prognostic features: patients were predominantly PS 1–2; the majority had multifocal disease with the largest lesion being >5 cm; and macroscopic vascular invasion, metastases, and AFP >1000 ng ml⁻¹, were each present in one-third of cases. The median time from application to funding decision was 17 days (range 3–260 days). For the primary ‘intention-to-treat'' analysis, median overall survival was 4.1 months when funding was declined, and 9.5 months when funding was approved (hazard ratio (HR) 0.48; 95% CI 0.3186–0.7267; P=0.0005).

Conclusion:

These data support the use of sorafenib for patients with advanced HCC as an effective intervention. In the United Kingdom, this applies to a relatively small group of patients, estimated to total ∼800 per year who, unfortunately, do not survive long enough to themselves lobby for the availability of this drug. These data provide a comparison of sorafenib with supportive care and demonstrate the potential detrimental impact on patient outcomes of rationing health-care resources on the basis of cost.     

A case of sorafenib-induced severe thrombocytopenia during treatment of unresectable hepatocellular carcinoma

An 81-year-old male with unresectable hepatocellular carcinoma underwent transarterial chemoembolization (TACE) combined with sorafenib. Platelet count was normal before and after TACE treatment, after which oral administration of sorafenib (400 mg po bid) was initiated. During this period, the patient experienced significant diarrhea, so the dosage was reduced to 200 mg po bid. Later, the patient showed obvious gingival bleeding with progressive exacerbation, and his blood routine examination showed a platelet count of 2 × 109 cells/L. The patient was clinically diagnosed with extreme severe thrombocytopenia. The patient was advised to stop taking sorafenib and was immediately treated with hemostasis, platelet transfusion, and suspended red blood cells. After the above treatment, the patient's symptoms improved, and he was discharged. Up to the date of follow-up, there was no further bleeding.     

甲胎蛋白对索拉菲尼治疗进展期肝细胞癌的预后评价

近几年来以索拉菲尼为代表的分子靶向药物已成为进展期肝细胞癌综合治疗的热点之一,但目前对于索拉菲尼治疗进展期肝细胞癌的预后评价指标尚没有统一的标准。甲胎蛋白作为重要的肝癌肿瘤标志物,已经广泛地应用于肝细胞癌的筛查诊断,并被认为具有潜在的预测肝细胞癌患者的预后价值。本文总结了近几年来关于甲胎蛋白对索拉菲尼治疗进展期肝细胞癌预后评价的相关文献。从总体上看,甲胎蛋白对于索拉菲尼治疗进展期肝细胞癌预后评价作用是值得肯定的,并可以作为传统的基于影像学资料的肝细胞癌预后评价标准的补充,具有广泛的研究前景和应用价值。     

Adverse events of sorafenib in hepatocellular carcinoma treatment

Sorafenib is an oral multikinase inhibitor approved by the US Food and Drug Administration for treatment of the patients with surgically unresectable hepatocellular carcinoma (HCC). Sorafenib mitigates angiogenesis by targeting vascular endothelial growth factor receptors and platelet-derived growth factor receptors in endothelial cells and pericytes. Moreover, it suppresses cell proliferation via blockage of B-RAF and RAF1 of the mitogen-activated protein kinase pathway in tumor cells. Sorafenib has been the standard molecular targeted medication in the treatment of advanced-stage HCC patients ineligible for potentially curative interventional (radiofrequency or microwave ablation) or palliative trans-arterial chemoembolization (TACE) therapies for over a decade. However, it only increases overall survival by less than 3 months, and systemic exposure to sorafenib causes clinically significant toxicities (about 50% of patients). Given the high frequency and severity of these toxicities, sorafenib dose must be often reduced or discontinued altogether. In this review, we discussed the mechanism of sorafenib-associated adverse events and their management during HCC treatment.     

The combination of transcatheter arterial chemoembolization and sorafenib is well tolerated and effective in Asian patients with hepatocellular carcinoma: Final results of the START trial

This phase II, investigator‐initiated, prospective single‐arm multinational study ( ClinicalTrials.gov registration NCT00990860) evaluated sorafenib in combination with doxorubicin‐based transarterial chemoembolization (TACE) in patients with intermediate‐stage, unresectable hepatocellular carcinoma (HCC). Patients with histologically or clinically diagnosed HCC received TACE with interrupted dosing of sorafenib (sorafenib discontinued for 3 days before and 4–7 days after TACE). TACE/sorafenib cycles were repeated every 6–8 weeks. Primary and secondary objectives were, respectively: to evaluate the safety and tolerability of TACE combined with sorafenib, and also their efficacy. The full analysis set comprised 192 patients (mean age 56.1 years). Most were male (87.0%), Eastern Cooperative Oncology Group (ECOG) score 0 (81.8%), Child‐Pugh A (91.8%) and Barcelona Clinic Liver Cancer (BCLC) stage B (81.5%); 81.2% had chronic hepatitis B. Combined TACE/sorafenib was well tolerated, with only 8.1% of patients discontinuing owing to adverse events (AEs). The most common grade ≥3 AEs were palmar‐plantar erythrodysesthesia syndrome (15.1%) and decreased platelet count (10.9%). Serious AEs (SAEs) occurred in 52 patients during the study; however, only four were considered related to sorafenib. A mean of 2.7 TACE cycles were administered and 52.6% of patients achieved complete response in target lesions; 16.8% achieved partial response, and 5.8% had progression of disease as their best response, evaluated by modified RECIST. Median progression‐free survival and time to progression were 384 and 415 days, respectively, and the estimated 3‐year overall survival was 86.1%. This study suggests that the combination of TACE and sorafenib is well tolerated and efficacious; the interrupted sorafenib dosing schedule may have contributed to a considerably lower AE profile than observed in other combination trials.     

Targeted agents for the systemic treatment of advanced hepatocellular carcinoma

The prognosis for advanced hepatocellular carcinoma is poor and systemic therapy has historically been of limited benefit. However, novel targeted agents offer promise in improving patient outcomes. In a recent phase III study the oral multi-tyrosine kinase inhibitor sorafenib was demonstrated to have a survival advantage over a placebo in advanced hepatocellular carcinoma. Similar efficacy was demonstrated in a phase III trial limited to an Asian–Pacific group of patients. While these results are encouraging, it is vital to make further progress. In this review we examine current knowledge of targeted agents in advanced hepatocellular carcinoma. We also address some of the key issues that require exploration regarding the use of targeted agents in advanced hepatocellular carcinoma.