Alterations Of The Glucolysis Process Influcence The Clinical Efficacy Of Sorafenib In The Treatment Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is one of the most frequent causes of malignancy related mortalities worldwide,which is much more serious here in China.Liver resection,liver transplantation and radiofrequency ablation were regarded as the curative treatments for patients at early stages.However,as few patients had specific symptoms and signs at the early stage,most patients were at advanced stages at their initial diagnosis and were thus not eligible for the curative treatments.The multikinase inhibitor sorafenib(NexavarR)was the first officially approved molecular target regiment for patients with advanced HCC by Food and Drug Administration(FDA).However,the survival benefit by sorafenib was relatively limited and the clinical effect was heterogeneous,due to the complex oncological characteristics of HCC and the resistance of HCC to sorafenib.It is quite essential and urgent to investigate the underlying mechanisms for the resistance to sorafenib in order to further improve the clinical prognosis of the HCC patients.The alternation of the extracellular signal-regulated kinase(ERK)1/2 pathway has long been regarded to influnce the initiaion and progression of many malignant cancers as well as the clinical outcomes of sorafenib treated HCC.As the new member of the Mitogen-activated protein kinase(MAPK)family,the role of ERK5 molecular pathway have been revealed to modulate the malignant behavior of several types of malignant tumors including leukemia,colorectal cancer and hepatocellular carcinoma.But to the best of our knowledge,no studies investigating the function of ERK5 pathway in the development of sorafenib resistance have been reported yet.Aerobic glycolysis is the mainstream of glucose metabolism in tumor tissues even though in the oxygen rich environment,which is also known as the "Warburg effect".In the expence of lower energy efficiency,aerobic glycolysis provided the tumor tissues with the energy of malignant biological behavior and the substrates of multiple biochemical reactions at a much more rapid pattern,in order to meet the urgency and massive needs of tumor growth,expansion and drug resistance.Aerobic glycolysis is thus regarded as the conerstone of malignant tumor initiation and progression.More and more attentions have been drawn to the realm of aerobic glysolysis in order to reveal the mechanisms of resistance to sorafenib treatment.The key enzymes of the aerobic glycolysis,which can modulate the rate of glucose metabolism as well as act as the kinase further regulating the downstream genes encoding for the proliferation,apoptosis,migration,et al,play a pivotal role in the molecular mechanism for the drug resistance.The pyruvate kinase M2(PKM2)is a key rate limiting glycolytic enzyme,which catalyzes the phosphoenolpyruvate to pyruvate.It is one of the frequently altered glycolytic enzymes in cancer cells.Lactate dehydrogenase(LDH),which is a key enzyme in the conversion of pyruvate to lactate under anaerobic environment,has been recognized as an indirect marker of the extent of tumor hypoxia,a key biological mechanism for the development of treatment resistance in cancer cells.PKM2 and LDHA have been reported to be correlated with the sorafenib resistance.Several reports have revealed that ERK-dependent phosphorylation and nuclear translocation of PKM2 is required for the autoregulation of PKM2 expression and PKM2-dependent expression of glycolytic genes,which are essential for the EGFR-promoted Warburg effect and tumorigenesis.The relationship between ERK1/2 and PKM2 have been found in glioma and hepatocellular carcinoma.Meanwhile the relationship between ERK5 and PKM2 has not been reported yet.The aim of the present study was to evaluate the relationship between ERK5 and PKM2 related glycolytic pathways,and its influence on the sorafenib resistance.Materials and methods1.We retrospectively analyzed the prognostic value of serm LDH level in 119 HBV related HCC patients treated by sorafenib.2.The expressions of LDHA in 38 hepatocellular carcinoma specimen treated by sorafenib and Human HCC cell lines were investigated by immunohistochemical staining.3.Three lentiviruses were constructed:an empty vector control(Control),stable knockdown of PKM2(shPKM2)and overexpressed PKM2(PKM2 OE).And the Huh-7 cell stably infected with shRNAs against PKM2 and PKM2 OE were established.4.The effects of ERK5 specific inhibitor XMD8-92 on the antitumor effects such as glucose consumption,lactate production,proliferation,apoptosis,cell cycle distrubition and migration of sorafenib were detected.The PKM2 and LDHA expression were detected at the mRNA and protein levels?5.The effects of PKM2 knockdown and overexpression on Huh-7 cell glucose consumption,lactate production,proliferation,apoptosis and migration were assessed.And the LDHA mRNA and protein expression were detected by RT-PCR and western blotting methods.6.The effcts of PKM2 overexpression on the Huh-7 cell glucose consumption,lactate production,proliferation,apoptosis and migration were assessed under the combined effects of XMD8-92 and sorafenib.And the LDHA mRNA and protein expression were detected by RT-PCR and western blotting methods.Results1.Pretreatment serum LDH level was an independent prognostic factor for both overall survival(OS)and progression-free survival(PFS).For patients whose pre-treatment LDH ? 221 U/L,increased LDH value after 3 months of sorafenib treatment predicted inferior OS and PFS.And patients with elevated pre-treatment LDH level predisposed to be featured with lower serum albumin,presence of macroscopic vascular invasion,advanced Child-Pugh class,advanced T category,higher AFP,and higher serum total bilirubin.2.The expression level of LDHA in the hepatocellular carcinoma tissues was higher than that in the paired peritumoral tissues.3.The Huh-7 cells stably infected with shPKM2 and PKM2-OE were successfully established.4.XMD8-92 could enhance the anti-tumor effects of sorafenib on HCC cells.And compared with sorafenib alone,sorafenib plus XMD8-92 significantly downregulated the expression level of PKM2 and LDHA in Huh-7 and Hep 3B cells.5.Downregulation/Overexpression of PKM2 sensitized/mitigated the anti-tumor effects of sorafenib,which was accompanied by the deceased/increased expression of LDHA.6.Overexpression of PKM2 reversed the growth-suppressive and proapoptotic effects of sorafenib as well as sorafenib plus XMD8-92 in Huh7 cells.And overexpression of PKM2 reversed the downregulation effect of XMD8-92 on LDHA.Conclusions:PKM2 related glycolysis could influence the sorafenib resistance,which could be modulated by the ERK5 specific inhibitor XMD8-92.Serum LDH level was a potentially prognostic factor in HCC patients treated by sorafenib in HBV endemic area.