Studies On The Effects And Mechanisms Of A Novel Drug Candidate CT-707 Targeting Intratumor Hypoxia Of HCC

Objectives:Intratumor hypoxia is a hallmark of hepatocellular carcinoma(HCC),as a negative prognostic factor,leading to therapeutic resistance and malignant progression.Various strategies have been developed to combat with the hypoxia microenvironment,aiming at achieving optimal anti-cancer activities against those hypoxic cancer cells.However,the present approaches like bioreductive prodrugs and hypoxia-inducible factors(HIFs)inhibitors only target hypoxia region inside the tumor mass,while sparing those normoxic tumor cells undisturbed,which may result in incomplete tumor regression and poor activity in clinical trials.If so,novel strategies should be exploited,in order to effectively target HCC cancer cells of intratumor hypoxic region,while suppress those normoxic cancer cells simultaneously.In the present study,we have screened a series of inhibitors,and CT-707 stood out by its superior anti-tumor effects under hypoxia in HCC cells.CT-707 was a novel FAK/IGF-1R/ALK inhibitor,which has been approved by China FDA for Phase Ⅰclinical trial.This study aimed to uncover the function and mechanism of CT-707 under hypoxia,providing a promising therapeutic strategy for HCC treatment.Methods:(1)SRB assay was used to detect anti-proliferation activity of chemotherapeutic agents on human HCC cells and normal liver cells.(2)Analysis of apoptosis was determined by propidium iodide staining.(3)Western blotting assay was used to detect the protein expression.(4)Real-Time PCR was used to detect the mRNA level.(5)Immunofluorescence analysis was used to detect the distribution of protein in cells and tumor tissues.(6)Plasmid or small interfering RNA was introduced to overexpress or silence related gene.(7)HepG2 xenografted athymic mice model was introduced to investigate the in vivo anti-cancer activity.Results:1.CT-707 exerts better anti-proliferative effects and induced apoptosis under hypoxia in HCC cell lines HepG2 and Bel-7402SRB assay was used to detect the cell viability under normoxia or hypoxia,and the results revealed that CT-707 exhibited better anti-proliferative effects under hypoxia;PI staining assay was employed to monitor apoptosis and showed CT-707 trigged apoptosis under hypoxia;the cleavage of PARP was analyzed by western blotting,and CT-707 resulted in a remarkable enhanced PARP cleavage under hypoxia than that under normoxia.2.HIF-la and FAK were not required for the improved anti-proliferative effects under hypoxiaWestern blot and real-time PCR results illustrated CT-707 failed to down-regulate the HIF-1α protein expression and the mRNA levels of HIF-1α target genes VEGF and Glut-1;we introduced HIF-1α plasmid or Cobalt dichloride(CoCl2)mimicking hypoxia to stabilize HIF-1α under normoxia,however,the anti-cancer efficacy of CT-707 was minimally influenced compared with that of control cells;then we suppressed the expression of HIF-1α using specific siRNA under hypoxia in HepG2 cells,and CT-707 still exhibited more potent anti-cancer efficacy in those HIF-1α-depleted cells under hypoxia.We further explored whether the superior anti-cancer activity of CT-707 under hypoxia was dependent on FAK.Western blot analyses revealed CT-707 exhibited similar inhibitory patterns against p-FAK under normoxia and hypoxia,in a concentration-dependent manner.Furthermore,CT-707 still exerted better anti-proliferative effects under hypoxia in FAK-depleted Bel-7402 cells.3.The effects under hypoxia by CT-707 is mediated by the kinase inhibition on IGF-1RWestern blot uncovered that CT-707 could inhibit the upregulation of p-IGF-1R protein expression triggered by IGF-1 or hypoxia.Then we used IGF-1 and insulin to stimulate IGF-1R activation under normoxia,thus mimicking the hypoxic activation of the pathway,and the IGF-1R activation could sensitize the normoxic cells to CT-707.4.Superior anti-proliferative effects of CT-707 was attributed to the nuclear translocation and activation of YAP mediated by IGF-1 R under hypoxiaWestern blot was determined to show that the total protein level of YAP was reduced by the knockdown of IGF-1R,which was accompanied by an upregulation of p-YAP levels,while in HepG2 cells,IGF-1 or insulin caused reduced the phosphorylation of YAP,implying IGF-1R might be a regulator upstreaming of YAP.The results of Western blot,immunofluorescence analysis and real-time PCR revealed that CT-707 could abate the loss of YAP phosphorylation,the accumulation of YAP protein in nuclear and the elevated mRNA levels of the YAP target genes CTGF,Cyr-61 and AREG caused by hypoxia.We then introduced YAP-5SA mutant,which lacks five serine phosphorylation residues,insensitive to phosphorylation,thus predominantly located in the nucleus.The exogenous mutant YAP-5SA significantly rescued the loss of viability of cells exposed to CT-707 under hypoxia.5.CT-707 arrests tumor growth on HCC xenograft models by inhibiting YAP pathwayCT-707 administration significantly inhibited growth of HepG2 xenograft tumors.Immunofluorescence analysis showed CT-707 could elicited apoptosis in hypoxic regions of the xenografted tumors.Furthermore,the treatment with CT-707 prevented the nuclear accumulation of YAP and attenuated the mRNA levels of AREG,CTGF and Cyr-61 in vivo.Conclusions:The present study showed the CT-707 possessed potent activities against HCC in vitro and in vivo models,and intriguingly,exhibited superior anti-tumor effects against those hypoxic HCC cells.The interference of hypoxia-activated IGF-1R-YAP axis,but not HIF-1α or FAK,was found to contribute to the enhanced anti-cancer activities in the hypoxic microenvironment,thus providing a promising therapeutic strategy for HCC patients,particularly those presenting intratumor hypoxia.