The Anti-tumor Effect Of Anti-HER2 Antibody H2-18 On Gastric Cancer And Its Mechanism

Human epidermal growth factor receptor 2(HER2),a tyrosine kinase receptor,is overexpressed in Multiple types of tumors.Its overexpression promotes proliferation,migration,invasion and angiogenesis of tumor,which is closely related to the poor prognosis of patients.Trastuzumab,an anti-ErbB2 humanized antibody which binds the extracellular segment structure domain ?,has acquired the approval of FDA in the treatment of ErbB2-overexpressing metastatic breast cancers and gastric cancers.However,clinical researches found that about 70% of patients with ErbB2-amplified breast cancer are resistant to Trastuzumab(primary Trastuzumab resistance).The majority of Trastuzumab-responsive cancers progress within one year after treatment initiation(acquired Trastuzumab resistance).This phenomenon is also found in the in the treatment of gastric cancer.Pertuzumab is another humanized antibody which targets the extracellular domain II of HER2.Due to the synergistic antitumor effect of Trastuzumab and Pertuzumab,the combination of these two drugs is approved by FDA as a first-line treatment for ErbB2-overexpressing breast cancer.Although clinical trials have confirmed that the combination of Trastuzumab and Pertuzumab can partially overcome Trastuzumab resistance,the objective response rate is only 25% and the total response rate is less than 8%.Therefore,it is an urgent need to overcome both primary and acquired resistance to Trastuzumab.The mechanisms of primary and acquired Trastuzumab resistance are different.Thus,the strategies to overcome primary Trastuzumab resistance may be unable to circumvent acquired Trastuzumab resistance.Previously,we have screened a novel fully human anti-ErbB2 antibody H2-18 by phage display technology,which recognizes extracellular domain I of HER2 and overcomes primary Trastuzumab resistance through inducing programmed cell death.In this study,we studied the anti-tumor effect of H2-18 in the acquired Trastuzumab resistant cancer cells,and the anti-tumor effect of the combination of H2-18 and Trastuzumab and Pertuzumab in gastric cancer.1.The anti-tumor effect of H2-18 on the acquired Trastuzumab resistant cancer cellsWe developed an acquired Trastuzumab resistance cell line NCI-N87-TraRT,which was derived from Trastuzumab sensitive gastric cancer cell line NCI-N87.As potent ability to induce programmed cell death(PCD)is the key mechanism of H2-18 to overcome primary Trastuzumab resistance,we examined cell death by using Annexin V and PI double staining.The results showed that in both NCI-N87 and NCI-N87-TraRT cell lines,H2-18 could induce PCD,whereas other antibodies could not.Moreover,H2-18 could induce PCD in a dose-dependent manner.H2-18 could induce more cells to death in NCI-N87-TraRT cells than that in NCI-N87 cells.Similar results were obtained in BT-474 and BT-474 TraR cells.Then,the in vitro cell proliferation inhibitory ability of H2-18 was determined by cell proliferation assays in both NCI-N87 and NCI-N87-TraRT cell lines.The results showed that no difference were observed in the growth inhibition of NCI-N87 and NCI-N87-TraRT cells.H2-18 could inhibit cell proliferation less effectively than Trastuzumab in NCI-N87 cells,whereas H2-18 could inhibit cell growth more effectively than Trastuzumab in NCI-N87-TraRT cells.Next,nude models bearing tumor xenografts were used to assess the in vivo anti-tumor efficacy of H2-18.We found that in NCI-N87 cells,H2-18 was comparable to Trastuzumab in inhibition of the growth of tumors,but was weaker than the combination of Trastuzumab and Pertuzumab.However,in NCI-N87-TraRT cells,H2-18 exhibited a more potent antitumor activity than both Trastuzumab and Trastuzumab plus Pertuzumab.To explore the possible mechanisms underlying the great antitumor ability of H2-18,Western Blot was used to examine the HER2 signaling pathways.The results showed that H2-18 could inhibit p-HER3,p-AKT and p-ERK in NCI-N87 cells,whereas H2-18 had little effect on phosphorylation of HER3,AKT and ERK in NCI-N87-TraRT cells.In addition,in both NCI-N87 and NCI-N87-TraRT cell lines,H2-18 could increase ROS levels with upregulation of p-JNK and p-c-jun.When JNK and c-jun were knocked down by using ROS scavengers or SiRNA,the ability of H2-18 to induce cell death was impaired significantly.Thus,we speculate that H2-18 could induce cell death through activating RIP1-ROS-JNK-c-jun pathway,resulting in overcoming acquired Trastuzumab resistance.2.The antitumor effect of H2-18 in combination with Trastuzumab and Pertuzumab in gastric cancerWe used Western Blot and flow cytometry to test the expression of HER2 in multiple gastric cancer cell lines.The results showed that only NCI-N87 and NCI-N87-TraRT cell lines overexpressed HER2.The in vitro cell proliferation assays showed that only in NCI-N87 and NCI-N87-TraRT cell lines,Trastuzumab plus Pertuzumab could inhibit cell growth more effectively than Trastuzumab,Pertuzumab and H2-18 alone.Trastuzumab plus H2-18 was more potent in cell growth inhibition than Trastuzumab plus Pertuzumab.Among all the antibody combiantions,Trastuzumab plus Pertuzumab plus H2-18 exhibited the maximum activity in cell growth inhibition.Moreover,only in NCI-N87 and NCI-N87-TraRT cell lines,Trastuzumab and H2-18 have synergistic antitumor effect,as well as H2-18,Trastuzumab and Pertuzumab.The colony formation assay showed that H2-18 plus Trastuzumab could inhibit colony formation similarly to Trastuzumab plus Pertuzumab in NCI-N87 cells,whereas H2-18 plus Trastuzumab inhibited colony formation more effectively than Trastuzumab plus Pertuzumab in NCI-N87-TraRT cells.In both NCI-N87 and NCI-N87-TraRT cell lines,Trastuzumab plus Pertuzumab plus H2-18 exhibited the most potent ability to inhibit the formation of colonies.The in vivo animal experiments showed that H2-18 plus Trastuzumab inhibited tumor growth more effectively than Trastuzumab plus Pertuzumab,whereas Trastuzumab plus Pertuzumab plus H2-18 increased tumor volumes to the maximum.Furthermore,The antibody combinations were superior to antibodies in inhibition of p-HER3,p-AKT and p-ERK.H2-18 plus Trastuzumab was comparable to Trastuzumab plus Pertuzumab in inhibiting p-HER3,p-AKT and p-ERK.Trastuzumab plus Pertuzumab plus H2-18 exhibited the most potent ability to inhibit the phosphorylation of HER3,AKT and ERK.More importantly,H2-18 and Trastuzumab plus Pertuzumab plus H2-18 could induce PCD with the increase of ROS and p-JNK and p-c-jun,whereas Trastuzumab,Pertuzumab and Trastuzumab plus Pertuzumab had little effect on ROS-JNK-c-jun pathways.Therefore,we conclude that Trastuzumab plus Pertuzumab plus H2-18 have synergistic antitumor effect,which is attributable to inhibition of both PI3K/AKT and RAS/MAPK pathways in gastric cancer and induction of PCD through binding domain I of HER2.In summary,Trastuzumab plus Pertuzumab plus H2-18 exihibits excellent antitumor activity,both in vivo and in vitro,suggesting the potential as new strategy to overcome Trastuzumab resistance.