The Anti-tumor Effect Of Anti-ErbB2 Antibody H2-18 In Combination With Small Molecule Inhibitors On Trastuzumab-resistant Breast Cancer And Mechanism Of Action

ErbB2(human epidermal growth factor receptor 2,HER2),a member of human epidermal growth factor receptor family,is overexpressed in 25-30% of breast cancer and4-50% of gastric cancer.Its overexpression is associated with great aggression and poor prognosis of these cancers.Trastuzumab,an anti-ErbB2 humanized antibody,has acquired the approval of FDA in the treatment of ErbB2-overexpressing metastatic breast cancers and gastric cancers.However,many ErbB2-amplified breast cancers are resistant to trastuzumab,and the majority of trastuzumab-responsive cancers progress within 1 year treatment initiation.Thus,it has been an urgent need to develop new strategies to circumvent trastuzumab resistance.Previously,we have developed a novel fully human anti-ErbB2 antibody H2-18,which could greatly induce trastuzumab-resistant breast cancer cells to undergone programmed cell death(PCD).In contrast,trastuzumab could not effectively induce PCD.Nude mice bearing breast cancer xenografts had been used to assess the in vivo anti-tumor effect of H2-18.The results showed that H2-18 could inhibit the growth of trastuzumab-resistant breast cancer xenografts significantly.Our further study suggested that the anti-tumor activity of H2-18 in trastuzumab-resistant breast cancers might be attributable to its potent PCD-inducing ability.Aberrantly sustained activation of PI3K/AKT signaling pathway,one important mechanism underlying trastuzumab resistance,could be caused by PTEN deficiency and PIK3 CA activation mutation,and could still be inhibited by PI3 K inhibitors.GDC-0941,a pan-PI3 K inhibitor,draws the attention for its great antitumor activity.Src,a non-receptor tyrosine kinase that belongs to the Src family kinases,is the key node downstream of several pathways involving Trastuzumab resistance.Saracatinib,a Src inhibitor,could resensitive trastuzumab-resistant cancer cells to trastuzumab.In this study,we aimed to further improve the anti-tumor efficacy of H2-18 in trastuzmab-resistant breast cancers,and investigated the anti-tumor effect of H2-18 plus GDC-0941 and H2-18 plus Saracatinib on breast cancer cells.Firstly,a 3D cell proliferation assay was performed to assess the cell growth inhibitiory activity of H2-18 plus GDC-0941 and H2-18 plus saracatinib in both trastuzumab-sensitive(BT-474 and SKBR-3)and-resistant(HCC-1954 and HCC-1419)breast cancer cells.The results showed that the combination of H2-18 and GDC-0941 exihibited a more potent antitumoractivity than either drug alone in all of these cell lines.According to the Compusynsoware,H2-18 and GDC-0941 could inhibit in vitro cell proliferation synergistically.Similar results were obtained with H2-18 plus saracatinib.Western blot assay was performed to evaluate the changes in ErbB2 signaling pathway of BT-474 and HCC-1954 cells treated with H2-18 plus GDC-0941 and H2-18 plus saracatinib.GDC-0941 could inhibit Akt activation significantly and could activate Erk very slightly.H2-18 showed a potent activity on pErk inhibition,JNK/c-jun activation,but not pAkt inhibition.The combination of H2-18 and GDC-0941 did not exhibit a greater effect on decreasing p-Erk and increasing p-JNK/p-c-jun than H2-18 alone,but showed a more potent activity in reducing pAkt.Next,an Elisa assay was used to evaluate the level of pAkt(Ser473).However,the results showed that there is no statistical significance in the changes in pAkt(Ser473)level between H2-18 plus GDC-0941 and single drug alone.Similarly,H2-18 plus saracatinib was not more effective in inhibiting the ErbB2 signaling pathway than either drug alone.An Annexin V/PI assay was used to investigate the effect of the H2-18 plus GDC-0941 and H2-18 plus saracatinib on BT-474,SKBR-3,HCC-1954 and HCC-1419 cells.H2-18 plus GDC-0941 showed significantly enhanced PCD-inducing activity compared with each drug used alone.Similarly,H2-18 plus saracatinib also could induce cell death more obviously than either drug.We also assessed cell cycle distribution.In BT-474 cells,H2-18 plus GDC-0941 could induce a G1-phase cell cycle arrest more effectively than either H2-18 or GDC-0941 alone.However,in HCC-1954 cells,H2-18 plus GDC-0941 did not display a capability to induce a G1-phase arrest.The combination of H2-18 and saracatinib exhibited a stronger ability to induce trastuzumab-sensitive BT-474 and SKBR-3 cells in G1-phase than either H2-18 or saracatinib alone.In trastuzumab-resistant cell lines HCC-1954 and HCC-1419,H2-18 plus saracatinib did not induce more cell arrest in G1-phase than saracatinib alone.In addition,ROS level in BT-474 and HCC-1954 cells was determined by flow cytometry.The results showed that in HCC-1954 cells and BT-474 cells,both H2-18 and GDC-0941 could increase ROS production,whereas H2-18 plus GDC-0941 did not induce more ROS than GDC-0941 or H2-18 alone.Similarly,the ROS level induced by H2-18 plus saracatinib was not more than that of H2-18 alone.In vivo experiments also showed that the antitumor efficacy of H2-18 plus GDC-0941 was markedly superior to that of single agent.Similar results were observed with H2-18 and saracatinib.Thus,we concluded that the enhanced anti-tumor effect of H2-18 plus GDC-0941 or that of H2-18 plus saracatinib might be mainly attributable to the synergeticcell death-inducing ability of the combinations.Moreover,the ability of these drug combiantions to block both PI3 K and MAPK pathways may also contribute to their anti-tumor efficacy.In conclusion,H2-18 plus GDC-0941 or H2-18 plus saracatinib exhibits a potent anti-tumor ability,both in vivo and in vitro,suggesting its potential as an efficient strategy to overcome trastuzumab resistance in ErbB2-positive breast cancer.