Antitumor Activities And Mechanism Of Pan-HER Inhibitor PF299804 In Gastric Cancer Stem Cells

Background and Objective:Gastric cancer is one of the most common malignant tumors that occurs in digestive tract,with high incidence and mortality,especially in China.Many gastric cancer patients were diagnosed as advanced disease at the time of first consultation.The effects of chemotherapy and targeted therapy in gastric cancer patients are still unsatisfied,and the postoperative recurrence and distal metastasis are the leading cause of death in gastric cancer patients.Nowadays,the theory of cancer stem cells had been proposed for the explanation of the origin,recurrence,distal metastasis and drug resistance of malignant tumors.The characteristics of cancer stem cells such as tumorigenicity,self-renewal and multiple differentiation potential have been the topics of this research field.Thus,gastric cancer stem cell is one of the most promising treatment targets for gastric cancer in the future.The most important therapeutic target of gastric cancer is HER2,while results of To GA trial had confirmed the effect of trastuzumab plus chemotherapy as the standard first-line treatment for HER2 positive advanced gastric cancer patients.However,many patients become resistance to the treatment of trastuzumab within 1 year.HER2is belong to the Human Epidermal Growth Factor Receptor(HER)family,which plays an important role on tumorigenesis,proliferation,invasion,signal transduction,angiogenesis and metastasis of malignant tumors.Overexpression of HER family was observed in many cancers,while the efficiency and safety of anti-HER therapy had been confirmed in many preclinical and clinical studies.It is becoming more and more distinct that therapeutic strategy targets two or more members of HER family(Pan-HER)has a superior efficiency than which target single receptor.Many Pan-HER inhibitors have also shown their specific superiority in clinical trials and clinical practice.Nowadays,a phaseⅡtrial of PF299804,a Pan-HER inhibitor which targets EGFR,HER2 and HER4,had demonstrated that PF299804 functions as a single agent in HER2 positive gastric cancer patients with an encouraging result.Our team had previously identified gastric cancer stem cells in primary tumor tissue from gastric cancer patients,while these cells showed high expression level of EGFR.In this research,we investigated the antitumor activities of PF299804 in gastric cancer stem cells in vitro and in vivo.Furthermore,high-throughput RNA-sequencing was used to explore the differential genes and signal pathways after PF299804 treated.Meanwhile,we analyzed the prognostic value of HBXIP and MYC expression in tumor tissue through IHC.Materials and Methods:Western Blot and IHC were applied to investigate the expression level of EGFR,HER2 and HER4 in gastric cancer cell lines and gastric cancer stem cells.Then online survival analysis was conducted in Kaplan Meier plotter website to investigate the relationship between EGFR and HER2 expression and prognosis of gastric cancer patients.For in vitro experiments,CCK-8 assay was used to compare the inhibitory effect of PF299804,gefitinib and trastuzumab in gastric cancer stem cells.Transwell model,soft agarose colony formation assay,flow cytometry and limiting dilution assay were applied to investigate the effect of PF299804 on the ability of invasion,colony formation,cell cycle,cell apoptosis and self-renewal of gastric cancer stem cells.Meanwhile,gastric cancer stem cells were subcutaneously injected in BALB/c nude mice in order to investigate the antitumor activities of PF299804 in vivo.Tumor volumes were measured per 2 days,at the end of treatment,tumor samples were collected and weighted.RT-PCR and immunofluorescence staining were performed to examine the expression level of EGFR and HER2 in tumor samples after PF299804treatment.In order to explore the molecular mechanism of antitumor activities of PF299804,the expression level and phosphorylation level of HER family and relevant signaling pathways after treatment of PF299804 were detected by Western Blot.CO-IP was also applied to detect the formation of HER family dimers in gastric cancer stem cells.High-throughput RNA-sequencing was performed to analyzed the differential genes and signal pathways after PF299804 treatment.Finally,the significantly differential genes were confirmed by Western Blot at the protein level.Among these genes,HBXIP served as the differential gene in the downstream of HER family.Stable transformants of HBXIP overexpression in 0622 and 0603 were constructed through lentivirus transfection.Then Western Blot was applied to confirm the efficiency of stable transformants.CCK-8 assay and flow cytometry was applied to investigate the effect of HBXIP on proliferation and cell cycle in 0622 and 0603.CCK-8 assay was used to confirm the effect of HBXIP on proliferation inhibition induced by PF299804.Moreover,the interaction between HBXIP and MYC was also detected by CO-IP.RT-PCR was used to detect the expression level of MYC targeted genes after PF299804 treatment.Furthermore,we analyzed the expression level of HBXIP and MYC in tumor tissue,as well as the relationship between expression level of HBXIP and MYC and prognosis of gastric cancer patients.Results:(1)EGFR and HER2 had high expression level in gastric cancer stem cells and were associated with poor prognosis of gastric cancer patients.We used Western Blot and IHC to examine the expression level of EGFR,HER2and HER4 in gastric cancer cell lines and gastric cancer stem cells.The results of Western Blot showed that gastric cancer stem cells had a higher expression level of EGFR and HER2 when compared with gastric cancer cell lines,while results of IHC showed positive expression of EGFR and HER2 in xenografts.At the same time,the results of online survival analysis among gastric cancer patients in Kaplan Meier plotter website showed high expression level of EGFR and HER2 were associated with poor prognosis of gastric cancer patients(p<0.01).(2)PF299804,a Pan-HER inhibitor,significantly decreased the proliferation,colony formation,invasion and self-renewal abilities of gastric cancer stem cells in vitro,also induced cell cycle arrest and cell apoptosis.CCK-8 assay demonstrated that the proliferation ability of 0622 and 0603 was significantly inhibited by PF299804 when compared with single target agents(p<0.001).Soft agarose colony formation assay demonstrated that compared with control group,the colony formation ability of 0622 and 0603 was significantly inhibited after 48 hours treatment of PF299804(p<0.05).The results of Transwell model showed that treatment of PF299804 significantly decreased the invasion ability of gastric cancer stem cells(p<0.05).The results of cell cycle analysis showed that PF299804 could induce cell cycle arrest in gastric cancer stem cells in vitro(p<0.05).Through the cell apoptosis analysis,we found that PF299804 could also induce cell apoptosis in vitro(p<0.01).Meanwhile,results of limiting dilution assay showed that PF299804 could significantly inhibit the self-renewal ability of gastric cancer stem cells,when compared with control group(p<0.001).(3)PF299804 had a superior antitumor activity in vivo.For the xenograft model,we found that PF299804 had a superior antitumor activity in vivo.After 14 days of oral gavage,tumor volume and tumor weight were significantly decreased in treatment group.In 0622 group,tumor volume was335.27±126.27mm~3 in treatment group while it was 713.89±199.93mm~3 in control group at the end of treatment,with a statistical significance(p<0.001).And the tumor weight in treatment group was also significantly lower than that of control group(0.316±0.088g vs.0.205±0.089g,p=0.0117).In 0603 group,tumor volume was331.99±179.04mm~3 in treatment group while it was 953.75±313.83mm~3 in control group at the end of treatment,with a statistical significance(p<0.001).And the tumor weight in treatment group was also significantly lower than that of control group(0.505±0.212g vs.0.209±0.135g,p=0.0049).RT-PCR and immunofluorescence staining were also applied to investigate the expression level of EGFR and HER2 in tumor samples between two groups.Results showed that the expression level of EGFR and HER2 were significantly decreased in treatment group,compared with control group(p<0.05).(4)Molecular mechanism about the antitumor effect of PF299804 in gastric cancer stem cells.The results of Western Blot showed that PF299804 could significantly inhibit the phosphorylation level of HER family and relevant signaling pathways in 0622 and0603.Meanwhile,the formation of EGFR-HER2,EGFR-HER3,HER2-HER3 and HER3-p85 dimers were significantly inhibited by PF299804.Furthermore,we used high-throughput RNA-sequencing to investigate the differential genes after PF299804treatment.We found that the expression level of many genes that related with tumorigenesis,cell proliferation,signal transduction and other cellular functions had been changed.Among these genes,HBXIP was confirmed as the downstream differential gene of HER family,while overexpression transformants of HBXIP were constructed through lentivirus transfection,then the efficiency of lentivirus transfection was confirmed by Western Blot.Results of CCK-8 assay and cell cycle analysis showed that overexpression of HBXIP could significantly enhance the ability of proliferation and cell cycle progress in vitro(p<0.05),while overexpression of HBXIP might partly reserve the PF299804-induced cell proliferation inhibition(p<0.001).Moreover,we found PF299804 could inhibit the interaction between HBXIP and MYC,as well as the expression levels of MYC targeted genes.(5)Relationship between protein expression level of HBXIP and MYC,clinicopathological characteristics and prognosis of gastric cancer patients.Through IHC,we found that HBXIP highly expressed in tumor tissue when compared with paired adjacent tissue(p<0.01).Meanwhile,high expression level of HBXIP was significantly associated with lymph node metastasis(p=0.003),distal metastasis(p=0.009)and poor prognosis(log rank p=0.0021,HR=2.966,95%CI=1.483-5.934)of gastric cancer patients.High expression level of MYC was also significantly correlated with lymph node metastasis(p=0.003),distal metastasis(p=0.010)and poor prognosis(log rank p<0.001,HR=3.540,95%CI=1.846-6.789)in gastric cancer patients.Furthermore,co-expression of HBXIP and MYC correlated with the worst prognosis in gastric cancer patients(p<0.05),while it was an independent prognostic factor of gastric cancer patients(p=0.044).Conclusions:1.EGFR and HER2 had high expression level in gastric cancer stem cells and were associated with poor prognosis of gastric cancer patients.2.PF299804,a Pan-HER inhibitor,could significantly inhibit the proliferation,colony formation,invasion and self-renewal abilities of gastric cancer stem cells,as well as induce cell cycle arrest and cell apoptosis.PF299804 also had a superior antitumor activity in xenograft models,and it could decrease the expression level of EGFR and HER2 in tumor tissue.3.PF299804 could significantly inhibit the phosphorylation level of HER family and relevant signaling pathways in gastric cancer stem cells through the inhibition of HER family dimers.It could also inhibit the interaction between HBXIP and MYC,as well as the expression level of MYC targeted genes.Overexpression of HBXIP might partly reserve the PF299804-induced cell proliferation inhibition.4.Co-expression of HBXIP and MYC was associated with poor prognosis while it was an independent prognostic factor of gastric cancer patients.