Effects And Mechanism Studies Of Combined Notch1 And EGFR Inhibition In Head And Neck Squamous Cell Carcinoma

Background:Head and neck squamous cell carcinoma(HNSCC)is the sixth most common cancer in the world.Recently,organ-preservation therapies for HNSCC with combined chemoradiation and surgeries have emerged.As an oral EGFR inhibitor,Erlotinib has been approved for treatment of patients with locally advanced or metastatic non-small cell lung cancer(NSCLCs)and advanced pancreatic cancer,which has shown great clinical promise.In a preliminary study of chemical treatment of HNSCC,improvements in symptoms were observed in 7 of 10 patients.Recently,EGFR inhibition was reported to promote epithelial-to-mesenchymal transition(EMT),increase the number of cancer-associated fibroblasts,and activate Notch expression.Thus,development of optimal combinatorial therapies to eliminate the resistance to Erlotinib is important.Notchl is overexpressed in HNSCC.Previously,our research revealed common mutations in the Notchl gene in HNSCC,which is observed in 43%of Asian patients.A considerable number of these mutations can activate Notchl pathway.Chemical inhibitors of γ-secretase can suppress proteolysis-dependent activation of Notchl.PF-03084014 is a potent and selective inhibitor of y-secretase activity and is generally safe and well tolerated by oral administration at doses equal to or less than 150 mg of BID in phase I clinical trials.Crosstalk between Notchl and EGFR signaling in cell proliferation and cancer expression has been observed in genomics.Notch1-dependent regulation of EGFR has been described in several types of cancers.Thus,we sought to determine how two signaling pathways interplay by assessing the effects of dual-pathway inhibition.We found that none of the pathway inhibition is functional for cell survival in HNSCC.Dual inhibition using Erlotinib and PF-03084014 resulted in a marked decrease in cell proliferation,an increase in cell death,and an induction of apoptosis in a xenograft model.These results suggest a correlation between the EGFR and Notchl signaling pathways in HNSCC and a rationale for combined blockade.Objective1.To observe the effects of single and combined application of PF-03084014 and Erlotinib on the proliferation of head and neck squamous cell carcinoma cells in vitro and in vivo;2.To determine the impact of single and combined use of PF-03084014 and Erlotinib on the invasive ability of head and neck squamous cell carcinoma cells.3.To clarify the effect of the combination of PF-03084014 and Erlotinib on the apoptosis of head and neck squamous cell carcinoma cells in vitro and in vivo;4.To investigate the interactions between Notch1 and EGFR pathways and the molecular biological mechanisms of combined treatment on head and neck squamous carcinoma cells.Methods1.The half maximal inhibitory concentration(IC50)of each cell lines was calculated after single drug administration.Western blot was used to verify the inhibitory effects of PF-03084014 and Erlotinib on Notch1 and EGFR pathways.2.CCK-8 and colony formation assays were used to detect changes on cell proliferation after combined administration.Transwell invasion assay was used to detect changes in cell invasive ability.Flow cytometry,Tunel staining and cleaved Caspase-3 immunofluorescence staining were used to detect early apoptosis.3.HNSCC xenograft tumor model was constructed to observe the changes of tumor growth after single and combined administration.Ki-67 and Tunel staining were used to observe the changes of tumor proliferation and apoptosis.Notch1 and p-EGFR immunohistochemistry staining was used to detect the status of Notchl and EGFR pathways after treatment.4.Western blot was used to determine the effect of the two inhibitors on EGFR downstream signaling pathway and to explore the interaction between the two novel pathways.Results1.HNSCC exhibited complete inhibition of Notchl and EGFR pathways after treated with Erlotinib or PF-03084014 at the concentrations of IC50.The half maximal inhibitory concentration(IC50)of Erlotinib or PF-03084014 ranged from 0.1 to 20μM or 20 to 35 μM,respectively.Compared to HN6 and CAL27 cells,HN4,HN13 and UMSCC38 cells showed relatively insensitive to Erlotinib.All five cell lines appeared to be resistant to PF-03084014.The signaling inhibitory efficiency of the two inhibitors was detected by assessing the expression of p-EGFR(phosphorylated form of EGFR)or NICD in HN6,CAL27,and UMSCC38 cell lines.We found that in HN6 and CAL27 cells,the expression of p-EGFR decreased apparently when the concentration of Erlotinib reached 0.μM;in UMSCC38 cells,the expression of p-EGFR did not present a decrease until the concentration of Erlotinib reached 5 μM.However,the expression of NICD in all the three cell lines dropped rapidly when the concentration of PF-03084014 reached 5μM.2.Combined treatment resulted in synergistic reduced cell proliferation and invasion and induced cell apoptosis in HNSCC.We then investigated whether dual inhibition showed any additive effects on cell proliferation.After 3 days’ treatment,the addition of PF-03084014 enhanced cell growth at doses from 10 to 20 μM in HN6 and CAL27 cells,but not evident in UMSCC38 cells.The addition of Erlotinib gradually inhibited cell proliferation as the concentration increased.However,this trend of enhanced cell growth induced by PF-03084014 was undetectable after cells had combined treatment with Erlotinib(0.1μM),suggesting the necessity of combined treatment of Erlotinib when treating cells with PF-03084014.To verify the invasive alteration in cells after treatment with Erlotinib,we performed Transwell assays in HN6 and CAL27 cells.Markedly,we found that low doses’treatment of Erlotinib(0.001 to 0.1 μM or 0.001 to 0.01 μM)enhanced migratory ability in HN6 or CAL27 cells.Likewise,we detected the effect of PF-03084014 treatment.We discovered increased invasion in both HN6 and CAL27 cell lines when concentration of PF-03084014 reached 2 to 20 μM,after which the invasive ability reduced.Importantly,combined treatment of Erlotinib and PF-03084014 dramatically attenuated invasive ability in both HN6 and CAL27 cells lines compared to the single treatment.To determine the function of Erlotinib and PF-03084014 in cell cycle,we performed Flow cytometry analysis in CAL27 cells after treated with Erlotinib or PF-03084014 for 3 days.The results revealed that Erlotinib predominantly induced cell cycle arrest in the G1 phase,whereas PF-03084014 slightly increased the number in the G2 phase.Moreover,combined inhibition resulted in growth arrest characterized by an increase of cells in the G1 phase and a decrease in the G2 phase.We then performed Annexin V-FITC/propidium iodide(PI)staining to detect the apoptosis rates.We found that when CAL27 cells were treated with both PF-03084014 and Erlotinib for three days,early apoptosis was substantially increased,whereas single drug treatment had minimal effects.Terminal deoxyribonucleotidyl transferse(TdT)-mediated biotin-16-dUTP nick-end labeling(TUNEL assay)was also utilized to investigate cell apoptosis.After 3 days’ combined treatment,cells exhibited enhanced positive TUNEL staining,compared to the cells treated with singe drugs.Activation of Caspase-3 in different cell types has been reported to be required for cell death.As a result,we examined Cleaved Caspase-3 by immunofluorescence in HN6 cells after treatment.As anticipated,the expression of Cleaved Caspase-3 in cells after combined treatment was much higher than the cells with single treatment,indicating the induction of early apoptosis induced by combined treatment of PF-03084014 and Erlotinib.Apart from the caspase family,as a member of the pro-survival subfamily,Bcl-2 also plays a pivotal role in cancer cell survival.The results of Western blot analysis also verified enhanced apoptosis as both Cleaved Caspase-3 and Bcl-2 protein expression was increased induced by combined therapy.3.Combined treatment induced tumor growth reduction and synthetic lethality in vivo.To further investigate the potential efficacy of combined inhibition of the two pathways,we performed a preclinical therapeutic drug trial in vivo using a xenograft tumor model of CAL27 cells in mice.After three weeks’ treatment,all mice were sacrificed and the tumors were dissected from mice.All mice in the control group(group 4,n=5)developed large tumors with an average size of 382 mm3 by day 21,whereas mice in the Erlotinib treatment group(group 1,n=5)developed much smaller tumors with an average size of 159 mm3.PF-03084014 treatment in group 3(n=5,average size=234 mm3)attenuated the tumor size.By contrast,combined treatment of PF-03084014 and Erlotinib effectively abrogated tumor growth(group 3,n=5,average size=71 mm3,***P<0.001)To assess the proliferative ability of cells in the four groups,the Ki67 staining was assessed in the tumor tissue sections.Combined treatment significantly inhibited mitogenesis as indicated by a reduction in Ki67 staining.Furthermore,we performed TUNEL assays to detect cell apoptosis in xenograft tumor tissues.The apoptosis quantification results demonstrated that tumors treated with PF-03084014 and Erlotinib exhibited enhanced apoptosis compared with tumors with PF-03084014 or Erlotinib single agent treatment,suggesting the synthetic lethality effects in HNSCC xenograft tumors.Tumor tissues were further analyzed by IHC to detect the efficacy of the inhibitors.In the control group gavaged with DMSO,both Notchl and p-EGFR were overexpressed.Nevertheless,we found that although Erlotinib treatment alone efficiently inhibited p-EGFR expression,it induced NICD translocation into the nucleus.In addition,PF-03084014 treatment alone decreased Notchl membranous expression and NICD translocation.By comparison,combination treatment decreased p-EGFR and Notch1 expression compared with the other groups.4.Dual pathway inhibition had synergistic inhibitory effects on the PI3K/AKT pathway.PI3K/AKT signaling is emerging as a central player in tumorigenesis in several cancer types,including HNSCC[33].Erlotinib[34]and Notchl inhibitor[35]could both function as anti-tumoral therapeutics by targeting the PI3K/AKT pathway.We therefore performed Western blot analysis to test whether combined inhibition effected PI3K/AKT pathway.As shown in HN6 and CAL27 cell lines(Figure 5A-B),the protein expression of EGFR and AKT remained almost unchanged.The levels of p-EGFR were downregulated after both single and combined treatment.However,p-AKT expression showed a drastic increase in two cell lines with PF-03084014 therapy.Furthermore,the inhibition of EGFR by Erlotinib reduced the expression of p-AKT,showing a synthetic inhibition in PI3K/AKT pathway.ConclusionsIn the present study,We found that none of the pathway inhibition is functional for cell survival in HNSCC.Dual inhibition using Erlotinib and PF-03084014 resulted in a marked decrease in cell proliferation,an increase in cell death,and an induction of apoptosis in a xenograft model.One mechanism has been found in our study that PF-03084014 alone could activate the PI3K-AKT pathway,the downstream of EGFR signaling.Meanwhile,Erlotinib alone could activate Notch1 downstream translation.Combined treatment of PF-03084014 and Erlotinib reversed PF-03084014-or Erlotinib-induced signaling activation.These results suggest a correlation between the EGFR and Notch1 signaling pathways in HNSCC and a rationale for combined blockade.