| Objective:As the new targeting drugs for the treatment of HER2-positive advanced gastric cancer patients, the clinical trial’s preliminary result of lapatinib is not satisfactory, bypass-pathway activation is an important mechanism of targeting drug-resistant. In this study, we explored the effect of HER3activation to the cell growth and signalling transduction pathway in the lapatinib treatment of HER2-positive gastric cancer cells in vitro, and investigated the sensitivity to lapatinib by blocking HER3signaling in these cells and its molecular mechanism.Methods:(1) The baseline of HER families expression was assayed by western blot in HER2-positive gastric cancer cells NCI-N87、SNU-216and HER2-positive breast cancer cell SKBR3. The sensitivity of cancer cells to lapatinib after the growth factor NRG1activating the HER3receptor was assayed by western blot.(2)The effect of lapatinib and pan-HER inhibition AZD8931to growth of HER2-positive cancer cells NCI-N87was assayed by CCK-8kit. The combined effect of the two agents was tested by two-way ANOVA.The technology of siRNA gene silencing was performed to knock down the HER3gene to observe the sensitivity of HER2-positive cancer cells to lapatinib.(3) The molecular mechanism of HER3activation inducing HER2-positive gastric cancer cells resistant to lapatinib was assayed by western blot.(4) From154patients, who underwent surgery between2007-2010at Fudan University Shanghai cancer center, HER3、p-HER3was assessed by immunohistochemistry in tissue microarrays.Results:(1)Western blot assay showed that the expression of HER3and p-HER3proteins were upregulated at the baseline in HER2-positive gastric cancer cells NCI-N87、SNU-216and breast cancer cell SKBR3. Colony formation assays and quantification analyses revealed that with the stimulation of HER3there is a significant increase in the emergence of lapatinib-resistant colonies (P<0.0001Two-way ANOVA),AZD8931in combination with lapatinib could reverse the HER3activation-induced resistance.(2) The growth inhibition of lapatinib and AZD8921to these cancer cells was dose-dependent. The combined effect of the two agents was synergistic in NCI-N87(P<0.001Two-way ANOVA).Compared with scrambled siRNA negative control, the CCK-8assays confirmed that HER3genes silencing significantly enhanced lapatinib effectiveness (P<0.001Two-way ANOVA).(3) Western blot assay showed that lapatinib can downregulate the expression of P-HER2、p-Akt、p-Erk proteins, while lapatinib had no influence on the expression of HER2、HER3、p-HER3、Akt、Erk1/2proteins. In HER2-positive cancer cells,p-HER3、 p-Akt protein treated by the HER3activitor NRG1was significantly upregulated,p-Erk was also upregulated in breast cancer cell SKBR3. When these cancer cells treated by lapatinib and/or AZD8931, p-HER2、p-HER3、p-Akt proteins was downregulated more significantly, while p-Erk downregulated correspondingly.(4) The expression of HER3、p-HER3in HER2-positive tumor sites was significantly higher than that in HER2-negative tissues (P<0.0001). It was found that HER3、p-HER3ratio were96.3%and81.5%for the group with HER2-positive tissues.Conclusions:The HER3receptor can activate the downstream signaling nope p-Akt inducing lapatinib resistance in HER2-positive gastric cancer, but had no significant influence on Ras-MAPK pathway. Blocking the HER3receptor increased the sensitivity of lapatinib in HER2-positive cancer cells, and effectively inhibited the activation of PI3K-Akt pathway. Most HER2-positive gastric cancer patients exist the activation of HER3bypass pathway. |
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