| Gastric cancer is the second most common cancer world-wide, with an estimated 900,000 new cases per year. Although the incidence and mortality of gastric cancer have decreased markedly in most areas of the world over the past several decades, control of gastric cancer at the advanced stage remains difficult.Trastuzumab, a therapeutic monoclonal antibody directed to the human epidermal growth factor receptor-2(HER2), has been used as standard therapy in advanced HER2-positive breast cancers. It is also indicated in HER2-positive advanced gastric cancers in combination with chemotherapy. The international phase III trial of trastuzumab for gastric cancer showed a clinically and statistically significant benefit in terms of response rate, median progression-free survival, and median overall survival. Currently, trastuzumab is approved for the clinical treatment of gastric cancer patients with HER2-positive tumors.However, resistance to trastuzumab is common in both breast and gastric cancers. HER2 is overexpressed in ~25% of gastric cancer patients, who would supposedly benefit from trastuzumab therapy. Unfortunately, the response rate to trastuzumab among the patients is only 12.8%, which is much lower than that previously reported in breast cancer. Currently, much of what we know about the resistant mechanisms associated with trastuzumab, including hyperactivation of the phosphatidylinositol-3-kinase(PI3K) pathway, deficiency of phosphatase and tensin homolog, an inhibitor of the PI3K/AKT pathway, and mutation in the catalytic subunitof PI3 K, comes from the researches in breast cancer. However, the molecular mechanisms of intrinsic or acquired trastuzumab resistance in gastric cancer have not been extensively explored.In this study, we established NCI-N87-R and MKN-45-R cells by treating gastric cancer cells with increasing doses of trastuzumab. Compared with NCI-N87 cells, NCI-N87-R cells exhibited remarkable resistance to trastuzumab in vitro. The morphology of the resistance cells was obviously changed. The expression of the epithelial marker E-cadherin was dramatically downregulated and a mesenchymal marker vimentin, which was negative in the parental cells, upregulated in the resistant cells. In addition, the important EMT regulators, E-cadherin transcriptional repressors ZEB1, Slug, and Snail were also upregulated, suggesting that trastuzumab resistance is associated with EMT in gastric cancer cells. By wound healing assays, three-dimensional cell culture, colony formation in soft agar, we demonstrated that the trastuzumab resistant cells possessed high potentials of migration and invasion. In addition, the resistant cells also exhibited a significant resistance to anoikis. We evaluated the tumorigenic potential of the resistant cells in an animal model. Our data show that the resistant cells acquired higher potentials of tumorigenesis and spontaneous liver metastasis in nude mice. Similar data were also observed in MKN-45 cells.These data suggest that trastuzumab resistant cells acquired malignant traits of EMT.EMT endows cells with stem-cell like characteristics, such as self-renewal, differentiation, and resistance to chemotherapy or radiotherapy. Several previous studies demonstrated that CD44 positive gastric cancer cells possessed the features of CSC. We analyzed the expression of CD44 in NCI-N87 and NCI-N87-R cells by real-time RT-PCR and flow cytometry. NCI-N87-R cells expressed significantly higher level of CD44 at both m RNA and protein levels. To determine in vitro self-renewal capacity of NCI-N87-R cells, we performed spheroid colony formation assays by culturing NCI-N87-R cells under nonadherent conditions with serum-free media. NCI-N87 R cells generated significantly larger and more spheroid colonies than NCI-N87 cells. We also examined other stemness markers CD133 and OCT-4. The results showed that the NCI-N87-R cells expressed higher levels of CD133 and OCT-4 than parental cells. Enhanced expression of OCT-4 was also observed in MKN-45-R cells. In nude mice the NCI-N87-R cells yielded tumors with as few as 5 × 103 cells. These data clearly indicate that trastuzumab resistant NCI-N87-R cells acquire the phenotype of cancer stem-like cells. To explore the signaling mechanisms of trastuzumab resistance in gastric cancer, we examined the phosphorylation status of AKT, ERK and STAT3, which are well known to be major cell survival pathways mediated by Her2. We found phosphorylated STAT3 level was dramatically increased in NCI-N87-R cells, but the phosphorylation of AKT was almost completely disappeared. In addition, the STAT3 transcription activities in NCI-N87-R cells were strongly enhanced in luciferase assays. Combined treatment with trastuzumab and WP1066(an inhibitor of Jak2/STAT3) resulted in a dramatic growth inhibition of the resistant cells. The data indicates that STAT3 plays a predominant role in the survival of NCI-N87-R cells.The pivotal role of STAT3 in tumor progression has been well known. Its constitutively activation is intimately with the development, conversion, invasion, and metastasis of cancer cells. Inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, activate STAT3 either directly or indirectly and induce EMT. By real-time RT-PCR and ELISA assays, we demonstrated that the expression of the IL-6 m RNA was dramatically increased by more than 100-folds and the secretion level of IL-6 as high as 7000 pg/ml in NCI-N87-R cells. Addition of recombinant IL-6 caused a marked increase of phosphorylated STAT3 in both NCI-N87 and NCI-N87-R cells. The anti-proliferation effect of trastuzumab was significantly abrogated by IL-6 in parental NCI-N87 cells. Treatment with WP1066 alone dramatically caused apoptosis of NCI-N87 R cells and combination of trastuzumab with WP1066 induced a much stronger pro-apoptosis effect. Together, these results suggest that IL-6 autocrine is involved in development of trastuzumab resistance in NCI-N87 cells.The oncogenic functions of the Notch signaling have been well documented and the roles of the Notch signaling in various stem and early progenitor cells have also been recognized. Several recent studies indicate that IL-6 is a novel Notch target in breast cancer cells and enhanced Notch signaling upregulates the IL-6 expression, leading to activation of autocrine and paracrine Jak/STAT signaling. We observered that the Notch activities were significantly enhanced in the resistant cells companied by upregulation of the Notch ligand Jagged-1 and the Notch responsive genes Hey1 and Hey2. Inhibiting the endogenous Notch pathway reduced the IL-6 expression and restored the sensitivities of the resistant cells to trastuzumab. Blocking of the STAT3 signaling by γ-secretase inhibitor abrogated IL-6-induced Jagged-1 expression, effectively inhibited the growth of the trastuzumab resistant cells, and enhanced the anti-tumor activities of trastuzumab in the resistant cells. In 3D or cell sphere culture systems, the invasion phenotypes of NCI-N87-R cells were obviously decreased and the apoptosis ratio was increased, by simultaneously blocking STAT3 and Notch signal pathways. These data demonstrate that IL-6/STAT3 and Jagged-1/Notch pathways synergize in induction of trastuzumab resistance in gastric cancer cells.Taken together, our findings reveal a novel mechanism that the activitation of IL-6/STAT3/Jagged-1/Notch axis may be an important reason for the trastuzumab resistance in gastric cancer cells and that combination of the Notch or STAT3 inhibitors with trastuzumab may prevent or delay clinical resistance and improve the efficacy of trastuzumab in gastric cancer. |
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