Activation Of PI3K/AKT Pathway Promotes Trastuzumab Resistance In HER2-positive Gastric Cancer

BackgroundThe human epidermal growth factor receptor family contains four members, including HER1 (EGFR, ErbBl), HER2 (Neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4). They are structurally similar. Among these members, HER2 plays a key role in activing other members. HER2 is located at the long arm of human chromosome 17. It encodes a 185KDa single-chain transmembrane glycoprotein, including an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic domain which possesses tyrosine kinases activity. When any of the other three receptors heterodimerise with HER2, dimerization leads to the auto-phosphorylation of tyrosine residues within the intracellular domain of HER2 and starts a variety of signaling pathways. Signaling pathways initiated by the activation of HER2 include STAT, PI3K/AKT/mT0R, Ras/Raf/MAPK and so on, which serve as promoting cell growth and opposing apoptosis.As the increasingly intense studies have been conducted about the mechanisms underlying the promotion and progression of tumor, over-expression of the HER2 protein occurs in many kinds of malignant tumors and also correlates with a poor prognosis. It is the same situation in gastric cancer. Trastuzumab (trade name Herceptin) is a humanized monoclonal antibody that inhibits HER2-positive tumor proliferation by interfering with the HER2 receptor. Now days it’s mainly used to treat HER2-positive metastatic breast cancer and gastric cancer and have shown great therapeutic efficacy. Several possible mechanisms by which trastuzumab suppresses H2 signaling include 1) blockade of PI3K/AKT signaling 2) induction of the G1 phase arrest of the cell cycle by upregulating the CKIs like p27kip1 3) suppression of shedding of the HER2 extracellular domain 4) inhibition of HER2 receptor dimerization 5) mediation of antibody-dependent cell-mediated cytotoxicity (ADCC). In spite of the effectiveness of trastuzumab, most trastuzumab-responsive patients develop resistance within one year after treatment initiation.According to the multiple trastuzumab resistance mechanisms proposed in recent studies, the development of resistance probably result from 1) Loss of PTEN:PTEN (phosphatase and tensin homolog deleted on chromosome ten) is one of the extensively studied suppressor genes. PI3K/AKT signaling pathway plays a significant role in conveying growth signals from outside the cell to inside the cell. It acts as a central point for regulation of the cell growth, division, metabolism, and tumorigenesis. PTEN was identified as a tumor suppressor by negatively regulating PI3K/AKT signaling pathway. Mutations and deletions of PTEN at high frequency are found in many tumor types such as breast cancer. Besides, it has been reported that the PTEN expression is not only crucial to the development of tumors, but also the reactions to the anti-tumor treatments. Based on the probable mechanisms of action of trastuzumab, it’s easily to infer that the continuous activation of PI3K/AKT could promote the trastuzumab resistance. Besides, recent studies have already determined that the constitutive expression of AKT in HER2-positive breast cancer impaired the inhibition of PI3K and promotion of CKI p27kip1 from trastuzumab, and thus weakened the anti-tumor effectiveness.2) PIK3CA mutations:the mutations of PIK3CA, a class Ⅰ PI3K catalytic subunit, also upregulate the activity of AKT signaling which contributes to the trastuzumab resistance.3) activation of the insulin-like growth factor 1 (IGF-1R) bypass pathways:except for the loss of PTEN and PIK3CA mutations, growth factor mediated AKT activation promotes the resistance developing as well. It has been reported that stable expression of IGF-1R make the trastuzumab-responsive HER2-positive cell lines into the resistant ones. However, inhibition of IGF-1R by inhibitors repressed the activity of PI3K/AKT signaling and restored the sensitivity to trastuzumab.As is well known, signal transduction pathways are multi-factor, multi-link, and cross-talking network systems. And tumor cell population, with the instable genome, show a high growth fraction. Therefore, with the constant progression of tumor cell population, inter-active network systems and influences from all sorts of endogenous and exogenous factors in the tumor microenvironment, molecular targeted therapies develop drug resistance attribute to new signaling activation.Objectives and significancesAlthough trastuzumab has shown promising therapeutic efficacy when used in combination with chemotherapeutics to treat HER2-positive breast cancer and gastric cancer, many trastuzumab-responsive patients develop resistance during the continuous treatment. Hence, in order to overcome the trastuzumab resistance it should attach a great importance to the studies on the underlying mechanisms. Previous studies on trastuzumab resistance have mainly focused on breast cancer. However, few reports on the mechanisms in gastric cancer has been published. In this study, we employed the human gastric cancer cell NCI-N87 with high expression of HER2 protein to create a trastuzumab-resistant cell line (NCI-N87/TR) for the first time, analyzed the characteristics of NCI-N87/TR cells and investigated the detailed mechanisms regulating the in vitro resistance of gastric cancer to trastuzumab.Materials and Methods1. HER2-positive human gastric cancer cell lines screeningIn order to determine HER2-positive gastric cancer as the study subject, western blotting was conducted to detect HER2 protein levels in all 4 gastric cancer cell lines (SGC7901, MKN45, NCI-N87, and MKN28).2. Induction of trastuzumab-resistant cellsWe employed the HER2-overexpressed human gastric cancer cell line NC1-N87 to establish trastuzumab-resistant NCI-N87/TR cell line by stepwise exposure to increasing doses of trastuzumab. Resistance index (RI) was completed via MTT assay.3. Sensitivity and cross drug resistance of NCI-N87/TR cells to chemotherapeutic drugsThe cross resistance to Taxol, DDP and 5-Fu of NCI-N87/TR cells and resistance index (RI) were analyzed via MTT test.4. Expression of AKT and PTEN in trastuzumab-resistant gastic cancer cellsWestern blotting were performed to detact the expression of AKT, p-AKT and PTEN proteins in NCI-N87/TR cells. The expression of PTEN gene was analyzed by real-time PCR.5. The expression of AKT/p-AKT after inhibition of PI3K and siRNA transfectionNCI-N87 and NCI-N87/TR cells were incubated with the PI3K inhibitor LY294002 and siRNA transfection, respectively. Western blotting were performed to detact the expression of AKT proteins.6. The expression of AKT after transfection of PTENBy the construction of the PTEN gene expression vector pBabe-puro-PTEN, transformation of E. coli strain DH5a, identification by colony PCR and enzymes digestion, positive clones were confirmed. Lipofectamine 2000 was used for cells transfection in NCI-N87 and NCI-N87/TR. Western blotting were performed to detact the expression of AKT/p-AKT and PTEN proteins.7. Statistical analysisAll statistical analyses were performed by SPSS 21.0 software (IBM Corporation, Armonk, NY, USA). All experiments were repeated at least 3 times and mean values were calculated. Data were represented as the mean±standard deviation. The significance of differences was analyzed using one-way ANOVA or Student’s t-test. P values less than 0.05 were considered statistically significant.Results1. Human gastric cancer cell lines screeningExpression of HER2 protein was detected in all 4 gastric cancer cell lines (SGC7901, MKN45, NCI-N87, and MKN28), with the highest level being observed in NCI-N87 cells (P< 0.05).2. Induction of trastuzumab-resistant cellsWe employed NCI-N87 cells for further studies because of their high HER2 protein expression. When the concentration of trastuzumab reached 3500 μ g/ml, the IC50 of NCI-N87 and NCI-N87/TR cells was 19.762 p. g/ml and 227.523 μ g/ml respectively, and the RI of NCI-N87/TR cells for trastuzumab was 11.513. Sensitivity and cross drug resistance of NCI-N87/TR cells to chemotherapeutic drugsThe NCI-N87/TR cells also showed CDR to Taxol and DDP (RI= 2.03 and 2.69, both P=0.000), while there was no resistance to 5-FU (RI=0.97, P=0.725).4. Expression of AKT, p-AKT and PTEN in trastuzumab-resistant gastic cancer cellsCompared with NCI-N87 cells, PTEN gene and protein expression both showed a signifcant decrease in NCI-N87/TR cells. Expression of AKT and P-AKT proteins in NCI-N87/TR cells was also markedly increased.5. The expression of AKT and p-AKT after inhibition of PI3K by inhibitor and siRNAAfter cells were incubated with the PI3K inhibitor LY294002, as well as transfected with siRNA targeting PI3Kpl 10, western blotting showed that the p-AKT protein expression decreased in both NCI-N87 and NCI-N87/TR cells, and the descent of AKT protein phosphorylation indicating that the inhibitor LY294002 or siRNA targeting PI3Kp1 10 effectively blocked the PI3K-AKT signaling pathway. After being treated with the PI3K inhibitor LY294002 or siRNA targeting PI3Kp1 10, the IC50 of NCI-N87/TR cells decreased from 172.31 μg/ml to 97.01 μg/ml and 107.07 μg/ml, the RI decreased from 7.09 to 3.99 and 4.40 respectively, indicating an increase of sensitivity to trastuzumab6. The expression of AKT and p-AKT after transfection of PTEN Western blotting showed that transfection with pBabe-puro-PTEN resulted in the up-regulation of PTEN protein expression and the down-regulation of P-AKT protein expression in NCI-N87 and NCI-N87/TR cells compared with transfection of the blank vector implying that PTEN gene transfection might inhibit activation of the downstream PI3K-AKT signaling pathway. Drug sensitivity testing showed that a er transfection with the PTEN gene, the IC50 of NCI-N87/TR cells decreased from 172.3lμg/ml to 100.04μg/ml and RI decreased from 7.09 to 4.12, indicating an increase of sensitivity to trastuzumabConclusions1. We successfully induced trastuzumab-resistant NCI-N87/TR cells by Exposure to increasing doses of trastuzumab. The RI of NCI-N87/TR cells for trastuzumab was 11.51. The NCI-N87/TR cells also showed cross drug resistance to Taxol and DDP (RI=2.03 and 2.69), while there was no resistance to 5-FU.2. We showed that phosphorylation of AKT protein was up-regulated in trastuzumab-resistant gastric cancer cells, indicating that activation of the PI3K/AKT signaling pathways might be one of the major resistance mechanisms of gastric cancer to trastuzumab.3. PTEN expression was decreased significantly in trastuzumab-resistant NCI-N87/TR cells at both the gene and protein level, implying that down-regulated PTEN might lose the inhibition of the downstream PI3K/AKT signaling pathway. With PTEN gene transfection, the activity of PI3K/AKT signaling was significantly decreased and restored the sensitivity to trastuzumab, indicating that the down-regulation of PTEN protein promote the activation of PI3K/AKT signaling pathway and PTEN gene might be a promising target for overcoming the trastuzumab resistance in HER2-positive gastric cancer.