A New Mechanism Of Trastuzumab Resistance In Gastric Cancer:MACC1 Promotes The Warburg Effect Via Activation Of The PI3K/AKT Signaling Pathway

Backgroud:Gastric carcinoma is the fifth most common cancer and the third leading cause deaths worldwide. All over the world, more than 1,000,000 new cases occur each year, of which more than 70% are diagnosed in developing countries, particularly in East Asia. In China alone, the crude mortality rate of GC is 2.5 per 1000 people, which accounts for 23.2% of all cancer deaths and is the leading cause. Due to the limitation of screeing methods, over 60% diagonosed gastric patients were in advanced period in our courtry. Because of its serious side-effect, the traditional cytotoxic chemotherapy has not substantially improved survival rates, morehope has been placed in the potential of newer targeted therapies that will provide improvements in outcomes similar to those they have achieved in other cancers. Molecular targeted therapy for tumors occurred in the process of a series of molecular events, using monoclonal antibodies or small molecule drugs, blocking cancer cells abnormal activation or inhibiting the important relative signaling pathway, so as to achieve the goal of treatment. For its highly selectiveness, low toxicity and gradually efficient, targeted-therapies have become an important part of clinical treatment of tumor.Human epidermal growth factor receptor 2 (HER2) is a member of the receptor family associated with tumor cell proliferation, adhesion, migration, and differentiation. Her2 is overexpressed in GC and predictes poor prognosis of GC patients. Trastuzumab, a humanized monoclonal antibody that targets HER2, inhibits the HER2-mediated signaling pathway and induces antibody-dependent cellular cytotoxicity. For HER2-positive advanced GC, combining chemotherapy with trastuzumab is significantly superior to chemotherapy alone with regard to efficacy and safety. Trastuzumab combined with chemotherapy has been approved by the U.S. Food and Drug Administration (FDA) as the first line therapy for inmetastatic, unresectable HER2 positive gastric and gastroesophageal junction cancers. Despite the significant clinical benefit observed in the patient population, not all individual subjects responded equally despite having HER2-positive cancers. Indeed, less than 60% of the patients did exhibit an objective response. Although the response rates to this combination are far higher than those of chemotherapy alone, the effects are usually transitory, suggesting a high incidence of either primary or acquired resistance in 6-12months after first use. Thus, more effective predictors of trastuzumab response in HER2-positive cancer, except for HER2, are required for personalized clinical treatment.Cancer cells prefer anaerobic breakdown of glucose for energy rather than mitochondrial oxidative phosphorylation, this phenomenon termed the "Warburg effect". Tumor disordered metabolism and its malignant transformation were promoter and result for each other, and the Warburg effect has been closely correlated with drug resistance in cancer cells. However, many glycolytic related proteins have been evaluated to be important factors in anti-cancer therapy resistance, such as GLUT, HK, LDHA and etc. Targeting the Warburg effect improves the HER2 positive breast cancer cells response to trastuzumab, and combined targeting Warburg effect may represent a promising strategy to overcome drug resistance in cancer therapy.One of the most important mechanisms underlying anti-tumor therapy resistance and tumor metabolism is the PI3K/AKT pathway, which is the most important signaling pathway in tumor malignant phenotypes. PI3K/AKT signaling pathway is one of the major mechanisms leading to resistance of HER2-positive breast cells to trastuzumab. We previously research have found that activation of the PI3K/AKT signaling pathway is one of the mechanisms leading to resistance of HER2-positive GC cells to trastuzumab. Therefore, it is reasonable to hypothesis that activation of AKT might induce enhancement of Warburg effect and resulted in trastuzumab resistance in GC cells.Metastasis associated in the colon cancer 1 (MACC1) gene contributed to poor prognosis and metastasis of colon cancer, which was also found high-espressed in hepatocyte carcinoma, pancreat carcinoma, non-small-cell lung cancer, ovarian cancer, and etc. MACC1 could promote tumor malignant phenotypes such as proliferation, invasion and metastasis via AKT/cc-Met signaling pathway. Previously, we found that MACC1 contributed to poor prognosis of GC by promoting tumor cells proliferation and invasion as well as epithelial-to-mesenchymal transition. Moreover, we discovered that MACC1 was upregulated by metabolic stress in GC via adenosine monophosphate-activated protein kinase signaling, which increased the resistance to metabolic stress by promoting the Warburg effect and consequently facilitated tumor progression. What’s more, MACC1 was reported as regulator of Met/AKT signaling pathway, and this relgulating axis was also been approved by our research. In view of the relationship between tumor malignant phenotypes and disorderd metabolisom, we suspect that MACC1 also involved in the anti-tumor drug resistant mechanisms. MACC1 might participate in the regulated mechanism by regulating the Warburg effect via the PI3K/AKT signaling pathways. Therefore, we proposed the research of MACC1 and Warburg effect in gastric cancer cells, moreover the possible regulatory mechanisms underlining the MACC1 inducing trastuzumab resistance in human HER2 positive gastric cancer patients.PURPOSE:This project used human HER2 positive gastric cancer cell lines as the reresearch objects. By using stepwise stimulateon of trastuzumab method, we have constructed the stable drug resistance. MACC1 over-expression and down-regulation stable transfected cell lines were constructed by constructed plasmids. Under the experiements in vitro and in vivo, we discussed the mechanism of MACC1 gene involved in regulating human HER2 positive gastric cancer cells resistance to transtuzumab. We aimed to found out the new effctively predictors of trstuzumab resistance in GC patients, in order to do better selection of the targeted population, moreover, to reverse the trastuzumab resistance in clinical therapy.Main Methods:1. Screening of cell lines:Western blot and MTT assays were conducted to screen human gastric carcinoma (GC) cells with HER2 positive and MACC1 over-expression. The most sensitivity and moderate sensitivity to Trastuzumab cells were selected as the research objects.2. Induction of trastuzumab stable resistant cells and analysis of its characteristics:The trastuzumab stable resistant cells were induced by stepwise exposure to increasing doses of trastuzumab. The MTT assay was conducted to determine the IC50 (half inhibitory concentration) of trastuzumab and the drug resistance index (RI).3. MTT assay:Cells were seeded onto 96-well plates at a density of 3×103 cells/well.24 hours later, the cells were treated with drugs at the indicated concentrations and incubated for specific time periods. Cell viability was determined 3 days after treatment using MTT assay according to the manufacturer’s protocol. Experiments were performed in triplicate.4. Establishment of stably transfected cell lines:pLVX-shMACCl and pLVX-MCMV-ZsGreen-PGK-Puro-MACC1 palsmid were constructed before transfection.Cell lines were transfected with these constructed plasmids. Stably transfected cell lines were selected with 0.5 mg/mL (a minimum lethal dose) puromycin at 48 hours after infection.5. Cell-based assay for glucose uptake:The levels of glucose uptake were measured using a Amplex(?) Red Glucose/Glucose Oxidase Assay Kit. Cells were seeded in 96-well plates at a density of 5×103 cells/well. After 24 h, glucose uptake assays were performed according to the manufacturer’s protocol.6. Cell-based assay for lactate assay:The levels of lactate production were examined using a Lactate Colorimetric/Fluorometric Assay Kit. Cells were plated onto in 96-well plates at a density of 5×103 cells/well. After incubation for 24 h, the culture medium was replaced with FBS-free DMEM. After an additional 8 hours, lactate assays were performed with culture media collected from each sample according to the manufacturer’s protocol.7. Inhibition of AKT signaling pathway:The AKT1/2/3 inhibitor MK2206 and PI3Kα/δ/β inhibitor LY294002 were used to inhibit the AKT signaling pathway.8. Activation of AKT signaling pathway:The AKT continuously activated plasmid Myr-Akt was used to activate the AKT signaling pathway.9. CalcuSyn software 2.0:Using the ColcuSyn(?) (Paramus, N.J., USA) software, the combination index (CI) was calculated for cells receiving combination therapy according to the Chou and Talalay mathematical model for drug interactions. The resulting combination index (CI) theorem of Chou-Talalay offers quantitative definition for an additive effect (CI=1), synergism (CI< 1), and antagonism (CI> 1) in drug combinations.10. Flow cytometric analysis of apoptosis:Annexin V-FITC Apoptosis Detection Kit was used to analysis of cell apoptosis.11. BALB/c Nude mice cancer xenograft models:GC cells were pre-treated with different plasmids or NC. The cells were suspended in 100 μl of PBS at a concentration of 5×107 cells/ml and injected into either flank of the same BALB/C female athymic nude mouse at 5-6 weeks of age (6 mice for each group, n=10). Xenografts were injected by tail vein with trastuzumab (10 mg/kg, i.p.,2 times/wk × 3 weeks), oxamate (750 mg/kg, i.p., daily for 21 days), or a combination of the agents after tumor formation. IHC staining、Western blot were used to analyses expression levels of protein.12. microPET/CT:BALB/c Nude mice were imaged and analyzed with 18F-FDG for in vivo glucose uptake.13. TUNEL:TUNEL assays were performed on sections using an DeadEndTM Colorimetric TUNEL assay kit principally according to the supplier’s instruction.14. Protein and gene level detaction:Western blotting and Real-time PCR were used to detected the cell and tissure protein and gene expression level.15. Statistical analysis:All data were represented as the mean of at least triplicate samples ± standard deviation. Statistical analysis included one-way ANOVA, or Student’s t-test using SPSS 20.0. P values less than 0.05 were considered statistically significant.Contents:1. Relationship between MACC1 and the trastuzumab resistance in HER2-positive GC cells1.1 Screening of cell lines:Western blot was used to tested the expression of HER2 and MACC1 protein level in BGC823, MKN28, MKN45, NCI-N87 and SGC7901 cells. Based on the previously researches, MKN45 cells were chosen as the relatively sensitive cells to trastuzumab.1.2 Induction of trastuzumab stable resistant cells and analysis of its characteristics:1.2.1 The trastuzumab stable resistant cells were induced by stepwise exposure to increasing doses of trastuzumab. The final concentration of trastuzumab is 2500ug/ml(MKN45/TR), IC50 is 296.26ug/ml, RI=9.78.1.2.2 The MTT assay and CalcuSyn software 2.0 was conducted to determine the IC50 (half inhibitory concentration) of trastuzumab and the drug resistance index (RI).1.3 MACC1 was downregulated by siRNA in trastuzumab-resistant NCI-N87/TR and MKN45/TR cell lines and treated with trastuzumab. Western blot was used to tested the expression of MACC1 protein level, MTT was conducted to determine the inhibition of cell viability under treatment of trastuzumab.1.4 Colonies of ectopic-MACC1 and shMACC1 and their respective controls were used to transfect NCI-N87、MKN45 cells and treated with trastuzumab. Western blot was used to tested the expression of MACC1 protein level, MTT was conducted to determine the inhibition of cell viability under treatment of trastuzumab.2. Relationship between MACC1 and the Warburg effect in HER2-positive GC cells2.1 The levels of glucose uptake and lactate production were measured 24 hours after plating in 96 wells of stable transfection NCI-N87 and MKN45 cells (MACC1, vector, shRNA and scramble) by using the Amplex(?) Red Glucose/Glucose Oxidase Assay Kit and Lactate Colorimetric/Fluorometric Assay Kit.2.2 The expression levels of GLUT1, HK2 and LDHA in MACC1, vector, siMACCl and scramble transfected cells were detected by Western blot.2.3 After MACC1 gene was been sclienced, the expression levels of GLUT1, HK2 and LDHA in NCI-N87/TR and MKN45/TR and their MACC1 downregulate cells were detected by Western blot.3. Synergistic effection of combined use of Trastuzumab and the glycolysis inhibitors3.1 NCI-N87 and MKN45 cells were treated with trastuzumab for 48 or 72 hours, GLUT1, HK2 and LDHA expression were measured. Meanwhile, the glucose uptake and lactate production were measured.3.2 The expression levels of GLUT1, HK2 and LDHA in NCI-N87/TR and MKN45/TR and their parantel cells were detected by Western blot.3.3 2-DG or OX, combined with trastuzumab to treat both parental GC cells and trastuzumab stable resistant cells, MTT and CalcuSyn software 2.0 was conducted to determine the inhibition of cell viability AND CI.3.4 2-DG or OX, combined with trastuzumab to treat both parental GC cells and trastuzumab stable resistant cells, Amplex(?) Red Glucose/Glucose Oxidase Assay Kit and Lactate Colorimetric/Fluorometric Assay Kit were used to measure the levels of glucose uptake and lactate production.4. Mechanism of MACCl promoted the Warburg effect via the PI3K/AKT signaling pathway and induced trastuzumab resistance4.1 After treated by trastuzumab for 48,72hours, the expression levels of p-AKT, and AKT in NCI-N87 and MKN45 cells were detected by Western blot.4.248 hours after transfected by MACC1, vector, siMACC and siCtrl, the expression levels of p-AKT, and AKT in NCI-N87 and MKN45 cells were detected by Western blot.4.3 MACC1 promoted the Warburg effect via the PI3K/AKT signaling pathway and induced trastuzumab resistance.4.3.1 MK2206 was used to treat MACC1-overexpressing NCI-N87 and MKN45 cell lines for 24 hours(48 hours after transient transfection). Wetern blotwas used to detecte the expression of GLUT1, HK2 and LDHA and glucose uptake and lactate production were measured. MTT was conducted to determine the inhibition of cell viability under 72 hours treatment of trastuzumab.4.3.2 Myr-AKT and MACC1 siRNA were applied to co-transfect NCI-N87 and MKN45 cells(48 hours after transient transfection). Wetern blotwas used to detecte the expression of GLUT 1, HK2 and LDHA and glucose uptake and lactate production were measured. MInTT was conducted to determine the inhibition of cell viability under 72 hours treatment of trastuzumab.4.3.3 LY294002 was used to treat MACC1-overexpressing NCI-N87 and MKN45 cell lines for 24 hours(48 hours after transient transfection).Annexin V-FITC Apoptosis Detection Kit was used to analysis of cell apoptosis 48 hours after treatment of trastuzumab.5. MACC1 induced trastuzumab resistance and enhanced Warburg effect In vivo5.1 NCI-N87 MACC1-overexpressiing, downregulated and their control cells were used to establish a BALB/c nude mouse xenograft model. The tumor volume was measured twice a week until it reached the average volume (120 mm3). MACC1 expression was confirmed in xenografts using IHC staining Western blotting analyses. Xenografts were treated with PBS as control or trastuzumab after the tumors had formed.5.2 Tumor voloum were measured 2 times a week. Voloum= (L×W2)/2 (mm3)5.3 Animal PET scanning detected the tumor cell 18F-FDG accumulation.5.4 TUNEL assays were performed to detecte the level of tumor cell apoptosis.6. Synergistic effection of combined use of Trastuzumab and the glycolysis inhibitors in vivo6.1 NCI-N87 MACC1-overexpressiing, downregulated and their control cells were used to establish a BALB/c nude mouse xenograft model. The tumor volume was measured twice a week until it reached the average volume (120 mm3). MACC1 expression was confirmed in xenografts using IHC staining Western blotting analyses. Xenografts were treated with PBS, trastuzumab, OX, trastuzumab plus OX after the tumors had formed.6.2 Tumor voloum were measured 2 times a week. Voloum= (L×W2)/2 (mm3)6.3 Animal PET scanning detected the tumor cell 18F-FDG accumulation.6.4 TUNEL assays were performed to detecte the level of tumor cell apoptosis.Results:1. MACC1 contributed to the resistance of HER2-positive GC cells in response to trastuzumab1.1 The sorting of expression level of HER2 is NCI-N87, MKN45, SGC7901, MKN28, BGC823 cells. MKN45 cells were chosen as the relatively sensitive cells to trastuzumab; MACC1 were expression in all cell lines; NCI-N87(IC50:24.41 μg/mL) and MKN45(IC50:30.30 μg/mL) were selected as research objects.1.2 The trastuzumab resistant cells were induced by stepwise exposure to increasing doses of trastuzumab. MKN45/TR:IC50:296.26ug/ml, RI=9.78. NCI-N87/TR:IC50:243.98ug/ml,RI=10.00.1.3 Comparied to the parental cells, MACC1 was upregulated in MKN45/TR and NCI-N87/TR cells;After downregulated MACC1 in the resistant cells, inhibition of cell viability after treated with trastuzumab was incresead(P<0.05);After transfection, overexpression of MACC1 significantly increased the cell viability after treatment with trastuzumab, while, downregulation of MACC1 significantly inhibited the viability (P<0.05).2. MACCl enhanced the Warburg effect in GC cells.2.1 The levels of glucose uptake and lactate production, obviously increased in MACC1-overexpressing cells (P<0.05), While, decreased notably in MACC1-downregulated cells (P<0.05). In addition, the expression of GLUT 1, HK2 and LDHA, decreased in MACC1-downregulated cells (P<0.05), whereas, increased in MACC1-overexpressing cells (P<0.05).2.2 After MACC1 was silenced in NCI-N87/TR and MKN45/TR cells, the expression of GLUT1, HK2 and LDHA proteins were also downregulated, and the levels of glucose uptake and lactate production were obviously decreased.3. Synergistic inhibition of cell growth and glycometabolism via the combination of trastuzumab and glycolysis inhibitors3.1 NCI-N87 and MKN45 cells were treated with trastuzumab for 48 or 72 hours, GLUT1, HK2 and LDHA expression were downregulated. Meanwhile, the glucose uptake and lactate production were decreased (P<0.05).3.2 Compaired to the parental cells, the expression level of GLUT 1, HK2 and LDHA were highlight in the NCI-N87/TR and MKN45/TR cells.3.3 The combination of trastuzumab and 2-DG/OX showed a strongly increased inhibitory efficacy in the cell viabilities and glycometabolism of MKN45, NCI-N87 parental cells and MKN45/TR, NCI-N87/TR cells compared to the agents individually. All the data were analyzed by the CalcuSyn software (CI<1). Moreover, the synergistic effect in trastuzumab resistant cells were more robust than in parental cells.4. MACC1 promoted the Warburg effect via the PI3K/AKT signaling pathway and induced trastuzumab resistance in vitro4.1 After been treated with trastuzumab, the p-AKT was downregulated in NCI-N87 and MKN45 cell lines.4.2 Compaired to the control cells, the expression level of p-AKT were highlight in the MACC1-overexpressing cells and declining in the MACC1-downregulated cells.4.3 MACC1-overexpressing and vector transfected cells incubation with MK2206 for 24 hours, expression levels of GLUT 1, HK2 and LDHA as well as the level of glucose uptake and lactate production were decreased (P<0.05). Moreover, the inhibition of cell viability after treated with trastuzumab was increased (P<0.05).4.4 Myr-AKT and MACC1 siRNA were applied to co-transfect NCI-N87 and MKN45 cells, expression levels of GLUT1, HK2 and LDHA as well as the level of glucose uptake and lactate production were increased (P<0.05). Moreover, the inhibition of cell viability after treated with trastuzumab was decreased (P<0.05).5. MACC1 induced trastuzumab resistance and enhanced the Warburg effect in vivo5.1 Compared to the vector group,18F-FDG accumulation was highlight in MACC1-overexpressing group, wherease, decreasing in MACC1-downregulated group rather than in scramble group.5.2 In trastuzumab treatment group, tumors with MACC1 overexpression were much bigger than the vector group (day 12,15,18,22,25 aftert beginning treatment P<0.05). whereas, were obvious smaller in MACC1-downregulated tumors than the scramble groups(day 8,15,18,22,25 after beginning treatment P<0.05).5.3 In MACC1-overexpressing group, compared with PBS group, the tumor growth,18F-FDG accumulation was not obviously inhibited by trastuzumab, as well as the cell apoptosis was not notably highlight(P>0.05).5.4 In MACC1-downregulated group, compared with PBS group, tumor growth,18F-FDG accumulation was obviously inhibited by trastuzumab, as well as the cell apoptosis was notably highlight(P<0.05).6. Combination of trastuzumab and oxamate effectively inhibited cell growth and the Warburg effect in MACCl-overexpressing xenografts6.1 In MACC1-overexpresing group, tumors treatement with combined drugs were obviously smaller than the singal treatment groups (day 15,18,22,25 aftert beginning treatment P<0.05), whereas, in vector, MACCl-downregulated and scamble group, this difference were not obvious (P>0.05).6.2 In MACC1-overexpresing group, 18F-FDG accumulation was obviously inhibited in combined treatement group than in the sigal treatment groups, whereas, in vector, MACC1-downregulated and scamble group, this difference were not obvious.6.3 In MACC1-overexpressing group, compared with singal treatment, cell apoptosis was not obviously increased in combined treatement group (P<0.05), while in vector, MACC1-downregulated and scramble group, cell apoptosis was not significantly highlight by combined treatement (P>0.05).Conclusion:1. MACC1 participate in the trastuzumab resistance of HER2-positive GC cells: ① Compared with parental cells, the expression of MACC1 was significantly increased in trastuzumab resistant cells, and downregulating MACC1 reversed the trastuzumab resistance; ② Overexpression of MACC1 significantly increased the cell viability after treatment with trastuzumab. while, downregulation of MACC1 significantly inhibited the cell viability.2. MACC1 enhanced the Warburg effect in HER2-positive GC cells:① Glucose uptake and lactate production obviously increased in MACC1-overexpressing cells, while, decreased notably in MACC1-downregulated cells; ②The expression of GLUT1, HK2 and LDHA decreased in MACC1-downregulated cells. whereas, increased in MACC1-overexpressing cells; ③ Silenced MACC1 in NCI-N87/TR and MKN45/TR cells, the expression of GLUT1, HK2 and LDHA were downregulated.3. Synergistic inhibition of cell growth and glycometabolism via the combination of trastuzumab and glycolysis inhibitors:① The combination of trastuzumab and glycolysis inhibitors showed a synergistic inhibitory efficacy in the cell viabilities and glycometabolism of MKN45, NCI-N87 parental cells and MKN45/TR, NCI-N87/TR cells compared to the agents individually; ② The synergistic effect in trastuzumab-resistant cells were more robust than in parental cells.4. MACC1 promoted the Warburg effect via the PI3K/AKT signaling pathway and induced trastuzumab resistance in vitro: ① Trastuzumab inhibited the AKT phosphorylation in NCI-N87 and MKN45 cell lines; ② MACC1 overexpression the activated AKT signaling pathway; ③ MK2206 suppressed the expression of GLUT1, HK2 and LDHA and weaked Warburg effect which were increased due to MACC1 overexpression, moreover, after activities of AKT signaling pathway was inhibited, the drug resistance was reversed; ④Myr-AKT increased expression of GLUT 1, HK2 and LDHA, reversed the decreased Warburg effect as well as the sensitiveness to trastuzumab caused by MACC1 downregulation.5. MACC1 induced trastuzumab resistance and enhanced the Warburg effect in vivo:5① Animal PET scanning demonstrated that 18F-FDG accumulation was markedly enhanced by MACC1 overexpression; ② In trastuzumab treatement group, compaired to the cintrol, the tumor voloum were much bigger in MACC1-overexpressing group, wherease much were smaller in the MACC1-downregulated group.③In MACC1-overexpressing group, there was no obvious difference in tumor growth, Warburg effect and cell apoptosis bwtween PBS and trastuzumab treatement; ④ In MACC1-downregulated groups; compaired to the PBS, trastuzumab treatement inhibited tumor growth, Warburg effect more strongly, and induced cell apoptosis more effectively.6. Combination of trastuzumab and Warburg effect inhibitor effectively inhibited cell growth and the Warburg effect in MACC1-overexpressing xenografts:①In MACC1-overexpressing group, compaired to the trastuzumab or oxamate singal used, the combined treatement inhibited tumor growth, Warburg effect more strongly, and induced cell apoptosis more effectively; ②In vector、MACC1-downregulated and scramble groups; there was no obvious difference in tumor growth, Warburg effect and cell apoptosis between singal and combined treatement.