Study On The Role Of PRAS40 Phosphorylation At Thr246 In The Development Of Trastuzumab Resistance In HER2-positive Breast Cancer

BackgroundTrastuzumab resistance is common in the treatment of HER2-positive breast cancers, which is mainly result from the aberrant activation of PI3K-AKT pathway, but the exact mechanisms remain unclear. Accumulating evidence support abnormal PI3K-AKT pathway activation would play important roles in trastuzumab resistance. Recent studies indicate that p-PRAS40-Thr246 involves in lipid accumulation, and also acts as downstream regulator of PI3K-AKT pathway. Our previous study also found that phosphorylated PRAS40-Thr246 overexpressed in HER2-positive breast cancers, and is an independent prognostic factor for breast cancers. Therefore, we proposed that p-PRAS40-Thr246 might act as a potential modulatory factor and a candidate potential target for trastuzumab resistance. In this project, we plan to:1. From the clinical level validation with a larger sample size, in order to explore the correlations between p-PRAS40-Thr246 and trastuzumab resistance in positive HER2 breast cancer, and study on the relationship clinical pathological parameters and the prognosis of patients with breast cancer;2. From the cellular level validation,study whether p-PRAS40-Thr246 is the key factor of the PI3K-AKT pathway leading to trastuzumab resistance;3. Through the establishment of breast cancer trastuzumab resistance nude mice model,in order to explore whether the utility of inhibiting p-PRAS40-Thr246 can reverse breast cancer of trastuzumab resistance.This project is supposed to provide a basis for the combination treatment with trastuzumab and MK-2206, and to establish new mechanisms about trastuzumab resistance.Part One Clinical study on the correlation between the role of PRAS40 phosphorylation at Thr246 and trastuzumab resistance in HER2-positive breast cancerObjectiveTo investigate the correlation between p-PRAS40-Thr246 expression level in breast cancer tissue and its clinical pathology parameters,and to investigate the correlation between p-PRAS40-Thr246 expression level in breast cancer tissue and PI3K-AKT signaling pathways activated state,and to analyze whether P-PRAS40-Thr246 can be a new type of molecular biomarker which can predict the response of trastuzumab therapy in breast cancer by the analysis of breast cancer tissue’s detection and.its characteristicsMaterials and MethodsFormalin fixed, paraffin embedded (FFPE) HER2 overexpressing primary breast carcinoma specimens were retrospectively collected from 55 patients and had received trastuzumab treatment. Complete data on tumor characteristics, treatment details and follow up results of disease progression were obtained for all cases, and expression data were retrospectively reviewed and extracted from the medical records. According to standard clinical instructions, the expression of p-PRAS40-Thr246 and PTEN was evaluated by IHC; PIK3CA mutations were detected by the allele specific polymerase chain reaction. The association between p-PRAS40-Thr246 expression and other clinical or pathological characteristics was determined using the χ2 test. The Kaplan Meier method was used to plot the TTP data. The differences between the groups were analyzed using the log rank test. The effect between the expression of p-PRAS40-Thr246 and trastuzumab treatment for breast cancer was performed using Cox’s proportional hazard regression.Results1. There is no significant correlation between p-PRAS40-Thr246 expression level in breast cancer tissue and its clinical pathology parameters. In the present study, IHC was used to detect p-PRAS40-Thr246 expression in HER2 positive metastatic breast cancer tissues. The specificity and suitability of the p-PRAS40-Thr246 antibody used for the IHC has been validated by previous studies. As revealed in figure 1, phospho-PRAS40Thr246 was expressed diffusely throughout the cytoplasm of the breast cancer cells at varying intensities, and demonstrated an occasional nuclear location. Based on previous studies, an H score of> 100 was used as the criterion for the positive expression of p-PRAS40-Thr246. Using this range, a total of 22 cases (40%) of HER2 positive breast cancer tumors were defined as p-PRAS40-Thr246 positive. The clinicopathological variables of the patients were not significantly associated with the p-PRAS40-Thr246 expression levels (Table 1)..2. There was a significant correlation between p-PRAS40-Thr246 expression level in breast cancer tissue and PI3K-AKT signaling pathways activated state. In addition, the PI3K pathway activation status of the same set of HER2 positive breast cancer tumors was investigated. In total, PTEN loss was demonstrated in 34.5%(19/55) of the HER2 positive breast cancer tissues. In agreement with previous findings, seven mutations in exon 20 (H1047R) and three in exon 9 (E542K and E545K) were identified, which corresponded to a PIK3CA mutation frequency of 18.2%. According to the previously established criteria, patients were classified as having an activated (with either PIK3CA mutants or low PTEN expression; n=27) or an unactivated PI3K pathway status (with PIK3CA mutant type and high PTEN expression; n=28). A significant association was identified between p-PRAS40-Thr246 expression and PI3K pathway status, and PTEN loss alone, but not with PIK3CA mutation alone (Table 2)..3. P-PRAS40-Thr246 may be a new type of molecular biomarker which can predict the response of trastuzumab therapy in breast cancer. To further explore the prognostic value of p-PRAS40-Thr246, the correlation between p-PRAS40-Thr246 expression and TTP was analyzed using Kaplan-Meier survival curves. patients with positive p-PRAS40-Thr246 expression exhibited a significantly shorter TTP following trastuzumab based treatment than those with negative expression (figure 2). Univariate analysis was used to determine the association between disease progression and PI3K-AKT pathway activation status following trastuzumab based treatment in HER2 positive metastatic breast cancers, as determined by different marker sets. As revealed in Table Ⅱ, patients with PIK3CA mutations and/or PTEN loss were at an increased risk for disease progression compared with other subgroups. The hazard ratios (HR), which were based on multivariate Cox regression analysis, and were adjusted for other significant predictors (grade), revealed that positive p-PRAS40-Thr246 expres-sion was an independent and significant risk factor for disease progression (HR,2.081; 95% confidence interval,1.113 3.890; P=0.022; Table 2)Conclusions:From the clinical level validation, which showed P-PRAS40-Thr246 expressed in breast cancer tissues, there was a significant correlation between its expression level and the activated state of PI3K-AKT signaling pathways. P-PRAS40-Thr246 is related to the development of trastuzumab resistance in breast cancer. P-PRAS40-Thr246 is the key factor in PI3K-AKT pathway who leading to trastuzumab resistance. Clinical detection of p-PRAS40-Thr246 can predict the effect of trastuzumab therapy in breast cancer.Part Two Cytological study on the role of PRAS40 phosphorylation at Thr246 in the development of trastuzumab resistance in HER2-positive breast cancer cellsObjectiveThis study aimed to investigate the cytotoxicity and reversing effects of MK-2206 on SKBR3/TDR breast cancer cells at the cellular level, to verify whether antagonizing the expression of p-PRAS40-Thr246 in breast cancer cells can reverse the resistance to trastuzumab. The findings described herein provide a basis for the clinical therapy of breast cancer.Materials and MethodsThrough the method of induction,we cultured SKBR3/TDR cells which are HER2 positive breast cancer cell lines and trastuzumab resistance. SKBR3 and SKBR3/TDR cells were treated with different concentrations of MK-2206, to observe the inhibition of cell proliferati of MK-2206 on SKBR3 and SKBR3/TDR. The multiple drug resistance and reversing rates were determined by CCK-8 assay, to observe the trastuzumab resistance reversal when treated with MK-2206 and Trastuzumab together. Cell apoptosis and the accumulation of trastuzumab were evaluated by flow cytometry (FCM). The expression of p-PRAS40-Thr246�'�p-(Thr308)AKT were detected by Western blot,to observe the effect of MK-2206 to SKBR3 and SKBR3 /TDR.Results1. The CCK-8 test results suggest that trastuzumab and MK-2206 can inhibit the proliferation of SKBR3 and SKBR3/TDR cells in a dose-dependent manner(figure 3, figure 4).The IC50 of SKBR3 and SKBR3/TDR from trastuzumab are respectively (57.32±1.64) ug/ml and (248.24±1.92) ug/ml, the resistance ratio is 4.33 times. The IC50 of SKBR3 and SKBR3/TDR from MK-2206 are respectively (40.74±1.66) nmol/L and (43.66±1.43) nmol/L, the resistance ratio is 1.07 times, which shows that SKBR3/TDR have no resistance to MK-2206. When treated with different concentrations of MK-2206 and trastuzumab together,the IC50 of SKBR3 and SKBR3/TDR are all decreased in varying degrees, especially of the SKBR3/TDR cells, the differences was statistically significant(P<0.01)(the results are shown in table 3).2. According to the results of FCM, MK-2206 can reverse trastuzumab resistance in breast cancer cells. When treated with MK-2206 and trastuzumab together, the IC50 of SKBR3/TDR can significantly reduce (P<0.01). Annexin V-FITC double staining showed that when 0,2,4,8 nmol/L MK-2206 were used to treat SKBR3 and SKBR3 /TDR cells for 24 h, all can increase the rate of cell apoptosis, especially of the SKBR3/TDR cells. The difference between experimental group and the control group was statistically significant (P<0.01), (the results are shown in table 4, figure 5). FCM method was used to detect the contents of trastuzumab in SKBR3/TDR cells, results show that after treated by MK-2206 with different concentrations of 0,2,4,8,16 nmol/L, the fluorescence intensity of cells are respectively (2980±14.57)、(3400 ±16.78)、(3000±12.49)、(2800±10.74) and (3800±18.40). The difference was not statistically significant between experimental group and control group (P>0.05) (figure 6). Which shows MK-2206 has no effect on the accumulation of trastuzumab..3. Western Blot test results show that MK-2206 can decrease the expression of P-PRAS40-THR246 and P-(Thr308)AKT protein. Compared with the control group, different concentrations of MK-2206 after treating SKBR3/TDR and SKBR3 cells, both can decrease the expression of p-PRAS40-Thr246 and P-(Thr308) AKT protein in a dose-dependent manner (figure 7 and figure 8), differences between the experimental group and control group was statistically significant (P<0.01).Conclusions:From the cellular level validation, which showed MK-2206 could decrease the expression of P-PRAS40-Thr246, inhibiting the expression of P-PRAS40-Thr246 could reverse trastuzumab resistance in breast cancer and increase its sensitivity to trastuzumab, which could be a reversal to SKBR3/TDR cells.Part Three The establishment of breast cancer trastuzumab resistance nude mice model and Study on the reversal of drug resistance through antagonism PRAS40 phosphorylation at Thr246 in HER2-positive breast cancer in vivoObjectiveStudies in vitro have shown that resistance of SKBR3/TDR relate to activation of PI3K-AKT pathway.So MK-2206,specific inhibitors to AKT,can reverse resistance to trastuzumab meanwhile enhance apoptosis and autophagy effectively.In order to verify the effectiveness and safety of this treatment further, we conduct an experiment in vivo similarly. In this study, our aim is to explore the efficacy of antagonizing the expression of p-PRAS40-Thr246 in reversing the resistance to trastuzumab in treating implant breast carcinoma of nude mice. We expect to provide new way for the treatment of drug resistance in breast through this studyMaterials and MethodsFemale nude mice were purchased,feeding mice 1 week for accommodation. SKBR3/TDR tumors were established by subcutaneously injecting cells to axilla of nude mice. For efficacy studies, mice were randomized among control and treated groups (n=4) when tumors were well-established (about 150mm3). Trastuzumab was administered by intraperitoneal injection at 4 mg/kg,5 times per week. MK2206 was formulated in 30% captisol and given orally at 120mg/kg,3 times per week. Control mice received a vehicle solution. Tumor dimensions were measured using a caliper and tumor volumes were calculated as V(mm3)=π/6x larger diameter x (smaller diameter)2. After 20 days of continuous observation, cut the tumor to weigh. Tumors were excised and snap frozen in -80℃ and then processed for Western blot analysis as described previously.Results1. growth state:5×106 SKBR3/TDR cell suspension were subcutaneously injected to nude mice,3 weeks later the tumors formated(as shown in figure 9,10,11,12,13), then after latent period, Gray nodules were visible in the axillary subcutaneous vaccination site of nude mice. The tumors appeared circular or oval, and tumor size were measured once every five days.2. through antagonism PRAS40 phosphorylation at Thr246 in nude mice breast cancer can inhibit the growth of tumor. The combined treatment of trastuzumab and antagonism PRAS40 phosphorylation at Thr246 can effectively reverse trastuzumab resistance in nude mice model, which suggest it’s a potential and safe therapy for breast.The retarding effect of trastuzumab on tumor growth was not obvious, which was not distinctly different compared with the control group. MK-2206 single treatment group could retard the growth of tumor, but the tumor still increased significantly. On the contrary,the combined treatment group of trastuzumab and MK-2206 could obviously inhibit tumor’s growth, which could also reduce tumor size relatively (as shown in figure 14, and table 5), and the difference was statistically significant. In addition, the nude mice could tolerate drug therapy. During treatment the death or sharp weight loss did not occur among nude mice, while only the change of tumor volume could be seen. Nude mice were killed after 20 days, and the tumors were removed and weighed up. By comparison, the tumor weight of combined treatment group decreased greatly than control group, a statistically significant difference. While compared with control group, the single treatment group had a certain degree of tumor weight loss, but the decrease was not obvious (as shown in figure 15, and table 6).3. Western Blot test results show that the combined treatment group of trastuzumab and MK-2206 can decrease the expression of p-PRAS40-Thr246 and P-(Thr308)AKT protein in breast cancer of nude mice (figure 16). Compared with the control group, MK-2206 single treatment group can decrease the expression of P-(Thr308)AKT protein and the expression of p-PRAS40-Thr246, but trastuzumab single treatment group cann’t decrease the expression of p-PRAS40-Thr246, differences between the experimental group and control group was statistically significant (P<0.01), these results show the effectiveness of the combined treatment group of trastuzumab and MK-2206.Conclusions:Through the establishment of breast cancer trastuzumab resistance nude mice model and the relevant experiment, which showed through antagonism P-PRAS40-Thr246 in nude mice breast cancer could inhibit the growth of tumor. The combined treatment of trastuzumab and MK-2206 could decrease the expression of P-PRAS40-Thr246. antagonism P-PRAS40-Thr246 can effectively reverse trastuzumab resistance in nude mice model, which suggest P- PRAS40-Thr246 is a new therapy target for breast cancer and is helpful to the research on the reversal of trastuzumab resistance in breast cancer.