| BACKGROUND AND OBJECTBreast cancer is the most common cancers in women. The genesis of breast cancer is associated with the activation of oncogenes and deactivation of cancer-suppression genes, especially some genes related to cell growth and endocrine such as HER2, ERK, ERα, etc. Therefore, some drugs which target to these genes such as trastuzumab, tamoxifen have good effect in clinic. However, drug resistance more and more limits their application in clinic.Trastuzumab is a kind of drugs target to HER2 in clinic. It binds with HER2 receptor which locate in the membrane of breast cancer cells and blocks the formation of HER2 dimer. And so the HER2-mediated signaling pathways are suppressed. However, the effect of trastuzumab alone on breast cancer is only 12-34%, and drug resistance is found in nearly 50% patients. Therefore, exploring the associated resistant mechanisms and designing some ways to overcome them may represent an important science problem in breast cancer therapy.Her2 receptor can activate several intracellular signaling pathways include Ras/MAPK, PI3K/AKT/mTOR, JAK/STAT, PLC-γ, etc. These signaling pathways are closely related to the growth and proliferation of breast cancer cells. So the resistance of trastuzumab is often linked to these signaling pathways.Previous studies showed that the change of HER2-mediated signaling pathway can result in trastuzumab resistance. The associated mechanisms include the enhanced insulin-like growth factor 1 receptor signaling, the accumulation of truncated HER2 mutant p95-HER2, over-activation of PI3K/AKT/mTOR signaling. Beside these mechanisms, the activation of JAK/STAT signaling may represent another important way to resist trastuzumab.JAK is a kind of non-recepor tyrosine kinase, include JAK1、JAK2、JAK3 and TYK2. Activated JAK can phosphorylate and activate STAT. JAK/STAT signaling pathway is over-activated in many tumor tissues. The levels of total STAT3 and phosphor-STAT3 in breast tumor tissue are significantly higher than that in normal breast tissue. However, the relation of STAT3 activation and trastuzumab resistance remains unclear. In this study, we first investigate whether trastuzumab can activate STAT3 in HER2-positive breast cancer cells. Then, the effect of trastuzumab on HER2-positive breast cancer cells is checked after STAT3 knockdown by siRNA or suppressing by inhibitor. We hope to find some clue between STAT3 activation and trastuzumab resistance in HER2-positive cancer cells. The associated results may provide evidence for clinical treat.AG490 is a kind of specific inhibitor of JAK/STAT signaling pathway. AG490 binds with tyrosine kinase JAK2 and inhibits its activity, which result it STAT3 can’t be phosphorylated and regulate cell proliferation by binding with DNA elements. At present, AG490 is used as anti-tumor drugs target to many cancers in clinic. However, whether AG490 can increase the sensitivity of trastuzumab in breast cancer is unknown.Due to activated STAT3 regulates the expression of many genes, such as it up-regulates the expression of anti-apoptosis genes Bcl-xL、Bcl-2 and Mcl-1, VEGF, cyclin D1/D2, c-myc and survivin, and down-regulates the expression of p53. Recently, cancer suppression gene SARI was also found involve in the JAK/STAT mediated signaling. SARI, is also called BATF2, has higher level in normal human tissue,while lower level in associated tumor tissues. Extopic expression of SARI suppress the proliferation of tumor cells while has no effect on normal cells. But the mechanisms of SARI-mediated anti-cancer are not fully clear, and its upstream signaling is also not clear. Moreover, whether AG490 suppresses breast cancer by activating SARI need further study.METHODSCCK-8 assay was used to detect the effect of trastuzumab on HER2-positive breast cancer cell SK-BR2 proliferation. Trypan blue staining was used to analyze the effect of trastuzumab on HER2-positive breast cancer cell SK-BR2 death. Colony formation assay was used to assess effect of trastuzumab on HER2-positive breast cancer cell SK-BR2 colony formation. Immunoblotting was used to detect the protein level of phosphor-STAT3. siRNA was used to knocked down the expression of STAT3. AG490 was used to inhibit the JAK/STAT pathway. The combination of AG490 and trastuzumab was used to elevate the effect on anti-breast cancer cells. By the same way, CCK-8 assay was used to detect the effect of AG490 on the proliferation of HER2-positive breast cancer cells and HER2-negative breast cancer cells. Trypan blue staining was used to analyze the effect of AG490 on total cell death of HER2-positive breast cancer cell and HER2-negative breast cancer cells. Colony formation assay was used to assess effect of AG490 on the colony formation ability of HER2-positive breast cancer cell and HER2-negative breast cancer cells. Immunoblotting was used to detect the protein level of phosphor-SARI. siRNA was used to knocked down the expression of SARI. The STAT3 binding sites located in SARI promoter region were analyzed online. The fragments include predicted STAT3 binding site or not were obtained by PCR from genome of breast cancer cells. Then these fragments were inserted into pGL3 report vector. The luciferase activity of reporter gene was detected after tranfection.RESULTSTrastuzumab suppressed the proliferation of SK-BR3 cells in dose-dependent manners. The suppression rate is 76.22%,72.97%,61.08%,42.16%and 23.78% at dose of 0.25,0.5,1.0,2.0,4.0 mg/ml respectively. Trastuzumab also promoted the total cell death of SK-BR3 cells in dose-dependent manners. The cell death rate is 10.35±1.66%,18.37±2.49%,31.97±3.08%,46.78±5.67%and 63.77±7.81%at dose of 0.25,0.5,1.0,2.0,4.0 mg/ml respectively. Compared to control group, trastuzumab also inhibited the colony formation of SK-BR3 cells in dose-dependent manners. The rate of colony formation is 64.79±8.38%,35.21±13.29%,26.03±10.79%, 16.29±10.34%and 6.18±6.06%at dose of 0.25,0.5,1.0,2.0,4.0 mg/ml respectively. Trastuzumab also increased the level of phospho-STAT3 in dose-dependent manners. Knockdown of STAT3 by siRNA significantly increased the trastuzumab-induced proliferation suppression and cell death on HER2-positive breast cancer cells.AG490 suppressed the proliferation of SK-BR3 cells and MDA-MB-231 cells in dose-dependent manners. The IC50 values of trastuzumab in these two cell lines are 43.281 μM and 28.327μM respectively. AG490 also promoted the total cell death of SK-BR3 cells and MDA-MD-231 cells in dose-dependent manners. The cell death rate is 5.78±1.67%,16.89±2.67%,31.08±4.65% in SK-BR3 cells and 8.05±2.05%, 27.26±2.89%,44.18±4.98% in MDA-MB-231 cells at dose of respectively. Compared to control group, AG490 also inhibited the colony formation of SK-BR3 cells and MDA-MB-231 cells in dose-dependent manners. The colony number of colony formation is 559±52,361±38,06±29,117±15 in SK-BR3 cells and 389±42,211±23, 149±17,68±13 in MDA-MB-231 cells at dose of 25,50 and 100μM respectively. AG490 also increased the level of SARI both at mRNA and protein levels in dose-dependent manners in SK-BR3 and MDA-MB-231 cells. Knockdown of SARI by siRNA significantly decreased the AG490-induced proliferation suppression in MDA-MB-231 cells. The analysis of bioinformatics finds a classic STAT3 binding site in the promoter region -1787 to -1797 of SARI. The promoter fragments contain STAT3 binding site or not were inserted into reporter vector pGL3-basic. After transfection, the luciferase activity had not obvious change in the cells with the reporter vector without STAT3 binding site while the same item significantly increased in the cells with the reporter vector with STAT3 binding site. These data indicate that AG490 increases the expression of SARI through STAT3 suppression-mediated transcriptional activation.CONCLUSION1. Trastuzumab suppressed the proliferation, colony formation and promoted total cell death in SK-BR3 cells in dose-dependent manners.2. Trastuzumab treatment activates STAT3 in HER2-positive breast cancer cells. Knockdown the expression or suppression the activity of STAT3 significantly increase the sensitivity of HER2-positive breast cancer cells to trastuzumab.3. AG490 suppressed the proliferation, colony formation and promoted total cell death in both HER2-positive breast cancer cells and HER2-negative breast cancer cells in dose-dependent manners.4. AG490 treatment up-regulates the expression of SARI in both HER2-positive breast cancer cells and HER2-negative breast cancer cells. Knockdown the expression of SARI significantly increase the sensitivity of breast cancer cells to AG490.5. There are STAT3-binding sites in promoter region of SARI gene. The promoter activity of fragment without STAT3-binding site is lower that of fragment with STAT3-binding site upon AG490 treatment.SIGNIFICANCE1. The activation of STAT3 attributes to the trastuzumab resistance in HER2-positive breast cancer cells.2. AG490 significantly enhances the sensitivity of HER2-positive breast cancer cells to trastuzumab.3. The anti-tumor effect of AG490 in breast cancer is closely related to AG490-induced SARI up-regulation. |
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