Dissertation > Medicine, health > Oncology > Genitourinary tumors > Breast tumor

Inhibition of Metadherin Sensitizes Breast Cancer Cells to AZD6244by Regulating ERK/FOXO3a Pathway

Author KongXiaoLi
Tutor YangQiFeng
School Shandong University
Course Surgery
Keywords Breast cancer MTDH AZD6244 FOXO3a ERK1/2
CLC R737.9
Type PhD thesis
Year 2012
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BACKGROUND AND OBJECTBreast cancer is one of the most common female cancers in the world. Recent studies indicate that cancer is the result of some signal pathways disorders caused by abnormal expression of oncogenes and tumor suppressor genes. In the past few decades, massive efforts in cancer research have led to the identification of a seemingly exhaustive list of oncogenes and tumor suppressor genes which are potential targets for anticancer therapeutics, such as ER, HER2, ERK1/2etc. Some gene targeted drugs have been widely used in clinical treatment, such as Tamoxifen, Herceptin and AZD6244, all of which have been demonstrated effectiveness for patients who have failed in the traditional treatments. However, the resistance to drugs is still one obstacle leading to treatment failure for some patients.AZD6244, a novel potent oral inhibitor of MEK1/2, is an effective inhibitor of the RAF/MEK/ERK pathway, and has the potential to inhibit cell proliferation, survival, metastasis and invasion. AZD6244was reported to be relatively safe, efficacious and demonstrated promising results in solid tumors. The results of some clinical trials showed that AZD6244obtained the expected target effects in various tumors including colorectal cancer, lung cancer, acute myelogenous and breast cancer. Some other trials indicated AZD6244has an efficacy comparable to capecitabine in terms of disease-free progression and progression-free survival. AZD6244selectively targets cancer cells and has thus, treatment related toxicity is reported to be low. Though the effectiveness of AZD6244is promising, the resistance to it is needed to be solved urgently. Reporters have shown that FOXO3a can be induced by AZD6244and make cells more sensitive to this drug. FOXO3a is a novel tumor suppressor in regulating cell cycle and cell apoptosis. It can be regulated by AKT and ERK1/2, and both the two pathways were reported to be regulated by MTDH in breast cancer. It has been reported that MTDH can regulate FOXO3a in esophageal squamous cell carcinoma and prostate cancer but in breast cancer whether MTDH can regulate the expression and activity of FOXO3a is still unknown.MTDH (metadherin, AEG-1, lyric) is a novel multifunctional oncogene which was first cloned by subtraction hybridization in2002. It has been found to be overexpressed in various cancers and involved in multiple molecular pathways including PI3K/AKT, MAPK, NFkB and so on to promote cancer cells proliferation, migration, tumor metastasis, angiogenesis and contributes to multiple drug resistance. In breast cancer, our group previously reported that more than40%of tumors overexpress MTDH, and this finding is associated with prognosis. MTDH can enhance the invasiveness of cells by inducing epithelial to mesenchymal transition (EMT), and can induce multiple drug resistance including doxorubicin, paclitaxel, cisplatin in breast cancer. But whether MTDH can affect the cell sensitivity to AZD6244in breast cancer via influencing FOXO3a is still unknown.Our study aims to find the relationship between MTDH and FOXO3a as well as the candidate pathways through interfering the expression of MTDH, and then study the role of MTDH in cells sensitivity to AZD6244whether via regulating the expression and activity of FOXO3a.METHODSTwo paired of60nt oligos which can transcripted into short hairpin RNA (shRNA) targeting MTDH gene were designed and inserted into the pSUPER.retro.puro(prpn) plasmid to conctruct the interference vector. The two interference vectors and control vector were transfected into MDA-MB-231cells. Stable transfected cell lines were selected by puromysin and the effeciency of interference was measured by real time PCR at the RNA levels while western blot at the protein levels. The cell line which has better interference effeciency called231-prpM was used for the experiments with the cell line231-prpn as control. The FOXO3a expression at protein level was examined by western blot, both ERK1/2and AKT activity were measured by the level of p-ERK1/2and p-AKT. The expression of p-FOXO3a (Ser294) was measured by western blot and the phosphorylation level of FOXO3a was measured by the ratio of p-FOXO3a/FOXO3a. To demonstrate whether MTDH could regulate the activity of FOXO3a, we tested the expression levels of FOXO3a in both nuclear and cytoplasma of231-prpn and231-prpM cells to study the change of its nuclear translocation.We used MTT to quantitate changes of viabilities in the two selected cell lines after treated with various concentrations of AZD6244., and used Flow Cytometry to tested the changes of G0/G1-phase arrest and apoptosis induced by AZD6244.To demonstrate the role of FOXO3a in MTDH mediated cells sensitivity to AZD6244, we knockdown FOXO3a in both231-prpn and231-prpM cells using siRNA. We tested the knockdown efficiency by western blot and then used MTT assay to measure the changes of sensitivity to AZD6244with the negative siRNA transfected cells as control.RESULTSThe interference vectors were successfully constructed and were verified by sequencing. Stable cell lines that express control vector or inference vector were successfully selected by puromycin and the expression level of MTDH was tested by Qrt-PCR and western blot, both of which showed well effect of interference.The results of western blot showed that MTDH knockdown inhibite the activity of ERK1/2, activate AKT and upregulate the expression of FOXO3a. Although the expression of p-FOXO3a(Ser294, phosphorylated by ERK1/2) was not changed significantly but the ratio of p-FOXO3a/FOXO3a was decreased after MTDH knockdown (p<0.01). These results indicate that MTDH knockdown can increase the expression of FOXO3a via inhititing the activity of ERK1/2. Besides we found MTDH knockdown make the expression of FOXO3a decreased in cytoplasma while increased in nuclear in breast cancer cells which indicated that MTDH knockdown activated the FOXO3a.We found MTDH knockdown make breast cancer cells more sensitive to AZD6244(IC50from6.04±0.08μM in cells with control vector to0.52±0.04μM in cells with MTDH knockdown)(p<0.01). Also the reduced expression of MTDH led to more accumulation of cells GO/G-phase (70.64%to42.98%versus57.99%to50.08%)(p<0.01) and more sub-G1phase (32.15%versus16.62%)(p<0.01) induced by AZD6244treatment.We used siRNA to knockdown FOXO3a in231-prpn and231-prpM cells with the negative siRNA transfected cells as control. Western blot results showed perfect knockdown efficiency. MTT assays showed that cells become more resistant to AZD6244after FOXO3a knockdown in231-prpM cells, which was not found in231-prpn cells.CONCLUSION1. MTDH functions as a potential oncogene in breast cancer and may promote cancer cells proliferation, invasion and migration via regulating FOXO3a.2. MTDH knockdown can increase the expression and activation of FOXO3a via MTDH/ERK1/2/FOXO3a pathway but not MTDH/AKT/FOXO3a pathway.3. MTDH knockdown make breast cancer cells more sensitive to AZD6244.4. The increased sensitivity to AZD6244by MTDH knockdown in breast cancer cells is mediated by activating FOXO3a.SIGNIFICANCE1. We revealed the relationship and pathway between MTDH and FOXO3a in breast cancer.2. We demonstrated that MTDH influences the cells sensitivity to AZD6244and reported the related pathway.3. We provided a new candidate target for the treatment of breast cancer.

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