MTDH Mediates Estrogen-independent Growth And Tamoxifen Resistance In MCK-7Breast Cancer Cells

BACKGROUND AND AIMSThere are about1.2million women with breast cancer occurrence each year in the world. Breast cancer has become the most common tumor in women right now. It has been demonstrated that breast cancer is related with estrogen levels and genetic factors. Some oncogenes have been shown to closely associated with the development of breast cancer such as ER, HER2, VEGF and so on.At present, breast cancer has been divided into different molecular types according to the status of estrogen receptor and HER2gene amplification and given different treatment including endocrine treatment and targeting HER2drugs. Endocrine treatment is an important part of ERa positive breast cancer treatment including estrogen receptor antagonist tamoxifen, toremifene and aromatase inhibitors such as anastrozole, as well as estrogen receptor degradation drug fulvestrant. Tamoxifen, which can target ERa and block the action of estrogen on breast cancer, is still a first-line endocrine therapy for the management of all stages of ERa-positive breast cancer. Five years of tamoxifen treatment was considered the standard duration, however,10years treatment is recommended recently. It can reduce the risk of recurrence by41%on average in patients with ERa-positive tumors. It was improved to greatly prolong disease-free survival and induce remission in over half of patients with ER-positive breast cancer. Unfortunately, almost50%of patients with advanced disease do not respond to first line treatment with tamoxifen. Furthermore, a significant percentage of patients experience tamoxifen resistance relapse, despite an initial positive drug response. The mechanisms of tumor resistance to tamoxifen therapy continue to pose a significant challenge to both clinicians and researchers. It might reside in the expression of specific molecules involved in different signaling pathways, which eventually could be used as predictive biomarkers of resistance. Moreover, these markers may be used to select patients might benefit from additional targeted treatments aside from ERa. Unfortunately, the molecular mechanisms of such resistance remain a poor understanding and serious clinical problems.Some oncogene activation and suppression of tumor suppressor gene are related with resistance to endocrine treatment.MTDH is a versatile newly discovered oncogene overexpression in a variety of malignancies including breast cancer and associated with an aggressive phenotype and poor prognosis. MTDH involved in regulating of PI3K/AKT,NF-kB and other signal pathways in tumor migration and invasion. Our previous study showed that MTDH is over expressed in more than40%breast cancer patients. It has been proved that MTDH can induce EMT (epithelial to mesenchymal transition) to enhance the invasiveness of breast cancer and mediates chemotherapy resistance to5-FU, doxorubicin. There is no evidence to support whether MTDH works in endocrine resitance and estrogen dependence. It was still unclear that the role and the mechanism of MTDH in tamoxifen sensitivity.In this study, gene transfection technique was used to transfect MCF-7breast cancer cells with MTDH gene, and get MTDH gene overexpression cell line. We further detected the role of MTDH in estrogen-dependence and tamoxifen sensitivity in ER positive breast cancer cells and explored its mechanism.MethodsWe used the RT-PCR methods to detect the expression of MTDH at the RNA level in different breast cancer cell lines including ERa-negative cell line MDA-MB-231, MDA-MB-468and ERa-positive cell lines MCF-7and T47D. The expression of MTDH in MDA-MB-231is the highest and MCF-7is the lowest. We chose ERa-positive cell lines MCF-7to study the role of MTDH m estrogen dependence and tamoxifen sensitivity.The cDNA representing the complete open reading frame of MTDH was cloned into the BamHI-XhoI vector fragment derived from the pcDNA3.1vector to generate pcDNA3.1-MTDH (3.1-m) cells. The expression plasmid was verified by sequencing of both strands and was used to transfect the MCF-7cells using lipofectamine2000transfection reagent according to the manufacturer’s protocol. The expression vectors and control vector were transfected into MCF-7breast cancer cells. Effeciency of expression was measured by real time PCR and western blot at the RNA levels and the protein levels. The cell line, which is called MCF7-3.1m, with better expression efficiency of MTDH, and the cell line were used as a control in experiment named MCF7-3.1n.We adopted MTT to detect the changes of viabilities in these two cell lines after treated with different concentrations of estrogen and used tumor formation in nude mice to further explore the relationship between MTDH and estrogen independent growth in vivo. We used cell proliferation assay by MTT and clone formation in different concentrations of tamoxifen to measure cells sensitivity to tamoxifen in MCF7-3.1m and MCF7-3.1n. To further demonstrated the mechansm of tamoxifem resistance induced by MTDH we used Flow Cytometry to test the changed apoptosis rate by measure sub G1-phase cells after treated by tamoxifen.To clarify the mechanism that MTDH mediated resistance to tamoxifen, we exzamined ERa, AKT,BCL-2,PTEN protein expression by western blot and found that AKT,BCL-2Was up-regulated while PTEN was down regulated.To demonstrate the role of PTEN in MTDH mediated cells independent to estrogen and tamoxifen resistance we knockdown PTEN in MCF7-3.1n cells using siRNA. Western blot and MTT assay was used to measure the interference efficiency and the changes of sensitivity to tamoxifen with the negative cells as control.RESULTSWe detected the expression of MTDH in a series of breast cancer cells including ERa negative cell lines MDA-MB-231, MDA-MB-468and ERa positive cell lines MCF-7and T47D. The RT-PCR result showed that the expression of MTDH in MDA-MB-231is the highest and MCF-7is the lowest. We chosed the ERa-positive cell lines MCF-7to study MTDH in estrogen dependence and tamoxifen sensitivity. We used RT-PCR and western blot to detect the efficiency after transfection and demonstrated that the stable cell lines overexpress MTDH was selected. After the MTT test with different concentration estrogen and nude mouse experiment, we found that MTDH expression make breast cancer cells MCF-7are independent of estrogen both in vitro and in vivo. As MTDH was indicated to play an important role in estrogen-independent growth, we concluded that MTDH might regulate the sensitivity to the selective estrogen receptor modulator tamoxifen. To demonstrate it we used cell proliferation assay by MTT and clone formation in different concentrations of tamoxifen to measure cells sensitivity to tamoxifen in MCF7-3.1m and MCF7-3.1n. The results showed that the cells over-expressing MTDH were more resistant to tamoxifen. We study the mechanisms of the resistance induced by MTDH by testing apoptosis rate. The results showed the apoptosis rates of MCF7-3.1m cells were lower than that of MCF7-3.1n after treated with tamoxifen (p=0.005). These indicated that MTDH could inhibit cell apoptosis induced by tamoxifen.We examined ERα, AKT (p-AKT, Ser473), BCL-2and PTEN expression by western blot and found that MCF-3.1m cells showed the same level of ERα as MCF-3.1n cells. The results showed that MTDH could increase expression of p-AKT and BCL2while down-regulated PTEN expression by western blot analysis which might contribute to tamoxifen resistance directly. The most direct and important upstream factor of AKT is PTEN. Our results showed that MTDH induce tamoxifen resistance by regulating PTEN/AKT pathway.To clarify the mechanism that MTDH mediated resistance to tamoxifen, we further silencing the expression of PTEN in MCF7-3.1n cells. Then MTT assay and western blot were all performed with cells transfected with siRNA of PTEN (MCF7-3.1n-siPTEN) and its negative control (MCF7-3.1n-con). After silencing the expression of PTEN, we repeated the MTT assay to test the cell sensitivity to tamoxifen. The results indicated that when PTEN was down-regulated, MCF7-3.1n-siPTEN cells were more resistant to tamoxifen than the control cells. Results of western blot exhibited that the p-AKT levels were up-regulated. So we concluded that MTDH could lead to tamoxifen-resistance by suppression of PTEN. All the results above showed that MTDH mediated estrogen-independent growth and tamoxifen resistance by regulating PTEN/AKT pathway.CONCLUSION1. MTDH could mediate estrogen-independent growth and induce resistance to tamoxifen in ERa-positive breast cancer cells.2. MTDH could reduce the expression of PTEN, up-regulate AKT and BCL2and inhibit the apoptosis induced by tamoxifen.3. MTDH induced tamoxifen resistance by regulating PTEN/AKT pathway in breast cancer.SIGNIFICANCE1. We found that MTDH can induce tamoxifen resistance in estrogen positive breast cancer and would be a new target to reverse endocrine resistance.2. We demonstrated that MTDH induced the MCF-7cells estrogen independent growth.3. MTDH could be a prognosis factor in ER positive breast cancer patients for treatment with tamoxifen.