A Study On The Impact Of ERβ Level To Tamoxifen In Breast Cancer Cell Lines

BackgroundBreast cancer is one of the most common malignant tumors in female，according to the statistics globally，there are approximately1.2millon womenpatients with breast cancer，and with nearly0.5millon women die of breastcancer each year. In Western Europe，North America and other developedcountries and regions，the incidence of breast cancer is the highest in malignanttumor of female. In our country，the incidence of breast cancer is continuouslyrising，and the new patients are more and more younger in recent years. As akind of tumor induced by hormone，there is a history of over100years about theendocrine therapy to the patients of breast cancer. Targeting the estrogenreceptor (estrogen receptor，ER) as prognostic factors in breast cancer patientsas well as therapeutic targets have important clinical significance. And also thetumor biopsy in the detection of ER has become the conventional treatmentoptions. At present，the antagonists of estrogen receptor，such as tamoxifen andother drugs，are seen as the first-line therapy for the ER positive breast cancerpatients. Unfortunately， approximately30%of patients who received thetreatment of endocrine therapy such as tamoxifen have not obtained obvioustherapeutic effects，and the reasons of its drug resistance are still unclear， theresistance mechanisms have been the study hotspot. After the discovery of the first estrogen receptor ERα later about10years，in1996，people successfullycloned and identified the second estrogen receptor β. Since that ERβ testing inbreast cancer，the potential application value in breast cancer occurrence，development，prognosis and response to endocrine therapy in terms of researchhave received increasing attentions. But at present，some of the shortages arewhen the clinical laboratory use immunohistochemistry assay for detection ofbreast cancer by ER expression levels，use antibodies mostly only for ER alpha oruneffectively distinguish between the ER alpha and ERβ. There are a few reportsabout ERβ in patients with breast cancer for tamoxifen resistance in the research，but the experimental group research result and conclusion is consistent. Thisproject is designed to use more sensitive and efficient methods to change the levelof ERβ in breast cancer cell lines，and a series of experimental techniques toobserve the reactivity of these cells to tamoxifen. And then on this basis we intendto be clear about the possible roles of tamoxifen endocrine therapy resistance onthe level of ERβ，and attempt to clarify the possible molecular mechanism. Andwe hope that our research may provide some direct and valuable references inorder to get more reasonable and effective clinical treatments for patients ofbreast cancer about endocrine antagonist.ObjectiveThis study aims at designing to use more sensitive and efficient methods tochange the level of ERβ in breast cancer cell lines，and a series of experimentaltechniques to observe the reactivity of these cells to tamoxifen. And then on thisbasis we intend to be clear about the possible roles of tamoxifen endocrinetherapy resistance on the level of ERβ，and attempt to clarify the possiblemolecular mechanism. And we hope that our research may provide some direct and valuable references in order to get more reasonable and effective clinicaltreatments in patients of breast cancer about endocrine therapy.Methods1. To construct the recombinant lentiviral expression vector containinghuman ERβ gene at different levels：we cloned the ERβ gene by RT-PCRmethod and linked it into the pENTRTM3C carrier to create an entry clone，andthen generated an expression clone by performing an LR recombinantionreaction between the entry clone and a Gateway destination vector(pLenti6.3/V5-DEST). Finally we identified the expression clone(pLenti-ERβ)by sequencing and PCR. The short hairpin RNA (pLKO-shRNA) effectivelysilencing ERβ gene was directly bought from Sigma company，and theeffectiveness of the silencing ERβ gene have also been verified by the company.2. To establish a stable reduced ERβ gene on the expression of ERβ mRNAand protein by lentiviral vector-mediated short hairpin RNA(pLKO-shRNA) inhuman breast cancer cells：three plasmid of lentiviral packaging system（thepackaging plasmid psPAX2， envelope plasmid pMD2.G and interferenceplasmid）. Then the virus supernatant was used to infect the breast cancer celllines T47D and MCF-7cells. After transfecting for48h,we intended torespectively select two effect target point at ERβ gene by PCR and western-blot.After a certain concentration of puromycin (1mg/L) continuous pressure filterfor4W，the positive clones have been selected. And then we again identifiedthe ERβ gene expression on the level of mRNA and protein. In the end，thestable down-regulation of ERβ gene in breast cancer cell lines T47D and MCF-7have been established successfully.3. On the basis of the construction of two stable down-regulation of ERβgene in two breast cancer cell lines，we observed the changes of biological behavior of breast cancer cells to tamoxifen response from the aspects of cellproliferation and apoptosis. The methods included MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，four methyl azo triazole blue)，EdU(5-ethynyl-2’ deoxyuridine,5-ethynyl–2，BrdU)，FCM （flow cytometry）and TUNEL(TdT-mediated dUTP Nick-End Labeling，TdT mediated DNA threephosphoric acid uridine nick-end labeling method). On this basis，we focused onmolecules relative to apoptosis(such as caspase-3) by Western-blot mainly，andwe hope to get the possible molecular mechanism on ERβ gene levels intamoxifen resistance to breast cancer.Results1. Recombinant lentiviral expression vectors containing ERβ gene havebeen validated by agarose gel electrophoresis，PCR and gene sequencing，andthe results showed that the human recombinant lentiviral expression vector(pLenti-ERβ) was successfully constructed. Besides,the pLKO-shRNA lentiviralexpression vector targeted to down-regulat ERβ gene has been verified by theSigma company.2. Firstly，the most obvious two targets of down-regulated effects to ERβwere successfully selected for each cell line：T47D(pLKO-shRNA-3326/3327)and MCF-7(pLKO-shRNA-3325/3326). Secondly，we continuously added acertain concentration of puromycin to the cells for screening about4W. Lastly，the ERβ expression were verified on the levels of mRNA and protein byRT-PCR and western-blot，and the results showed that：comparing with thecontrol group， the ERβ mRNA expression of experimental groups weresignificantly decreased and their lowered rates in two cells lines were(61.12±3.66)%，(76.47±3.16)%in T47D and (62.42±0.07)%，(42.49±1.96)%inMCF-7,but there was no significant difference between negative control groups and control groups. Also the expression of ERβ protein by western-blot in twobreast cancer cell lines showed that，comparing with the control groups，the ERβprotein expression of experimental groups were also significantly reduced：inT47D-ERβ-s1/s2，their protein levels were decreased to (60.83±3.07)%and(53.31±3.00)%. In MCF-7-ERβ-s1/s2，their protein levels were cut down to(83.69±5.07)%and (73.16±13.21)%. There were no significant differencesbetween negative control groups and blank control groups.3. Based on the two breast cancer cell lines with stablely knockdown thelevel of ERβ gene，we detected the biological characteristics of breast cancercells on cell proliferation and apoptosis to the TAM reactivity：①MTT assay： T47D cells in different concentrations of TAM for48h，the experimental groups(s1and s2) from10μM TAM，the cells survival rateswere significantly lower than the control group (the scramble)，especially in15μM TAM (P <0.05)；in MCF-7cells，cells survival rates of experimentalgroups were lower than the control group (the scramble) from5μM TAM，andthere was a most obvious difference in10~15μM TAM (P <0.05). But therewere no significant differences between two experimental groups (P>0.05).These results indicated that the lower level of ERβ may improve the inhibitionof proliferation at a certain concentration TAM to breast cancer cells.②EdU assay：to observe the differences of celluar DNAproliferation eachgroup of cell in the two stable transfection breast cancer cell lines：the EdUmarker positive cells in T47D-s1/s2were more than those in the scramble groupwithout TAM. However，test results of part with15μM TAM for48h showedthat none of EdU marker-positive cells in two groups of T47D-s1/s2werefound，and was significantly less than in T47D-the scramble group(P <0.05).Interestingly，we obtained the similar results in MCF-7cells. These results indicated that to some extent reduceing ERβ can stimulate proliferation of breastcancer cell, but can increase cancer cell sensitivity to TAM drugs.③FCM assay：to observe the levels of apoptosis in each group cells byFCM detection，the results were as following：without TAM，there were nosignificant differences in the three groups cells of T47D (P>0.05)；but theresults showed that the apoptosis cells of T47D-s1/s2two groups weresignificantly more than the T47D-scramble group after15μM TAM for48h (P<0.05)，there were no significant differences between the two experimentsgroups (P>0.05). The similar results were also obtained in MCF-7cells. Theseresults suggested that it could enhance the apoptosis of breast cancer for TAMdrug-induced in on some lower level of ERβ.④TUNEL assay：after the drug action by TAM，the apoptotic cells of twoexperimental groups（T47D-s1and s2）were significantly more than the controlgroup(P <0.05)，and apoptotic cells in the group（MCF-7-s2） were more thancontrol group；there was a statistical difference between experimental groupsand control ones.⑤The detection results of moleculars related to apoptosis by western blotshowed that in all the three groups T47D-scramble/S1/S2，the molecularcaspase-3was significantly activated to the subunit cleved-caspase-3(17KD)，but not seen in the control group. Also，there was no significant differencesamong three groups without TAM.Conclusion1. In estrogen receptor (ER) positive breast cancer cell lines T-47D andMCF-7，down-regulation of the ERβ gene expression may cause the change ofbreast cancer anti-estrogen drug tamoxifen reactivity：①To some extent，itlower ERβ level that may stimulate the proliferation of breast cancer cells themselves；②The sensitivity to TAM drugs for breast cancer cells could beincreased by reduced ERβ level. The experimental results showed that cellsproliferation was inhibited and apoptosis was obviously induced in sameconcentration of tamoxifen.2. ERβ gene expression levels of breast cancer cells may influence theeffect of breast cancer endocrine therapy，and our results implied that thisprocess may be relevant with enhanced activation of some moleculars such ascaspase-3in apoptosis. According to our study，obtained results suggested thatERβ expression levels could affect the treatment of breast cancer on endocrinetherapy，and its overexpression may lead to tamoxifen resistance.