GOER-mediated Estrogenic Effects In Mammary Cancer Associated Fibroblasts And Its Potential Role In Tamoxifen Resistance Of Breast Cancer

Estrogens play a causative role in the development of breast cancer.Estrogen (G-protein coupled) receptor (GPER) is the third independentestrogen receptor (ER), which mediates estrogenic effects via modulationof second messengers and transactivation of epidermal growth factorreceptor (EGFR) and downstream signal pathways, resulting in cellularbiological behavior changes, such as spreading, proliferation and migration.GPER is widely expressed in breast cancer tissues and cell lines; and isinvolved in estrogen dependent proliferation, migration, invasion, andmetastasis; thus plays an important role in progression of breast cancer.Tamoxifen (TAM) still plays a central role in endocrine therapy of breastcancer, however, was demonstrated to activate GPER as an agonist toaffect biological behaviors of breast cancer cells. Therefore, GPER wasrecently recognized as an essential player in TAM resistance of breastcancer. Cancer associated fibroblasts (CAF) is a group of fibroblasts ofpersistent activity in tumor stroma. They are well known to be an importantcomediator of breast cancer progression in tumor microenviroment sincetheir products such as growth factors, proteases and hormones deeply affectthe proliferation, invasion and metastasis of cancer cells in a paracrinemanner. Early studies implied that GPER may have actions in CAF and similar mesenchymal cells. The present study intends to evaluate GPERmediated estrogenic effects in mammary CAF and its potential significancein TAM resistance of breast cancer. The report was divided into thefollowing sections:Section1. Expression of GPER in tumor stromal fibroblasts andparenchyma of breast cancerObjective: To evaluate expression of GPER in both tumor stromalfibroblasts (TSF) and parenchyma of primary breast cancers.Methods: Immunohistochemistry was utilized to detect GPER in141cases of paraffin-embedded specimens of primary breast invasive ductalcancer and expression of GPER in both TSF and parenchyma werecorrelated to clinicopathologic variables of tumors independently.Results: GPER was positive stained in TSF and cancer cells in acytoplasmic manner. It was detected in vascular smooth muscle cells andendothelial cells as well. Fifty-nine tumors(41.8%) were considered asGPER+in TSF. GPER was detected in TSF of40.5%(32/79) of ERα+and43.5%(27/62) of ERα-tumors respectively. However, GPER staining inTSF could not correlate to determinants analyzed including age, tumor size,pathologic lymph node status, staging, histological grading, Nottinghamprognostic index or expression of ERα, progesterone receptor (PR) andhuman epidermal growth factor receptor-2(Her-2). While,66.7%(94/141)of all the samples were positive stained with variant density in tumorparenchyma. Stromal expression of GPER was correlated to parenchymalexpression of it significantly (P<0.001). GPER and ERα were co-expressedin41.1%(58/141) of tumors parenchyma and significant association wasfound between the two ERs (P=0.041). Epithelial GPER was also detected in58.1%(36/62) of ERα-tumors. Parenchymal GPER expression wasexclusively associated with ERα expression among factors analyzed.Conclusion: GPER was detected in TSF of41.8%breast cancersamples and in parenchyma of66.7%tumors, probably mediate estrogeniceffects in both CAF and cancer cells independently, while, co-contributingto tumor progression in both ERα+and ERα-breast cancer.Section2. Isolation, cultivation, characterization and immortalizationof mammary CAFObjective: To prepare materials for the followed experiments,fibroblasts were isolated from breast cancer tissues and correspondinggrossly normal breast tissues for primary culture. Mammary CAF werethen characterized and immortalized.Methods: The collagenase digestion and culture method was appliedto proceed isolation and primary culture of fibroblasts from16cases ofbreast cancer tissues and some of coupled normal mammary tissues. CAFmarker proteins were detected by immunofluorescence. Cellularproliferation and migration were monitored by xCELLigence real time cellanalysis system. Human telomerase reverse transcriptase (hTERT)retroviruses eukaryon expression plasmid was transfected to PT67cells byliposome, and the viral product was collected to infect CAF forimmortalization of CAF.Results: Fibroblasts isolated from mammary tissues were identified asadherent, large spindle-shaped cells with stress fibers and well-developedfibronexus as described before. The CAF marker proteins, α-smoothmuscle actin (α-SMA) and fibroblast activation protein (FAP), weredetected in more than90%of fibroblasts isolated from breast cancer tissues, thus termed as CAF. While, the markers were absent in fibroblasts isolatedfrom coupled normal tissues, named normal fibroblast (NF). CAF showedmore potent in proliferation (P<0.001) and migration (P<0.001) comparingto NF throughout the real time monitoring of xCELLigence system. Twolines of immortal CAF were established successfully since they could bepassaged continuously and also expressed α-SMA, although dramaticallyreduced in size.Conclusion: Fibroblasts were isolated from mammary tissuessuccessfully by the collagenase digestion and culture method. Fibroblastsisolated from breast cancer tissues presented characteristics of CAF thustermed as CAF. Two lines of immortal CAF maintaining fundamentalfeatures of primary CAF were established successfully and thus could beused in the followed studies.Section3. Expression and activity of GPER in mammary CAFObjective: To evaluate phenotype of ERs and activity of GPER inmammary CAF.Methods: Reverse transcriptase, real-time fluorescent quantitativepolymerase chain reaction (PCR) and immunofluorescence technique wereused to detect GPER, ERα and ERβ at mRNA and protein levels in primaryCAF, immortal CAF, NF and MCF-7cell as positive control. Under theadministration of17-β-estradiol (E2) and GPER specific agonist, G1,intracellular calcium (Ca2+) labeled by fluorescence probe was monitoredby laser confocal microscopy; phosphorylation of extracellularsignal-regulated kinase (ERK) was evaluated by Western blotting and theactivation of GPER as a G-protein coupled receptor was confirmed byxCELLigence real time cell analysis system further. Results: GPER mRNA was detected in CAF and immortal CAF(CAF-hTERT), as0.2~1.01fold in MCF-7cells. It was also detected in NF.Moreover, GPER was identified at protein level in CAF, CAF-hTERT, NFand MCF-7cells by immunofluorescence. However, neither ERα nor ERβwas present in CAF at mRNA or protein level. E2and G1in variantconcentration stimulated sharp increase of intracellular Ca2+concentrationwithin1~2min, enhancement of phosphorylation of ERK within5~30minand significant, transient rise in cell index within an hour in CAF.Importantly, these changes could be blocked by pretreatment of G15(10-6M, same as followed), the specific antagonist of GPER. However,similar estrogenic reactions were absent in NF.Conclusion: GPER was exclusively expressed as an ER in CAF andNF. Interestingly, GPER mediated “rapid non-genomic” estrogenicresponses; thus presented receptor activity in CAF but NF.Section4. GPER-mediated estrogenic effects in CAFObjective: To evaluate GPER-mediated estrogenic effects and relatedpathways in CAF.Methods: Cell Counting Kit8(CCK8) was applied for cell viabilityassay. Cellular proliferation was monitored by xCELLigence real time cellanalysis system. Cell cycle was measured by flow cytometry. GPERspecific antagonist G15and kinase inhibitors including EGFR inhibitorAG1478, ERK inhibitor U0126and phosphatidyl inositol-3-kinase (PI3K)inhibitor Wortmannin were employed to evaluate the pathway triggered byE2to influence growth and proliferation in CAF. xCELLigence system,morphological observation and Transwell assay were used to assessinfluence of E2, and G1on cell adhesion, spreading and migration in CAFrespectively. Results: E2and G1promoted cell growth of CAF in aconcentration-dependent manner and the optimum concentration was10-8Mfor E2and10-6M for G1which increased the cell number to162.8%±12.1%(P=0.0018) and155.9%±9.9%(P=0.0019) of controlrespectively after72h cultivation. Via the xCELLigence system, E2(10-8M,same as followed) and G1(10-6M, same as followed) were observed topromote proliferation significantly in CAF (P<0.001). Actually, thenormalized cell index of control, E2group and G1group were2.585±0.186,4.212±0.361and3.794±0.312at the96thhour after agents were added; thedifferences between experimental groups and control were significant.Furthermore, cultivation with E2and G1for24h significantly increased theS stage fraction of CAF in cell cycle measurement (26.86%±5.64%and24.70%±6.22%vs.13.38%±3.28%， P=0.023,0.041). Importantly,pretreatment of G15, AG1478(10-5M, same as followed) and U0126(10-5M,same as followed) significantly blocked the above changes (P<0.05).However, Wortmannin (10-5M, same as followed) showed no notableimpact on these effects (P>0.05). In addition, the first peak of cell indexindicating the adhesion of resuspended cells presented earlier forapproximate3hours, with significant higher peak value after stimulatedwith E2and G1(2.272±0.069at5.97thhour for E2group and2.257±0.223at6.22thhour for G1group vs.1.982±0.059at9.15thhour for control,P=0.0004and0.0386). The E2-and G1-treated cells were spread moreunder microscopy at the12edhour after the manegment and migrated more(132.1±30.3and108.8±19.8vs.34.5±6.3, P=0.006and0.004) in Transwellassay at the24thhour after the manegment. The spreading and migrativeeffects were reduced significantly by G15pretreatment (P<0.05).Conclusion: E2triggered GPER-EGFR-ERK signaling to promote growth and proliferation of CAF. GPER also mediated enhancement ofadhesion, spreading and migration of CAF in response to E2. GPERprobably contribute to cancer progression in a CAF-dependent manner.Accordingly, GPER-targeted strategy is probably a valuable approach tobreast cancer endocrine therapy in the future.Section5. CAF-dependent significance of GPER in tamoxifenresistance of breast cancerObjective: To assess TAM-triggered GPER-mediated effects in CAFand the potential CAF-dependent significance of GPER in TAM resistanceof breast cancer.Methods: Under the administration of TAM, intracellular Ca2+labeled by fluorescence probe was monitored by laser confocal microscopy;phosphorylation of ERK was evaluated by Western blotting and theactivation of GPER as a G-protein coupled receptor was further confirmedby xCELLigence real time cell analysis system. CCK8was applied in cellviability assay. Cell cycle was measured by flow cytometry. Morphologicalobservation and Transwell assay were used to evaluate effect of TAM onspreading and migration of CAF. The substrate of estrogens, testosteronewas added for cultivation and E2was detected in medium bychemiluminescence. Reverse transcription and real-time quantitative PCRwere used to evaluate expression of aromatase gene CYP19A1at mRNAlevel. Managements of TAM, G1, G15, AG1478, U0126and Wortmanninwere applied to evaluate the mechanisms responsible for TAM-inducedeffects in CAF.Results: Similar to E2and G1, TAM stimulated, in a dose-dependentmanner, modulation of Ca2+, phosphorylation of ERK and rise of cell indexin xCELLigence system. And these changes were blocked by G15. TAM(10-8M, same as followed) promoted growth and proliferation of CAF,which could be inhibited by G15, AG1478and U0126but Wortmannin.TAM also promotes cell spreading and migration both of which could bereduced by G15. Intriguingly, testosterone related to concentration of E2inCAF medium in a dose-dependent manner. After cultivation with TAM for48h, E2concentration in CAF medium with testosterone(10-7M) wasincreased from302.5±40.8pg/ml of control to541.8±68.7pg/ml (P=0.011).Interestingly, pretreatment of G15, AG1478, U0126and Wortmanninreduced it to353.8±43.5pg/ml (P=0.022),345±52.8pg/ml (P=0.02),328.5±36.8pg/ml (P=0.017) and520.2±42.8pg/ml (P=0.676) respectively.Furthermore, cultivation with G1in presence or absence of aforementionedinhibitors affected the E2concentration in CAF medium with same trends.Importantly, mRNA of aromatase gene CYP19A1was upregulated by3.4±0.64times (P=0.003) by cultivation with TAM for18h in CAF. Again,management of G15, AG1478, U0126and Wortmannin decreased it to1.3±0.22(P=0.006),2.0±0.29(P=0.029),2.0±0.31(P=0.036) and3.3±0.85times (P=0.855), respectively. Moreover, cultivation with G1in presenceor absence of these inhibitors regulated CYP19A1gene expression with asimilar trend.Conclusion: TAM triggered GPER-EGFR-ERK signaling to promotecell growth, proliferation, spreading and migration in CAF similar to E2and G1. This pathway is also responsible for the upregulation of aromatasegene expression and E2synthesis in CAF. These effects probably causesecondary effect in cancer cells to induce TAM resistance in breast cancer.Accordingly, GPER-targeted strategy probably is a complementation orsupplement for current endocrine therapy of breast cancer.