Inhibition Of BTF3 Sensitizes Luminal/ER+ Breast Cancer To PI3K? Inhibition Through The Transcriptional Regulation Of ER?

Background and purpose:Estrogen receptor alpha(ERa)is a hormone-regulated transcription factor that is present in approximately 70%of breast cancers.Estrogen receptor alpha(ERa)-positive(ER+)breast cancers often respond well to endocrine therapies that block ERa signaling.However.these breast cancers also frequently relapse after prolonged endocrine therapies,justifying the need for the development of alternative effective treatment strategies.More than 30%of ER+breast cancer,representing the most common genomic alterations in this subtype of tumors.The high frequency of PIK3CA mutations in luminal/ER+cancers suggests that inhibitors of PI3K kinases or key nodes in this pathway may benefit ER+/PIK3CA mutant breast cancer patients.Intensive efforts have been made to test selective PI3Ka inhibitors in clinical trials in patients with advanced breast tumors harboring PIK3CA alteration.While clinical trials of these inhibitors have yielded promising results,not all the PIK3CA mutation-positive breast cancers respond to these agents,and even those that initially respond often relapse after months of therapy.Recent studies have reported that the blockade of ERa activity sensitizes luminal/ER+breast tumors to PI3K inhibition,in particular PI3Kct isoform specific inhibitors.Indeed,these studies reveal that increased ERa transcriptional activity accounts for the limited efficacy of PI3K inhibitors used as monotherapy in breast cancers of the luminal subtype.As such,the investigation of the molecular mechanisms that are involved in the modulation of ERa expression may open up new avenues for the development of biomarkers that help stratify patients most likely to benefit from PI3Ka targeted therapies.BTF3(Basic transcription factor 3)has been reported to be overexpressed and proposed to function as an oncogene in various tumor types including prostate cancer,Nevertheless,the correlation of BTF3 expression with breast cancer has not been reported.In this study,we aim to find the expression and function in the development of breast cancer of BTF3 in luminal/ER+breast cancer,and explore the related mechanism.The discovery of the role of BTF3 in luminal/ER+breast cancer may provide a rationale therapeutic target or provide a theoretical basis for the combination of breast cancer therapy.Methods and result:1.Analyzed BTF3 expression in the breast cancer data set of the Cancer Genome Atlas with PAM50 classification(TCGA),we found that higher expression levels of BTF3 were strongly associated with the luminal subtype of breast cancers.In contrast,BTF3 expression was significantly lower in the basal-like breast cancer samples than in the HER2-enriched breast tissues.We further analyzed the status of BTF3 expression in the TCGA dataset with an alternative categorization of the breast cancer subtypes that is routinely used in the clinic for diagnosis.BTF3 expression was significantly higher in the ER/PR+subtype than in the TNBC.It was also notable that even within the HER2+subtype,the ER/PR+tumors had a much higher BTF3 expression than the ER/PR-tumors.2.Considering the high expression of BTF3,we next want to explore the role of BTF3 in luminal/ER+breast cancer.We showed that BTF3 knockdown significantly reduced the growth of MCF7 and T47D cells cultured in monolaver and markedly attenuated the 3D spheroid growth of MCF7 and T47D cells.To examine if BTF3 affects cellular migration,we conducted the wound-healing assays.We found that the sap between the scratched area was larger in BTF3 knockdown group than in control group 48 hours post-wounding.Clonogenic survival assay found that restoring BTF3 expression rescued the growth inhibitory effect caused by BTF3 knockdown.Together,these results suggest that BTF3 has strong oncogenic ability in luminal/ER+breast cancer cells.3.As BTF3 promotes cell growth,we next assessed its effect on cell cycle progression.The flow cytometry data analysis revealed that the percentage of cells in G2/M phase was significantly higher in siBTF3-transfected MCF7 cells and T47D cells than in siNC transfected cells 48 hours after serum stimulation.We also examined the effect of BTF3 on cell survival and found that BTF3 knockdoxvn induced apoptotic cell death.Together,these results suggest a role of BTF3 in the regulation of G2/M transition and cell survival in luminal/ER+breast cancer cells4.Analysis of TCGA breast cancer database Desmedt breast database and Wang_breast database revealed that BTF3 expression is substantially elevated in ER+cohorts when compared to ER-cohorts.We also identified a highly significant correlation between BTF3 and ESR1 expression in these cohorts.Interestingly,immunoblotting analysis revealed that BTF3 is only overexpressed in breast cancer cell lines with high abundance of ERa.Together,these results prompted us to further investigate the relationship between BTF3 and ERa.Using siRNA to silence BTF3 we found BTF3 knockdown led to substantially reduced expression of ERa at mRNA and protein levels,and it also led to reduced expression of ERa-mediated transcriptional targets,PGR,GREB1 and IGFBP4.We ectopically expressed ERa in siBTF3 or siNC transfected MCF7 cells.Our results showed that ectopic expression of ERcc at least in part rescued the growth inhibitory effect caused by BTF3 silencing.Together,these data suggest that BTF3 contributes to the growth of luminal breast cancer cells,at least in part,through transcriptional regulation of ERa5.Recent studies reveal that blockade of PI3K signaling results in an induction of ERa-dependent transcriptional activity.As our data point to a role of BTF3 in the regulation of ERa transcription,we next tested if BTF3 may affect the response of luminal breast cancer cells to PI3Ka selective inhibitor BYL-719 treatment.Using western blotting to analyze the expression of intracellular protein.we found that BYL-719 nearly completely abolished phosphorylated AKT(pAKT)levels;BYL-719 treatment did lead to a striking increase in ERa protein expression.Interestingly while either BYL-71 9,or to a higher degree siBTF3,as single-agents resulted in a moderate increase in apoptotic signal,the combination of BTF3 knockdown and BYL-719 induced more substantial apoptosis.And we confirmed that combined use of siBTF3 and BYL-719 resulted in significantly attenuated 3D spheroid growth.Together,these results suggest BTF3 knockdown may sensitize the response of luminal/ER+breast cancer cells to PI3Ka inhibition by BYL-719 treatment.6.In vitro experiments have shown that targeting inhibition of BTF3 can sensitize luminal/ER+breast cancer cells to BYL-719.We would like to further explore whether there is a similar situation in vivo.We used siRNA-mediated BTF3 knockdown and B YL-719 in the well-established ER+/PIK3CA mutant MCF7 breast tumor xenograft model.We found that single-agent B YL-719 led only to tumor stasis and resulted in an induction of ERcx expression.Meanwhile,treatment with siRNA targeting BTF3 also resulted in tumor stasis but with a significant decrease in ERa expression.Moreover silencing BTF3 in combined with BYL-719 treatment resulted in dramatic tumor regressions.Together.these results suggest that BTF3 silencing sensitizes Luminal/ER+breast cancer cells to PI3Kcc inhibition in vivoConclusion:1.High expression levels of BTF3 exist in luminal/ER+breast cancer.2.BTF3 promotes the proliferation.survival and migration of Luminal/ER+breast cancer cells.3.BTF3 regulates ESR1 expression and ERa-dependent transcription.4.BTF3 knockdown abrogates the upregulation of ERa expression induced by BYL-719.5.BTF3 silencing sensitizes Luminal/ER+breast cancer cells to BYL-719 in vitro and in vivo.