The phenotypes of each breast cancer subtype are defined by their transcriptomes. However, the transcription factors that regulate differential patterns of gene expression that contribute to specific disease outcomes are not well understood. Herein, using gene silencing and overexpression approaches, RNA-Seq, and splicing analysis, we report that the transcription factor B-cell leukemia/lymphoma 11A (BCL11A), is highly expressed in triple-negative breast cancer (TNBC) and drives metastatic disease. Moreover, BCL11A promoted cancer cell invasion by suppressing the expression of muscleblind-like splicing regulator 1 (MBNL1), encoding a splicing regulator that suppresses metastasis. This ultimately increased the levels of an alternatively spliced isoform of integrin-α6 (ITGA6) that is associated with worse patient outcomes. These results suggest that BCL11A sustains TNBC cell invasion and metastatic growth by repressing MBNL1-directed splicing of ITGA6. Our findings also indicate that BCL11A lies at the interface of transcription and splicing and promotes aggressive TNBC phenotypes.
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Due to the lack of receptor expression, there are limited targeted therapies available for TNBC. As a result, TNBC patients are primarily treated with chemotherapies such as taxanes. Although TNBCs initially regress in response to taxane treatment, resistance is common. One mechanism of taxol unresponsiveness/resistance is an increase in chromosomal instability (CIN). Increased CIN can confer survival advantages to cancer cells and increase their aggressiveness. However, CIN levels can be leveraged using drugs that inhibit proteins important for chromosomal stability. Combining CIN-inducing drugs, such as taxanes, can improve treatment efficacy or re-sensitize tumor cells to certain drugs by shifting cells towards a state of maladaptive CIN that is incompatible with cell viability. We have found that the non-receptor SRC family kinase YES1 is crucial for chromosomal stability. YES1 is important for cell division, motility, adhesion, and survival in both normal and TNBC cells. Since taxol and YES1 silencing independently increase chromosomal instability, I hypothesized that combining a YES1 inhibitor (YES1i) with taxanes would shift cells toward an irreversible state of maladaptive CIN, decreasing their survival. I found that YES1 mRNA and protein are upregulated in taxane-resistant cells and that YES1 protein expression correlates with the paclitaxel IC50 in a panel of TNBC cell lines, suggesting that YES1 may drive taxane resistance. Furthermore, I found that a selective YES1 inhibitor (CH6953755) synergizes with paclitaxel and improves taxane response in both in vitro and in vivo TNBC models. In addition, the combination of YES1i and paclitaxel treatment increases phenotypes associated with chromosomal instability more than either drug alone. These data suggest that YES1 inhibition in combination with taxanes represents an innovative and novel drug treatment regimen that improves TNBC patient outcomes. Citation Format: Natasha Ingles, Katrina Piemonte, Salendra Singh, Kristen Weber-Bonk, Ruth Keri. YES1 is a targetable vulnerability for improving taxane response in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-11.
Cancer stem cells (CSC) contribute to the high incidence of metastatic recurrence in triple-negative breast cancer (TNBC) and elucidating the mechanisms controlling CSC properties should provide novel targets for therapeutic development that improves patient outcomes. The transcription factor BCL11A is highly differentially expressed in TNBC versus all other subtypes and controls breast CSC phenotypes such as marker expression and tumor initiation. However, the genes that BCL11A targets to control CSC biology and whether BCL11A promotes metastatic progression are unknown. To address these questions, we assessed the impact of transiently silencing the expression of BCL11A in TNBC cells using siRNA transfections. Reducing BCL11A expression did not impact TNBC cell viability or in vitro migratory capacity. However, silencing BCL11A significantly decreased invasion of TNBC cell lines, in vitro. Furthermore, stable shRNA-mediated silencing of BCL11A in the highly metastatic MDA-MB-231 cell line reduced metastatic outgrowth when xenografted into immunocompromised mice, suggesting that BCL11A is critical for metastatic progression of TNBC. To identify the BCL11A-regulated transcriptome in TNBC, we performed RNA-seq analysis of cells transiently transfected with non-targeting or BCL11A-targeted siRNAs in the MDA-MB-231 cell line. Consistent with the impact of BCL11A on invasion, numerous genes involved in adhesion and extracellular matrix were reduced with BCL11A silencing. The gene encoding the matrix metalloproteinase, MMP1, was the most significantly differentially expressed gene, with a 10-fold decrease in RNA expression following BCL11A suppression. We confirmed this down-regulation by both qRT-PCR and western blots of independent samples, as well as in the HCC1143 TNBC cell line. Current studies are assessing the functional impact of MMP1 regulation by BCL11A on invasion and metastatic progression of TNBC. Citation Format: Darcie D. Seachrist, Natasha N. Ingles, Molly M. Hannigan, Donny D. Licatalosi, Ruth A. Keri. BCL11A is necessary for the expression of extracellular matrix genes and metastatic progression of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 32.
Triple-negative breast cancer (TNBC) represents 15% of all breast cancers, yet conveys the poorest patient prognoses due to the lack of targeted therapies and the high incidence of metastatic recurrence. This prompt recurrence is thought to be due, in part, to the high proportion of cancer stem cells (CSC) within TNBC tumors. Therefore, understanding what regulates CSCs and metastasis will allow us to significantly reduce cancer-related deaths. One of the transcription factors that is upregulated in TNBC is BCL11A, which is critical for CSC biology. We have found that BCL11A is necessary for TNBC invasion and metastasis. However, the genes that BCL11A targets to control invasion and metastasis are unknown. To identify the BCL11A-regulated transcriptome, we performed RNA-seq analysis of MDA-MD-231 TNBC cells transiently transfected with BCL11A-targeted siRNA or a non-silencing control. Gene set enrichment analysis of the differentially expressed genes revealed enrichment of genes involved in extracellular matrix and matrisome genes. Notably, BCL11A silencing reduced expression of several matrix-metalloproteases, with MMP1 being the most significantly differentially expressed gene. Thus, we hypothesize that BCL11A promotes TNBC invasiveness and metastasis through regulation of MMP expression. We confirmed downregulation of MMP1 and MMP9 mRNA and protein in response to BCL11A silencing by both qRT-PCR and western blot in independent MDA-MB-231 cells, as well as in the HCC1143 TNBC cell line. MMP activity was also reduced when assessed by fluorokine assay and gelatin zymography, for MMP1 and MMP9, respectively. Furthermore, both BCL11A and MMP expression is increased in brain-seeking clones of MDA-MB-231 cells and MMP1 and 9 expression is also reduced with BCL11A silencing. Together, these data suggest that BCL11A regulates TNBC invasion by reducing the expression of MMP1 and MMP9. Current studies are directly assessing the functional impact of MMP regulation by BCL11A on invasion and metastatic progression of TNBC. Citation Format: Natasha N. Ingles, Darcie Seachrist, Ruth Keri. BCL11A regulation of extracellular matrix genes may be necessary for invasion of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 172.
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