The transcriptional repressor BCL11A promotes breast cancer metastasis.

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. Here, 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 promotes cancer cell invasion by suppressing the expression of muscleblind-like splicing regulator 1 (MBNL1), a splicing regulator that suppresses metastasis. This ultimately increases the levels of an alternatively spliced isoform of integrin-α6 (ITGA6), which 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.

Targeting BCL-xL improves the efficacy of bromodomain and extra-terminal protein inhibitors in triple-negative breast cancer by eliciting the death of senescent cells.

Inhibitors of bromodomain and extra-terminal proteins (BETi) suppress oncogenic gene expression and have been shown to be efficacious in many in vitro and murine models of cancer, including triple-negative breast cancer (TNBC), a highly aggressive disease. However, in most cancer models, responses to BETi can be highly variable. We previously reported that TNBC cells either undergo senescence or apoptosis in response to BETi, but the specific mechanisms dictating these two cell fates remain unknown. Using six human TNBC cell lines, we show that the terminal response of TNBC cells to BETi is dictated by the intrinsic expression levels of the anti-apoptotic protein B-cell lymphoma-extra large (BCL-xL). BCL-xL levels were higher in cell lines that senesce in response to BETi compared with lines that primarily die in response to these drugs. Moreover, BCL-xL expression was further reduced in cells that undergo BETi-mediated apoptosis. Forced BCL-xL overexpression in cells that normally undergo apoptosis following BETi treatment shifted them to senescence without affecting the reported mechanism of action of BETi in TNBC, that is, mitotic catastrophe. Most importantly, pharmacological or genetic inhibition of BCL-xL induced apoptosis in response to BETi, and inhibiting BCL-xL, even after BETi-induced senescence had already occurred, still induced cell death. These results indicate that BCL-xL provides a senescent cell death-inducing or senolytic target that may be exploited to improve therapeutic outcomes of TNBC in response to BETi. They also suggest that the basal levels of BCL-xL should be predictive of tumor responses to BETi in current clinical trials.

KLF4 defines the efficacy of the epidermal growth factor receptor inhibitor, erlotinib, in triple-negative breast cancer cells by repressing the EGFR gene.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by high rates of recurrence and poor overall survival. This is due, in part, to a deficiency of targeted therapies, making it essential to identify therapeutically targetable driver pathways of this disease. While epidermal growth factor receptor (EGFR) is expressed in 60% of TNBCs and drives disease progression, attempts to inhibit EGFR in unselected TNBC patients have had a marginal impact on outcomes. Hence, we sought to identify the mechanisms that dictate EGFR expression and inhibitor response to provide a path for improving the utility of these drugs. In this regard, the majority of TNBCs express low levels of the transcription factor, Krüppel-like factor 4 (KLF4), while a small subset is associated with high expression. KLF4 and EGFR have also been reported to have opposing actions in TNBC. Thus, we tested whether KLF4 controls the expression of EGFR and cellular response to its pharmacological inhibition. METHODS: KLF4 was transiently overexpressed in MDA-MB-231 and MDA-MB-468 cells or silenced in MCF10A cells. Migration and invasion were assessed using modified Boyden chamber assays, and proliferation was measured by EdU incorporation. Candidate downstream targets of KLF4, including EGFR, were identified using reverse phase protein arrays of MDA-MB-231 cells following enforced KLF4 expression. The ability of KLF4 to suppress EGFR gene and protein expression and downstream signaling was assessed by RT-PCR and western blot, respectively. ChIP-PCR confirmed KLF4 binding to the EGFR promoter. Response to erlotinib in the context of KLF4 overexpression or silencing was assessed using cell number and dose-response curves. RESULTS: We report that KLF4 is a major determinant of EGFR expression and activity in TNBC cells. KLF4 represses transcription of the EGFR gene, leading to reduced levels of total EGFR, its activated/phosphorylated form (pEGFR), and its downstream signaling intermediates. Moreover, KLF4 suppression of EGFR is a necessary intermediary step for KLF4 to inhibit aggressive TNBC phenotypes. Most importantly, KLF4 dictates the sensitivity of TNBC cells to erlotinib, an FDA-approved inhibitor of EGFR. CONCLUSIONS: KLF4 is a major regulator of the efficacy of EGFR inhibitors in TNBC cells that may underlie the variable effectiveness of such drugs in patients.

Follistatin is a metastasis suppressor in a mouse model of HER2-positive breast cancer.

BACKGROUND: Follistatin (FST) is an intrinsic inhibitor of activin, a member of the transforming growth factor-ß superfamily of ligands. The prognostic value of FST and its family members, the follistatin-like (FSTL) proteins, have been studied in various cancers. However, these studies, as well as limited functional analyses of the FSTL proteins, have yielded conflicting results on the role of these proteins in disease progression. Furthermore, very few have been focused on FST itself. We assessed whether FST may be a suppressor of tumorigenesis and/or metastatic progression in breast cancer. METHODS: Using publicly available gene expression data, we examined the expression patterns of FST and INHBA, a subunit of activin, in normal and cancerous breast tissue and the prognostic value of FST in breast cancer metastases, recurrence-free survival, and overall survival. The functional effects of activin and FST on in vitro proliferation, migration, and invasion of breast cancer cells were also examined. FST overexpression in an autochthonous mouse model of breast cancer was then used to assess the in vivo impact of FST on metastatic progression. RESULTS: Examination of multiple breast cancer datasets revealed that FST expression is reduced in breast cancers compared with normal tissue and that low FST expression predicts increased metastasis and reduced overall survival. FST expression was also reduced in a mouse model of HER2/Neu-induced metastatic breast cancer. We found that FST blocks activin-induced breast epithelial cell migration in vitro, suggesting that its loss may promote breast cancer aggressiveness. To directly determine if FST restoration could inhibit metastatic progression, we transgenically expressed FST in the HER2/Neu model. Although FST had no impact on tumor initiation or growth, it completely blocked the formation of lung metastases. CONCLUSIONS: These data indicate that FST is a bona fide metastasis suppressor in this mouse model and support future efforts to develop an FST mimetic to suppress metastatic progression.

TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis.

Breast cancer subtypes and their phenotypes parallel different stages of the mammary epithelial cell developmental hierarchy. Discovering mechanisms that control lineage identity could provide novel avenues for mitigating disease progression. Here we report that the transcriptional corepressor TLE3 is a guardian of luminal cell fate in breast cancer and operates independently of the estrogen receptor. In luminal breast cancer, TLE3 actively repressed the gene-expression signature associated with highly aggressive basal-like breast cancers (BLBC). Moreover, maintenance of the luminal lineage depended on the appropriate localization of TLE3 to its transcriptional targets, a process mediated by interactions with FOXA1. By repressing genes that drive BLBC phenotypes, including SOX9 and TGFß2, TLE3 prevented the acquisition of a hybrid epithelial-mesenchymal state and reduced metastatic capacity and aggressive cellular behaviors. These results establish TLE3 as an essential transcriptional repressor that sustains the more differentiated and less metastatic nature of luminal breast cancers. Approaches to induce TLE3 expression could promote the acquisition of less aggressive, more treatable disease states to extend patient survival. SIGNIFICANCE: Transcriptional corepressor TLE3 actively suppresses SOX9 and TGFß transcriptional programs to sustain the luminal lineage identity of breast cancer cells and to inhibit metastatic progression.

Focal Adhesion Kinase Provides a Collateral Vulnerability That Can Be Leveraged to Improve mTORC1 Inhibitor Efficacy.

The PI3K/AKT/mTORC1 pathway is a major therapeutic target for many cancers, particularly breast cancer. Everolimus is an mTORC1 inhibitor used in metastatic estrogen receptor-positive (ER+) and epidermal growth factor receptor 2-negative (HER2-) breast cancer. However, mTORC1 inhibitors have limited efficacy in other breast cancer subtypes. We sought to discover collateral sensitivities to mTORC1 inhibition that could be exploited to improve therapeutic response. Using a mouse model of breast cancer that is intrinsically resistant to mTORC1 inhibition, we found that rapamycin alters the expression of numerous extracellular matrix genes, suggesting a potential role for integrins/FAK in controlling mTORC1-inhibitor efficacy. FAK activation was also inversely correlated with rapamycin response in breast cancer cell lines. Supporting its potential utility in patients, FAK activation was observed in >50% of human breast cancers. While blocking FAK in mouse models of breast cancer that are highly responsive to rapamycin had no impact on tumor growth, FAK inhibition sensitized rapamycin-resistant tumors to mTORC1 inhibition. These data reveal an innate dependency on FAK when mTORC1 signaling is lost in tumors that are resistant to mTORC1 inhibitors. They also suggest a precision medicine approach to improving mTORC1 inhibitor efficacy in resistant cancers by suppressing FAK signaling.

LIN9 and NEK2 Are Core Regulators of Mitotic Fidelity That Can Be Therapeutically Targeted to Overcome Taxane Resistance.

A significant therapeutic challenge for patients with cancer is resistance to chemotherapies such as taxanes. Overexpression of LIN9, a transcriptional regulator of cell-cycle progression, occurs in 65% of patients with triple-negative breast cancer (TNBC), a disease commonly treated with these drugs. Here, we report that LIN9 is further elevated with acquisition of taxane resistance. Inhibiting LIN9 genetically or by suppressing its expression with a global BET inhibitor restored taxane sensitivity by inducing mitotic progression errors and apoptosis. While sustained LIN9 is necessary to maintain taxane resistance, there are no inhibitors that directly repress its function. Hence, we sought to discover a druggable downstream transcriptional target of LIN9. Using a computational approach, we identified NIMA-related kinase 2 (NEK2), a regulator of centrosome separation that is also elevated in taxane-resistant cells. High expression of NEK2 was predictive of low survival rates in patients who had residual disease following treatment with taxanes plus an anthracycline, suggesting a role for this kinase in modulating taxane sensitivity. Like LIN9, genetic or pharmacologic blockade of NEK2 activity in the presence of paclitaxel synergistically induced mitotic abnormalities in nearly 100% of cells and completely restored sensitivity to paclitaxel, in vitro. In addition, suppressing NEK2 activity with two distinct small molecules potentiated taxane response in multiple in vivo models of TNBC, including a patient-derived xenograft, without inducing toxicity. These data demonstrate that the LIN9/NEK2 pathway is a therapeutically targetable mediator of taxane resistance that can be leveraged to improve response to this core chemotherapy. SIGNIFICANCE: Resistance to chemotherapy is a major hurdle for treating patients with cancer. Combining NEK2 inhibitors with taxanes may be a viable approach for improving patient outcomes by enhancing mitotic defects induced by taxanes alone.

Mitotic Vulnerability in Triple-Negative Breast Cancer Associated with LIN9 Is Targetable with BET Inhibitors.

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR.