MEF2D Functions as a Tumor Suppressor in Breast Cancer.

The myocyte enhancer factor 2 (MEF2) gene family play fundamental roles in the genetic programs that control cell differentiation, morphogenesis, proliferation, and survival in a wide range of cell types. More recently, these genes have also been implicated as drivers of carcinogenesis, by acting as oncogenes or tumor suppressors depending on the biological context. Nonetheless, the molecular programs they regulate and their roles in tumor development and progression remain incompletely understood. The present study evaluated whether the MEF2D transcription factor functions as a tumor suppressor in breast cancer. The knockout of the MEF2D gene in mouse mammary epithelial cells resulted in phenotypic changes characteristic of neoplastic transformation. These changes included enhanced cell proliferation, a loss of contact inhibition, and anchorage-independent growth in soft agar, as well as the capacity for tumor development in mice. Mechanistically, the knockout of MEF2D induced the epithelial-to-mesenchymal transition (EMT) and activated several oncogenic signaling pathways, including AKT, ERK, and Hippo-YAP. Correspondingly, a reduced expression of MEF2D was observed in human triple-negative breast cancer cell lines, and a low MEF2D expression in tissue samples was found to be correlated with a worse overall survival and relapse-free survival in breast cancer patients. MEF2D may, thus, be a putative tumor suppressor, acting through selective gene regulatory programs that have clinical and therapeutic significance.

Genome-Wide Characterization of TAZ Binding Sites in Mammary Epithelial Cells.

The transcriptional co-activator with PDZ binding motif (TAZ) is a key effector of the Hippo signaling pathway. We and others previously reported that high expression levels of TAZ are positively associated with decreased survival rates and shorter times to relapse in basal-like breast cancer (BLBC) patients. The oncogenic activity of TAZ involves the regulation of diverse signal transduction pathways that direct processes such as cell proliferation, migration, and resistance to apoptosis, albeit through poorly characterized gene expression programs. Here, using a tet-inducible system in mammary epithelial MCF10A cells, we have characterized the TAZ-regulated transcription program using RNA sequencing in a temporal and spatial manner. We further identified global TAZ binding sites at different TAZ activation time points by chromatin immunoprecipitation (ChIP) sequencing analysis. We found that the vast majority of TAZ was rapidly localized in enhancer regions at the early TAZ activation time point and then gradually spread to promoter regions. TAZ bound to enhancer regions following a switch in potential TEAD and FOSL2 transcription factor motifs. Furthermore, the ATAC sequencing analysis indicated that TAZ activation led to chromatin structural alterations. Together, our results have revealed the landscape of genome-wide TAZ binding sites and may lead to improvements in the current understanding of how TAZ regulates the gene expression program that contributes to the development of breast cancer.

Comparative Proteomics Analysis of Exosomes Identifies Key Pathways and Protein Markers Related to Breast Cancer Metastasis.

Proteomics analysis of circulating exosomes derived from cancer cells represents a promising approach to the elucidation of cell-cell communication and the discovery of putative biomarker candidates for cancer diagnosis and treatment. Nonetheless, the proteome of exosomes derived from cell lines with different metastatic capabilities still warrants further investigation. Here, we present a comprehensive quantitative proteomics investigation of exosomes isolated from immortalized mammary epithelial cells and matched tumor lines with different metastatic potentials in an attempt to discover exosome markers specific to breast cancer (BC) metastasis. A total of 2135 unique proteins were quantified with a high confidence level from 20 isolated exosome samples, including 94 of the TOP 100 exosome markers archived by ExoCarta. Moreover, 348 altered proteins were observed, among which several metastasis-specific markers, including cathepsin W (CATW), magnesium transporter MRS2 (MRS2), syntenin-2 (SDCB2), reticulon-4 (RTN), and UV excision repair protein RAD23 homolog (RAD23B), were also identified. Notably, the abundance of these metastasis-specific markers corresponds well with the overall survival of BC patients in clinical settings. Together, these data provide a valuable dataset for BC exosome proteomics investigation and prominently facilitate the elucidation of the molecular mechanisms underlying primary tumor development and progression.

Loss-of-function of the hippo transducer TAZ reduces mammary tumor growth through a myeloid-derived suppressor cell-dependent mechanism.

TAZ, one of the key effectors in the Hippo pathway, is often dysregulated in breast cancer, leading to cancer stemness, survival, and metastasis. However, the mechanistic bases of these tumor outcomes are incompletely understood and even less is known about the potential role played by the non-malignant cellular constituents of the tumor microenvironment (TME). Here, we revealed an inverse correlation between TAZ expression and survival in triple-negative breast cancer (TNBC), but not other subtypes of breast cancer. We found that TAZ knockdown in two murine TNBC tumor cell line models significantly inhibited tumor growth and metastasis in immune competent but not immune deficient hosts. RNA-seq analyses identified substantial alterations in immune components in TAZ knockdown tumors. Using mass cytometry analysis, we found that TAZ-deficiency altered the immune landscape of the TME leading to significant reductions in immune suppressive populations, namely myeloid-derived suppressor cells (MDSCs) and macrophages accompanied by elevated CD8+ T cell/myeloid cell ratios. Mechanistic studies demonstrated that TAZ-mediated tumor growth was MDSC-dependent in that MDSC depletion led to reduced tumor growth in control, but not TAZ-knockdown tumor cells. Altogether, we identified a novel non-cancer cell-autonomous mechanism by which tumor-intrinsic TAZ expression aids tumor progression. Thus, our findings advance an understanding of the crosstalk between tumor-derived TAZ expression and the immune contexture within the TME, which may lead to new therapeutic interventions for TNBC or other TAZ-driven cancers.

Identification of TAZ-Dependent Breast Cancer Vulnerabilities Using a Chemical Genomics Screening Approach.

Breast cancer stem cells (BCSCs) represent a subpopulation of tumor cells that can self-renew and generate tumor heterogeneity. Targeting BCSCs may ameliorate therapy resistance, tumor growth, and metastatic progression. However, the origin and molecular mechanisms underlying their cellular properties are poorly understood. The transcriptional coactivator with PDZ-binding motif (TAZ) promotes mammary stem/progenitor cell (MaSC) expansion and maintenance but also confers stem-like traits to differentiated tumor cells. Here, we describe the rapid generation of experimentally induced BCSCs by TAZ-mediated reprogramming of human mammary epithelial cells, hence allowing for the direct analysis of BCSC phenotypes. Specifically, we establish genetically well-defined TAZ-dependent (TAZDEP) and -independent (TAZIND) cell lines with cancer stem cell (CSC) traits, such as self-renewal, variable resistance to chemotherapeutic agents, and tumor seeding potential. TAZDEP cells were associated with the epithelial to mesenchymal transition, embryonic, and MaSC signature genes. In contrast, TAZIND cells were characterized by a neuroendocrine transdifferentiation transcriptional program associated with Polycomb repressive complex 2 (PRC2). Mechanistically, we identify Cyclin D1 (CCND1) as a critical downstream effector for TAZ-driven tumorigenesis. Overall, our results reveal a critical TAZ-CCND1-CDK4/CDK6 signaling axis, suggesting novel therapeutic approaches to eliminate both BCSCs and therapy-resistant cancer cells.

USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer.

The Hippo signaling pathway is an evolutionarily conserved pathway that was initially discovered in Drosophila melanogaster and was later found to have mammalian orthologues. The key effector proteins in this pathway, YAP/TAZ, are often dysregulated in cancer, leading to a high degree of cell proliferation, migration, metastasis and cancer stem cell populations. Due to these malignant phenotypes it is important to understand the regulation of YAP/TAZ at the protein level. Using an siRNA library screen of deubiquitinating enzymes (DUBs), we identified ubiquitin specific peptidase 1 (USP1) as a novel TAZ (WWTR1) regulator. We demonstrated that USP1 interacts with TAZ and increases TAZ protein stability. Conversely, loss of function of USP1 reduces TAZ protein levels through increased poly-ubiquitination, causing a decrease in cell proliferation and migration of breast cancer cells. Moreover, we showed a strong positive correlation between USP1 and TAZ in breast cancer patients. Our findings facilitate the attainment of better understanding of the crosstalk between these pathways and may lead to potential therapeutic interventions for breast cancer patients.

Regulation of the Hippo signaling pathway by deubiquitinating enzymes in cancer.

Regulation of the Hippo signaling pathway is essential for normal organ growth and tissue homeostasis. The proteins that act to regulate this pathway are important for ensuring proper function and cellular location. Deubiquitinases (DUBs) are a family of proteases that act upon many proteins. While ubiquitinases add ubiquitin and target proteins for degradation, DUBs act by removing ubiquitin (Ub) moieties. Changes in ubiquitin chain topology results in the stabilization of proteins, membrane trafficking, and the alteration of cellular localization. While the roles of these proteins have been well established in a cancer setting, their convergence in cancer is still under investigation. In this review, we discuss the roles that DUBs play in the regulation of the Hippo signaling pathway for homeostasis and disease.

Targeting TAZ-Driven Human Breast Cancer by Inhibiting a SKP2-p27 Signaling Axis.

Deregulated expression of the transcriptional coactivator with PDZ-binding motif (WWTR1/TAZ) is a common feature of basal-like breast cancer (BLBC). Yet, how oncogenic TAZ regulates cell-cycle progression and proliferation in breast cancer remains poorly understood, and whether TAZ is required for tumor maintenance has not been established. Here, using an integrative oncogenomic approach, TAZ-dependent cellular programs essential for tumor growth and progression were identified. Significantly, TAZ-driven tumor cells required sustained TAZ expression, given that its withdrawal impaired both genesis and maintenance of solid tumors. Moreover, temporal inhibition of TAZ diminished the metastatic burden in established macroscopic pulmonary metastases. Mechanistic investigation revealed that TAZ controls distinct gene profiles that determine cancer cell fate through cell-cycle networks, including a specific, causal role for S-phase kinase-associated protein 2 (SKP2) in mediating the neoplastic state. Together, this study elucidates the molecular events that underpin the role of TAZ in BLBC and link to SKP2, a convergent communication node for multiple cancer signaling pathways, as a key downstream effector molecule. IMPLICATIONS: Understanding the molecular role of TAZ and its link to SKP2, a signaling convergent point and key regulator in BLBC, represents an important step toward the identification of novel therapeutic targets for TAZ-dependent breast cancer.

NTRK1 is a positive regulator of YAP oncogenic function.

Multiple cancer signalling networks take part in regulatory crosstalks with the Hippo tumour suppressor pathway through the transcriptional cofactor Yes-associated protein (YAP). Nevertheless, how YAP is controlled by pathway crosstalks in tumourigenesis remains poorly understood. Here, we performed a targeted kinase inhibitor screen in human cancer cells to identify novel Hippo pathway regulators. Notably, we identified the nerve growth factor (NGF) receptor tyrosine kinase (NTRK1), a molecule not previously associated with Hippo signalling. NTRK1 inhibition decreased YAP-driven transcription, cancer cell proliferation and migration. Furthermore, using a complementary functional genomics approach and mouse xenograft models, we show that NTRK1 regulates YAP oncogenic activity in vivo. Mechanistically, NTRK1 inhibition was found to induce large suppressor kinase 1 (LATS1) phosphorylation and to control YAP subcellular localization. Taken together, these results provide compelling evidence of crosstalks between the NGF-NTRK1 and Hippo cancer pathways.

Loss of KIBRA function activates EGFR signaling by inducing AREG.

The Hippo signaling pathway is a central regulator of organ size, tissue homeostasis, and tumorigenesis. KIBRA is a member of the WW domain-containing protein family and has recently been reported to be an upstream protein in the Hippo signaling pathway. However, the clinical significance of KIBRA deregulation and the underlying mechanisms by which KIBRA regulates breast cancer (BC) initiation and progression remain poorly understood. Here, we report that KIBRA knockdown in mammary epithelial cells induced epithelial-to-mesenchymal transition (EMT) and increased cell migration and tumorigenic potential. Mechanistically, we observed that inhibiting KIBRA induced growth factor-independent cell proliferation in 2D and 3D culture due to the secretion of amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand. Also, we show that AREG activation in KIBRA-knockdown cells depended on the transcriptional coactivator YAP1. Significantly, decreased expression of KIBRA is correlated with recurrence and reduced BC patient survival. In summary, this study elucidates the molecular events that underpin the role of KIBRA in BC. As a result, our work provides biological insight into the role of KIBRA as a critical regulator of YAP1-mediated oncogenic growth, and may have clinical potential for facilitating patient stratification and identifying novel therapeutic approaches for BC patients.

The Hippo Signaling Transducer TAZ Regulates Mammary Gland Morphogenesis and Carcinogen-induced Mammary Tumorigenesis.

Hippo signaling pathway is an evolutionarily conserved pathway that controls organ size by regulating cell proliferation, apoptosis and stem cell self-renewal. TAZ (transcriptional coactivator with the PDZ-binding motif) is a key downstream effector of the mammalian Hippo pathway. Here, using a transgenic mouse model with mammary-gland-specific expression of constitutively active TAZ, we found that TAZ induction in mammary epithelial cells was associated with an increase in mammary glandular size, which probably resulted from adipocyte hypertrophy. Consistent with its known oncogenic potential, we observed tumor formation in TAZ transgenic mice after administration of the carcinogen 7,12-dimethylbenzanthracene (DMBA) and demonstrated that tumorigenesis was reliant on the presence of TAZ. Our findings establish a previously unknown roles of TAZ in regulating both mammary gland morphogenesis as well as carcinogen-induced mammary tumor formation.

VGLL4 Selectively Represses YAP-Dependent Gene Induction and Tumorigenic Phenotypes in Breast Cancer.

Members of the mammalian Vestigial-like (VGLL) family of transcriptional cofactors activate genes in response to a wide variety of environmental cues. Recently, VGLL proteins have been proposed to regulate key signaling networks involved in cancer development and progression. However, the biological and clinical significance of VGLL dysregulation in human breast cancer pathogenesis remains unknown. Here, we report that diminished VGLL4 expression, but not VGLL1-3, correlated with both shorter relapse-free survival and shorter disease-specific survival of cancer patients with different molecular subtypes of breast cancer. Additionally, we further demonstrate that overexpression of VGLL4 reduces breast cancer cell proliferation, migration, intravasation/extravasation potential, favors cell death, and suppresses tumor growth in vivo. Mechanistically, VGLL4 negatively regulates the TEAD1-YAP1 transcriptional complex and exerts its growth inhibitory control through its evolutionary conserved TDU2 domain at its C-terminus. The results suggest that VGLL4 is a candidate tumor suppressor gene which acts by selectively antagonizing YAP-dependent tumor growth. VGLL4 may be a promising therapeutic target in breast cancer.

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation.

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP1, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. The Hippo tumor suppressor pathway has been suggested to inhibit the YAP1 function through serine phosphorylation-induced cytoplasmic retention and degradation. Here we report that the tyrosine188 (Y188) site of YAP1 isoform with 2 WW domains (known as YAP1-2) plays an important role in YAP1-induced cellular transformation. IP-Mass Spectrometry analysis of YAP1 identified the phosphorylation of Y188 but not other tyrosine residues. In contrast to the aberrant 3D acinus formation observed in YAP1-WT transduced cells, overexpression of YAP1-Y188F (non-phosphorylated mimic) displayed normal 3D structures. In addition, knockdown of the endogenous YAP1 in MDA-MB231 breast cancer cells inhibited cell proliferation and migration, which were then successfully rescued by the exogenous YAP1-WT and YAP1-Y188E but not Y188F. Mechanistically, we also demonstrated that YAP1-Y188F had a higher affinity to the upstream negative regulator PTPN14 and was extensively localized in the cytoplasm. Since the Y188 is located in the conserved aromatic core of the WW domain of YAP1, our finding has a wide implication for WW domain signaling in general, where Y phosphorylation may act as a common positive regulator of the complex formation via WW domains. In summary, our results indicate that tyrosine 188 plays an important role in the YAP1-induced cellular transformation and its phosphorylation may intriguingly serve as a positive indicator of YAP1 activation.

Characterization of TAZ domains important for the induction of breast cancer stem cell properties and tumorigenesis.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth and cell fate during development and regeneration. Conversely, deregulation of the Hippo pathway has been reported in several malignancies. Here, we used integrative functional genomics approaches to identify TAZ, a transcription co-activator and key downstream effector of the Hippo pathway, as an essential driver for the propagation of TNBC malignant phenotype. We further showed in non-transformed human mammary basal epithelial cells that expression of constitutively active TAZ confers cancer stem cell (CSC) traits that are dependent on the TAZ and TEAD interacting domains. In addition, to gain a better understanding of how TAZ functions, we performed genetic-function analysis of TAZ. Significantly, we identified that both the WW and transcriptional activation domains of TAZ are critical for the induced CSC properties as well as tumorigenic potential as manifested in vitro and in human breast cancer xenograft in vivo. Collectively, our data suggest that pharmacological inhibition of TAZ activity may provide a novel means of targeting and eliminating breast CSCs.

Molecular profiling and computational network analysis of TAZ-mediated mammary tumorigenesis identifies actionable therapeutic targets.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer (BC) cases and contributes disproportionately to BC mortality. TAZ, a key transducer of the Hippo pathway, has recently been demonstrated to confer breast cancer stem cell (CSC) traits. However, TAZ target genes and the underlying transcriptional regulatory pathways responsible for the CSC phenomenon remain unknown. Here, we demonstrate that the oncogenic activity of TAZ is essential for propagation of the malignant phenotype. We further show that constitutively active TAZ tumor-derived cells exhibit unique tumor-initiating properties, including increased self-renewal and metastatic seeding potential, acquired chemotherapy resistance and the ability to efficiently regenerate tumor formation in vivo. Combined digital RNA expression analysis and computational network approaches identify several signaling pathways that distinguish breast cancer tumor-initiating cells (T-ICs) from bulk tumor cells. We demonstrate the utility of this approach by repositioning the small molecule tyrosine kinase inhibitor, Dasatinib, which selectively targets T-ICs and inhibits TNBC growth in vivo.

6p22.3 amplification as a biomarker and potential therapeutic target of advanced stage bladder cancer.

Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type.