Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways.

The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1, a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.

Coordinated activation of c-Src and FOXM1 drives tumor cell proliferation and breast cancer progression.

Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we have shown that deletion of c-Src in a genetically engineered model mimicking the luminal B molecular subtype of breast cancer abrogated the activity of forkhead box M1 (FOXM1), a master transcriptional regulator of the cell cycle. We determined that c-Src phosphorylated FOXM1 on 2 tyrosine residues to stimulate its nuclear localization and target gene expression. These included key regulators of G2/M cell-cycle progression as well as c-Src itself, forming a positive feedback loop that drove proliferation in genetically engineered and patient-derived models of luminal B-like breast cancer. Using genetic approaches and small molecules that destabilize the FOXM1 protein, we found that targeting this mechanism induced G2/M cell-cycle arrest and apoptosis, blocked tumor progression, and impaired metastasis. We identified a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the luminal B subtype, which responds poorly to currently approved therapies. These findings revealed a regulatory network centered on c-Src and FOXM1 that is a targetable vulnerability in aggressive luminal breast cancers.

Integrated multi-omics analysis of adverse cardiac remodeling and metabolic inflexibility upon ErbB2 and ERRα deficiency.

Functional oncogenic links between ErbB2 and ERRα in HER2+ breast cancer patients support a therapeutic benefit of co-targeted therapies. However, ErbB2 and ERRα also play key roles in heart physiology, and this approach could pose a potential liability to cardiovascular health. Herein, using integrated phosphoproteomic, transcriptomic and metabolic profiling, we uncovered molecular mechanisms associated with the adverse remodeling of cardiac functions in mice with combined attenuation of ErbB2 and ERRα activity. Genetic disruption of both effectors results in profound effects on cardiomyocyte architecture, inflammatory response and metabolism, the latter leading to a decrease in fatty acyl-carnitine species further increasing the reliance on glucose as a metabolic fuel, a hallmark of failing hearts. Furthermore, integrated omics signatures of ERRα loss-of-function and doxorubicin treatment exhibit common features of chemotherapeutic cardiotoxicity. These findings thus reveal potential cardiovascular risks in discrete combination therapies in the treatment of breast and other cancers.

An ErbB2 splice variant lacking exon 16 drives lung carcinoma.

Lung cancer causes more deaths annually than any other malignancy. A subset of non-small cell lung cancer (NSCLC) is driven by amplification and overexpression or activating mutation of the receptor tyrosine kinase (RTK) ERBB2 In some contexts, notably breast cancer, alternative splicing of ERBB2 causes skipping of exon 16, leading to the expression of an oncogenic ERBB2 isoform (ERBB2ΔEx16) that forms constitutively active homodimers. However, the broader implications of ERBB2 alternative splicing in human cancers have not been explored. Here, we have used genomic and transcriptomic analysis to identify elevated ERBB2ΔEx16 expression in a subset of NSCLC cases, as well as splicing site mutations facilitating exon 16 skipping and deletions of exon 16 in a subset of these lung tumors and in a number of other carcinomas. Supporting the potential of ERBB2ΔEx16 as a lung cancer driver, its expression transformed immortalized lung epithelial cells while a transgenic model featuring inducible ERBB2ΔEx16 specifically in the lung epithelium rapidly developed lung adenocarcinomas following transgene induction. Collectively, these observations indicate that ERBB2ΔEx16 is a lung cancer oncogene with potential clinical importance for a proportion of patients.

Reduction of Global H3K27me3 Enhances HER2/ErbB2 Targeted Therapy.

Monoclonal antibodies (mAbs) targeting the oncogenic receptor tyrosine kinase ERBB2/HER2, such as Trastuzumab, are the standard of care therapy for breast cancers driven by ERBB2 overexpression and activation. However, a substantial proportion of patients exhibit de novo resistance. Here, by comparing matched Trastuzumab-naive and post-treatment patient samples from a neoadjuvant trial, we link resistance with elevation of H3K27me3, a repressive histone modification catalyzed by polycomb repressor complex 2 (PRC2). In ErbB2+ breast cancer models, PRC2 silences endogenous retroviruses (ERVs) to suppress anti-tumor type-I interferon (IFN) responses. In patients, elevated H3K27me3 in tumor cells following Trastuzumab treatment correlates with suppression of interferon-driven viral defense gene expression signatures and poor response. Using an immunocompetent model, we provide evidence that EZH2 inhibitors promote interferon-driven immune responses that enhance the efficacy of anti-ErbB2 mAbs, suggesting the potential clinical benefit of epigenomic reprogramming by H3K27me3 depletion in Trastuzumab-resistant disease.

An ErbB2/c-Src axis links bioenergetics with PRC2 translation to drive epigenetic reprogramming and mammary tumorigenesis.

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis.

Targeting EZH2 reactivates a breast cancer subtype-specific anti-metastatic transcriptional program.

Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to pathogenesis. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. Here we report that genetic or pharmacological targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behaviour in both a mouse model of breast cancer and patient-derived xenografts reflective of the Luminal B subtype. We further define a subtype-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, anti-invasive transcriptional program. We demonstrate that higher FOXC1 is predictive of favourable outcome specifically in Luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in Luminal B breast cancer, where options for targeted therapy are limited.

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.

Integration of Distinct ShcA Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance.

The commonality between most phospho-tyrosine signaling networks is their shared use of adaptor proteins to transduce mitogenic signals. ShcA (SHC1) is one such adaptor protein that employs two phospho-tyrosine binding domains (PTB and SH2) and key phospho-tyrosine residues to promote mammary tumorigenesis. Receptor tyrosine kinases (RTK), such as ErbB2, bind the ShcA PTB domain to promote breast tumorigenesis by engaging Grb2 downstream of the ShcA tyrosine phosphorylation sites to activate AKT/mTOR signaling. However, breast tumors also rely on the ShcA PTB domain to bind numerous negative regulators that limit activation of secondary mitogenic signaling networks. This study examines the role of PTB-independent ShcA pools in controlling breast tumor growth and resistance to tyrosine kinase inhibitors. We demonstrate that PTB-independent ShcA complexes predominately rely on the ShcA SH2 domain to activate multiple Src family kinases (SFK), including Src and Fyn, in ErbB2-positive breast cancers. Using genetic and pharmacologic approaches, we show that PTB-independent ShcA complexes augment mammary tumorigenesis by increasing the activity of the Src and Fyn tyrosine kinases in an SH2-dependent manner. This bifurcation of signaling complexes from distinct ShcA pools transduces non-redundant signals that integrate the AKT/mTOR and SFK pathways to cooperatively increase breast tumor growth and resistance to tyrosine kinase inhibitors, including lapatinib and PP2. This study mechanistically dissects how the interplay between diverse intracellular ShcA complexes impacts the tyrosine kinome to affect breast tumorigenesis.Implications: The ShcA adaptor, within distinct signaling complexes, impacts tyrosine kinase signaling, breast tumor growth, and resistance to tyrosine kinase inhibitors. Mol Cancer Res; 16(5); 894-908. ©2018 AACR.

LPP is a Src substrate required for invadopodia formation and efficient breast cancer lung metastasis.

We have previously shown that lipoma preferred partner (LPP) mediates TGFß-induced breast cancer cell migration and invasion. Herein, we demonstrate that diminished LPP expression reduces circulating tumour cell numbers, impairs cancer cell extravasation and diminishes lung metastasis. LPP localizes to invadopodia, along with Tks5/actin, at sites of matrix degradation and at the tips of extravasating breast cancer cells as revealed by intravital imaging of the chick chorioallantoic membrane (CAM). Invadopodia formation, breast cancer cell extravasation and metastasis require an intact LPP LIM domain and the ability of LPP to interact with α-actinin. Finally, we show that Src-mediated LPP phosphorylation at specific tyrosine residues (Y245/301/302) is critical for invadopodia formation, breast cancer cell invasion and metastasis. Together, these data define a previously unknown function for LPP in the formation of invadopodia and reveal a requirement for LPP in mediating the metastatic ability of breast cancer cells.

ERRα mediates metabolic adaptations driving lapatinib resistance in breast cancer.

Despite the initial benefits of treating HER2-amplified breast cancer patients with the tyrosine kinase inhibitor lapatinib, resistance inevitably develops. Here we report that lapatinib induces the degradation of the nuclear receptor ERRα, a master regulator of cellular metabolism, and that the expression of ERRα is restored in lapatinib-resistant breast cancer cells through reactivation of mTOR signalling. Re-expression of ERRα in resistant cells triggers metabolic adaptations favouring mitochondrial energy metabolism through increased glutamine metabolism, as well as ROS detoxification required for cell survival under therapeutic stress conditions. An ERRα inverse agonist counteracts these metabolic adaptations and overcomes lapatinib resistance in a HER2-induced mammary tumour mouse model. This work reveals a molecular mechanism by which ERRα-induced metabolic reprogramming promotes survival of lapatinib-resistant cancer cells and demonstrates the potential of ERRα inhibition as an effective adjuvant therapy in poor outcome HER2-positive breast cancer.

Inducible and coupled expression of the polyomavirus middle T antigen and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in mammary tumour progression.

INTRODUCTION: Effective in vivo models of breast cancer are crucial for studying the development and progression of the disease in humans. We sought to engineer a novel mouse model of polyomavirus middle T antigen (PyV mT)-mediated mammary tumourigenesis in which inducible expression of this well-characterized viral oncoprotein is coupled to Cre recombinase (TetO-PyV mT-IRES-Cre recombinase or MIC). METHODS: MIC mice were crossed to the mouse mammary tumour virus (MMTV)-reverse tetracycline transactivator (rtTA) strain to generate cohorts of virgin females carrying one or both transgenes. Experimental (rtTA/MIC) and control (rtTA or MIC) animals were administered 2 mg/mL doxycycline beginning as early as eight weeks of age and monitored for mammary tumour formation, in parallel with un-induced controls of the same genotypes. RESULTS: Of the rtTA/MIC virgin females studied, 90% developed mammary tumour with complete penetrance to all glands in response to doxycycline and a T50 of seven days post-induction, while induced or un-induced controls remained tumour-free after one year of induction. Histological analyses of rtTA/MIC mammary glands and tumour revealed that lesions followed the canonical stepwise progression of PyV mT tumourigenesis, from hyperplasia to mammary intraepithelial neoplasia/adenoma, carcinoma, and invasive carcinoma that metastasizes to the lung; at each of these stages expression of PyV mT and Cre recombinase transgenes was confirmed. Withdrawal of doxycycline from rtTA/MIC mice with end-stage mammary tumours led to rapid regression, yet animals eventually developed PyV mT-expressing and -non-expressing recurrent masses with varied tumour histopathologies. CONCLUSIONS: We have successfully created a temporally regulated mouse model of PyV mT-mediated mammary tumourigenesis that can be used to study Cre recombinase-mediated genetic changes simultaneously. While maintaining all of the hallmark features of the well-established constitutive MMTV-PyV mT model, the utility of this strain derives from the linking of PyV mT and Cre recombinase transgenes; mammary epithelial cells are thereby forced to couple PyV mT expression with conditional ablation of a given gene. This transgenic mouse model will be an important research tool for identifying synthetic viable genetic events that enable PyV mT tumours to evolve in the absence of a key signaling pathway.

Transgenic mouse models--a seminal breakthrough in oncogene research.

Transgenic mouse models are an integral part of modern cancer research, providing a versatile and powerful means of studying tumor initiation and progression, metastasis, and therapy. The present repertoire of these models is very diverse, with a wide range of strategies used to induce tumorigenesis by expressing dominant-acting oncogenes or disrupting the function of tumor-suppressor genes, often in a highly tissue-specific manner. Much of the current technology used in the creation and characterization of transgenic mouse models of cancer will be discussed in depth elsewhere. However, to gain a complete appreciation and understanding of these complex models, it is important to review the history of the field. Transgenic mouse models of cancer evolved as a new and, compared with the early cell-culture-based techniques, more physiologically relevant approach for studying the properties and transforming capacities of oncogenes. Here, we will describe early transgenic mouse models of cancer based on tissue-specific expression of oncogenes and discuss their impact on the development of this still rapidly growing field.

Mammary epithelial-specific disruption of c-Src impairs cell cycle progression and tumorigenesis.

The tyrosine kinase c-Src is activated in a large proportion of breast cancers, in which it is thought to play a key role in promoting the malignant phenotype. c-Src activity is also elevated in transgenic mouse models of breast cancer, including the widely used polyomavirus middle-T antigen (PyVmT) model, which provides an opportunity to study the importance of c-Src in mammary tumorigenesis. However, germline c-Src deletion in mammary epithelial and stromal compartments complicates the interpretation of in vivo tumorigenesis studies as a result of severe defects in mammary gland development. We have therefore engineered a mouse strain in which deletion of c-Src can be targeted to the mammary epithelium. We demonstrate that mammary epithelial disruption of c-Src impairs proliferation and tumor progression driven by PyVmT in vivo. Whereas related kinases substitute for c-Src in PyVmT signaling, c-Src ablation impairs cell cycle progression with decreased cyclin expression and elevated expression of cyclin-dependent kinase inhibitors. Our data indicate that c-Src has essential and unique functions in proliferation and tumor progression in this mouse model that may also be important in certain contexts in some human breast cancers.

Regulation of cell signalling by uPAR.

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation and tissue remodelling and in many human cancers, in which it frequently indicates poor prognosis. uPAR regulates proteolysis by binding the extracellular protease urokinase-type plasminogen activator (uPA; also known as urokinase) and also activates many intracellular signalling pathways. Coordination of extracellular matrix (ECM) proteolysis and cell signalling by uPAR underlies its important function in cell migration, proliferation and survival and makes it an attractive therapeutic target in cancer and inflammatory diseases. uPAR lacks transmembrane and intracellular domains and so requires transmembrane co-receptors for signalling. Integrins are essential uPAR signalling co-receptors and a second uPAR ligand, the ECM protein vitronectin, is also crucial for this process.

uPAR promotes formation of the p130Cas-Crk complex to activate Rac through DOCK180.

The urokinase-type plasminogen activator receptor (uPAR) drives tumor cell membrane protrusion and motility through activation of Rac; however, the pathway leading from uPAR to Rac activation has not been described. In this study we identify DOCK180 as the guanine nucleotide exchange factor acting downstream of uPAR. We show that uPAR cooperates with integrin complexes containing beta(3) integrin to drive formation of the p130Cas-CrkII signaling complex and activation of Rac, resulting in a Rac-driven elongated-mesenchymal morphology, cell motility, and invasion. Our findings identify a signaling pathway underlying the morphological changes and increased cell motility associated with uPAR expression.

Stimulation of the weak ATPase activity of human hsp90 by a client protein.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways. In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23. The role of these proteins in Hsp90-mediated assembly processes is poorly understood. It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro. Here we show, for the first time, that human Hsp90 has ATPase activity in vitro. The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli. Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C. Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold. This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity. In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity. We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90. In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate. Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further. In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis. Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding. We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo. We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur.