A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer.

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

p66ShcA functions as a contextual promoter of breast cancer metastasis.

BACKGROUND: The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. METHODS: We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. RESULTS: We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. CONCLUSION: This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

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.

Interplay between ShcA Signaling and PGC-1α Triggers Targetable Metabolic Vulnerabilities in Breast Cancer.

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose. ShcA signaling simultaneously increased the metabolic rate and flexibility of breast cancer cells by inducing the PGC-1α transcriptional coactivator, a central regulator of mitochondrial metabolism. Breast tumors that engaged ShcA signaling were critically dependent on PGC-1α to support their increased metabolic rate. PGC-1α deletion drastically delayed breast tumor onset in an orthotopic mouse model, highlighting a key role for PGC-1α in tumor initiation. Conversely, reduced ShcA signaling impaired both the metabolic rate and flexibility of breast cancer cells, rendering them reliant on mitochondrial oxidative phosphorylation. This metabolic reprogramming exposed a targetable metabolic vulnerability, leading to a sensitization of breast tumors to inhibitors of mitochondrial complex I (biguanides). Genetic inhibition of ShcA signaling in the Polyoma virus middle T (MT) breast cancer mouse model sensitized mammary tumors to biguanides during the earliest stages of breast cancer progression. Tumor initiation and growth were selectively and severely impaired in MT/ShcA-deficient animals. These data demonstrate that metabolic reprogramming is a key component of ShcA signaling and serves an unappreciated yet vital role during breast cancer initiation and progression. These data further unravel a novel interplay between ShcA and PGC-1α in the coordination of metabolic reprogramming and demonstrate the sensitivity of breast tumors to drugs targeting oxidative phosphorylation.Significance: This study uncovers a previously unrecognized mechanism that links aberrant RTK signaling with metabolic perturbations in breast cancer and exposes metabolic vulnerabilities that can be targeted by inhibitors of oxidative phosphorylation. Cancer Res; 78(17); 4826-38. ©2018 AACR.

The Shc1 adaptor simultaneously balances Stat1 and Stat3 activity to promote breast cancer immune suppression.

Tyrosine kinase signalling within cancer cells is central to the establishment of an immunosuppressive microenvironment. Although tyrosine kinase inhibitors act, in part, to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome is a hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold, a central regulator of tyrosine kinase signalling, as essential for promoting breast cancer immune suppression. Herein we show that the ShcA pathway simultaneously activates STAT3 immunosuppressive signals and impairs STAT1-driven immune surveillance in breast cancer cells. Impaired Y239/Y240-ShcA phosphorylation selectively reduces STAT3 activation in breast tumours, profoundly sensitizing them to immune checkpoint inhibitors and tumour vaccines. Finally, the ability of diminished tyrosine kinase signalling to initiate STAT1-driven immune surveillance can be overcome by compensatory STAT3 hyperactivation in breast tumours. Our data indicate that inhibition of pY239/240-ShcA-dependent STAT3 signalling may represent an attractive therapeutic strategy to sensitize breast tumours to multiple immunotherapies.

Tungsten targets the tumor microenvironment to enhance breast cancer metastasis.

The number of individuals exposed to high levels of tungsten is increasing, yet there is limited knowledge of the potential human health risks. Recently, a cohort of breast cancer patients was left with tungsten in their breasts following testing of a tungsten-based shield during intraoperative radiotherapy. While monitoring tungsten levels in the blood and urine of these patients, we utilized the 66Cl4 cell model, in vitro and in mice to study the effects of tungsten exposure on mammary tumor growth and metastasis. We still detect tungsten in the urine of patients' years after surgery (mean urinary tungsten concentration at least 20 months post-surgery = 1.76 ng/ml), even in those who have opted for mastectomy, indicating that tungsten does not remain in the breast. In addition, standard chelation therapy was ineffective at mobilizing tungsten. In the mouse model, tungsten slightly delayed primary tumor growth, but significantly enhanced lung metastasis. In vitro, tungsten did not enhance 66Cl4 proliferation or invasion, suggesting that tungsten was not directly acting on 66Cl4 primary tumor cells to enhance invasion. In contrast, tungsten changed the tumor microenvironment, enhancing parameters known to be important for cell invasion and metastasis including activated fibroblasts, matrix metalloproteinases, and myeloid-derived suppressor cells. We show, for the first time, that tungsten enhances metastasis in an animal model of breast cancer by targeting the microenvironment. Importantly, all these tumor microenvironmental changes are associated with a poor prognosis in humans.

p66ShcA promotes breast cancer plasticity by inducing an epithelial-to-mesenchymal transition.

Breast cancers are stratified into distinct subtypes, which influence therapeutic responsiveness and patient outcome. Patients with luminal breast cancers are often associated with a better prognosis relative to that with other subtypes. However, subsets of patients with luminal disease remain at increased risk of cancer-related death. A critical process that increases the malignant potential of breast cancers is the epithelial-to-mesenchymal transition (EMT). The p66ShcA adaptor protein stimulates the formation of reactive oxygen species in response to stress stimuli. In this paper, we report a novel role for p66ShcA in inducing an EMT in HER2(+) luminal breast cancers. p66ShcA increases the migratory properties of breast cancer cells and enhances signaling downstream of the Met receptor tyrosine kinase in these tumors. Moreover, Met activation is required for a p66ShcA-induced EMT in luminal breast cancer cells. Finally, elevated p66ShcA levels are associated with the acquisition of an EMT in primary breast cancers spanning all molecular subtypes, including luminal tumors. This is of high clinical relevance, as the luminal and HER2 subtypes together comprise 80% of all newly diagnosed breast cancers. This study identifies p66ShcA as one of the first prognostic biomarkers for the identification of more aggressive tumors with mesenchymal properties, regardless of molecular subtype.

The ShcA PTB domain functions as a biological sensor of phosphotyrosine signaling during breast cancer progression.

ShcA (SHC1) is an adapter protein that possesses an SH2 and a PTB phosphotyrosine-binding motif. ShcA generally uses its PTB domain to engage activated receptor tyrosine kinases (RTK), but there has not been a definitive determination of the role of this domain in tumorigenesis. To address this question, we employed a ShcA mutant (R175Q) that no longer binds phosphotyrosine residues via its PTB domain. Here, we report that transgenic expression of this mutant delays onset of mammary tumors in the MMTV-PyMT mouse model of breast cancer. Paradoxically, we observed a robust increase in the growth and angiogenesis of mammary tumors expressing ShcR175Q, which displayed increased secretion of fibronectin and expression of integrin α5/ß1, the principal fibronectin receptor. Sustained integrin engagement activated Src, which in turn phosphorylated proangiogenic RTKs, including platelet-derived growth factor receptor, fibroblast growth factor receptor, and Met, leading to increased VEGF secretion from ShcR175Q-expressing breast cancer cells. We defined a ShcR175Q-dependent gene signature that could stratify breast cancer patients with a high microvessel density. This study offers the first in vivo evidence of a critical role for intracellular signaling pathways downstream of the ShcA PTB domain, which both positively and negatively regulate tumorigenesis during various stages of breast cancer progression.