Co-dependency for MET and FGFR1 in basal triple-negative breast cancers.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks both effective patient stratification strategies and therapeutic targets. Whilst elevated levels of the MET receptor tyrosine kinase are associated with TNBCs and predict poor clinical outcome, the functional role of MET in TNBC is still poorly understood. In this study, we utilise an established Met-dependent transgenic mouse model of TNBC, human cell lines and patient-derived xenografts to investigate the role of MET in TNBC tumorigenesis. We find that in TNBCs with mesenchymal signatures, MET participates in a compensatory interplay with FGFR1 to regulate tumour-initiating cells (TICs). We demonstrate a requirement for the scaffold protein FRS2 downstream from both Met and FGFR1 and find that dual inhibition of MET and FGFR1 signalling results in TIC depletion, hindering tumour progression. Importantly, basal breast cancers that display elevated MET and FGFR1 signatures are associated with poor relapse-free survival. Our results support a role for MET and FGFR1 as potential co-targets for anti-TIC therapies in TNBC.

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.

A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer.

Therapies targeting epidermal growth factor receptor (EGFR) have variable and unpredictable responses in breast cancer. Screening triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), we identify a subset responsive to EGFR inhibition by gefitinib, which displays heterogeneous expression of wild-type EGFR. Deep single-cell RNA sequencing of 3,500 cells from an exceptional responder identified subpopulations displaying distinct biological features, where elevated EGFR expression was significantly enriched in a mesenchymal/stem-like cellular cluster. Sorted EGFRhi subpopulations exhibited enhanced stem-like features, including ALDH activity, sphere-forming efficiency, and tumorigenic and metastatic potential. EGFRhi cells gave rise to EGFRhi and EGFRlo cells in primary and metastatic tumors, demonstrating an EGFR-dependent expansion and hierarchical state transition. Similar tumorigenic EGFRhi subpopulations were identified in independent PDXs, where heterogeneous EGFR expression correlated with gefitinib sensitivity. This provides new understanding for an EGFR-dependent hierarchy in TNBC and for patient stratification for therapeutic intervention.

Identification of Interacting Stromal Axes in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a molecularly heterogeneous cancer that is difficult to treat. Despite the role it may play in tumor progression and response to therapy, microenvironmental (stromal) heterogeneity in TNBC has not been well characterized. To address this challenge, we investigated the transcriptome of tumor-associated stroma isolated from TNBC (n = 57). We identified four stromal axes enriched for T cells (T), B cells (B), epithelial markers (E), or desmoplasia (D). Our analysis method (STROMA4) assigns a score along each stromal axis for each patient and then combined the axis scores to subtype patients. Analysis of these subtypes revealed that prognostic capacity of the B, T, and E scores was governed by the D score. When compared with a previously published TNBC subtyping scheme, the STROMA4 method better captured tumor heterogeneity and predicted patient benefit from therapy with increased sensitivity. This approach produces a simple ontology that captures TNBC heterogeneity and informs how tumor-associated properties interact to affect prognosis. Cancer Res; 77(17); 4673-83. ©2017 AACR.

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation.

Mesodermal cells signal to neighboring epithelial cells to modulate their proliferation in both normal and disease states. We adapted a Caenorhabditis elegans organogenesis model to enable a genome-wide mesodermal-specific RNAi screen and discovered 39 factors in mesodermal cells that suppress the proliferation of adjacent Ras pathway-sensitized epithelial cells. These candidates encode components of protein complexes and signaling pathways that converge on the control of chromatin dynamics, cytoplasmic polyadenylation, and translation. Stromal fibroblast-specific deletion of mouse orthologs of several candidates resulted in the hyper-proliferation of mammary gland epithelium. Furthermore, a 33-gene signature of human orthologs was selectively enriched in the tumor stroma of breast cancer patients, and depletion of these factors from normal human breast fibroblasts increased proliferation of co-cultured breast cancer cells. This cross-species approach identified unanticipated regulatory networks in mesodermal cells with growth-suppressive function, exposing the conserved and selective nature of mesodermal-epithelial communication in development and cancer.

The prognostic ease and difficulty of invasive breast carcinoma.

Breast carcinoma (BC) has been extensively profiled by high-throughput technologies for over a decade, and broadly speaking, these studies can be grouped into those that seek to identify patient subtypes (studies of heterogeneity) or those that seek to identify gene signatures with prognostic or predictive capacity. The sheer number of reported signatures has led to speculation that everything is prognostic in BC. Here, we show that this ubiquity is an apparition caused by a poor understanding of the interrelatedness between subtype and the molecular determinants of prognosis. Our approach constructively shows how to avoid confounding due to a patient's subtype, clinicopathological profile, or treatment profile. The approach identifies patients who are predicted to have good outcome at time of diagnosis by all available clinical and molecular markers but who experience a distant metastasis within 5 years. These inherently difficult patients (~7% of BC) are prioritized for investigations of intratumoral heterogeneity.

Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer.

Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Met(mt)) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Met(mt) mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Met(mt), significantly increased tumor penetrance over Met(mt) or Trp53 loss alone. Unlike Met(mt) tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Met(mt) tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-to-mesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.

Stromal retinoic acid receptor beta promotes mammary gland tumorigenesis.

Retinoic acid is a potent differentiation and antiproliferative agent of breast cancer cells, and one of its receptors, retinoic acid receptor ß (RARß), has been proposed to act as a tumor suppressor. In contrast, we report herein that inactivation of Rarb in the mouse results in a protective effect against ErbB2-induced mammary gland tumorigenesis. Strikingly, tissue recombination experiments indicate that the presence of Rarb in the stromal compartment is essential for the growth of mammary carcinoma. Ablation of Rarb leads to a remodeling of the stroma during tumor progression that includes a decrease in angiogenesis, in the recruitment of inflammatory cells, and in the number myofibroblasts. In agreement with this finding, we observed that a markedly reduced expression of chemokine (C-X-C motif) ligand 12 (Cxcl12) in the stroma of Rarb-null mice is accompanied by a decrease in the CXCL12/chemokine C-X-C receptor 4 (CXCR4)/ErbB2 signaling axis in the tumors. Relevance to the human disease is underlined by the finding that gene-expression profiling of the Rarb-deficient mammary stromal compartment identified an ortholog RARß signature in human microdissected breast tissues that differentiates tumor from normal stroma. Our study thus implicates RARß in promoting tumorigenesis and suggests that retinoid-based approaches for the prevention and treatment of breast cancer should be redesigned.