Sean Cory

Distinct Loci Influence Radiation-Induced Alveolitis from Fibrosing Alveolitis in the Mouse

Thoracic radiotherapy may produce the morbidity-associated lung responses of alveolitis or fibrosing alveolitis in treated cancer patients. The genetic factors that influence a patients likelihood of developing alveolitis and the relationship of this inflammatory response to the development of fibrosis are largely unknown. Herein we use genetic mapping to identify radiation-induced lung response susceptibility loci in reciprocal backcross mice bred from C3H/HeJ (alveolitis response) and C57BL/6J (fibrosing alveolitis/fibrosis response) strains. Mice were treated with 18-Gy whole thorax irradiation and their survival, lung histopathology, and bronchoalveolar lavage cell types were recorded. A genome-wide scan was completed using 139 markers. The C3H/HeJ alveolitis response included mast cell infiltration and increased neutrophil numbers in the lavage compared with the level in the C57BL/6J strain, which developed fibrosis. In backcross mice, posttreatment survival was dictated by the development of an alveolitis response with increased mast cell, bronchoalveolar lavage total cell, and neutrophil numbers. Fibrosis was measured only in a subset of mice developing alveolitis and, in these mice, was associated with neutrophil count. Genotyping revealed coinheritance of C3H alleles (chromosomes 2, 4, 19, and X) and C57BL/6J alleles (chromosomes 1, 7, 9, and 17) to result in higher fibrosis scores in backcross mice. Mice that inherited C57BL/6J alleles at the putative alveolitis susceptibility loci were spared this response and lived to the end of the experiment. In this animal model, independent loci control the development of alveolitis from fibrosis, whereas fibrosing alveolitis occurs with the coinheritance of these factors.

Crk adaptor proteins act as key signaling integrators for breast tumorigenesis

IntroductionCT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo.MethodsThe clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer.ResultsA gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation.ConclusionsThese data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.

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 patients 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.

Dynamic Reprogramming of Signaling Upon Met Inhibition Reveals a Mechanism of Drug Resistance in Gastric Cancer

Understanding how inhibitors rewire the Met receptor pathway points to a strategy for more effective gastric cancer treatment. Met with Resistance in Gastric Cancer The hepatocyte growth factor receptor Met is associated with poor prognosis in various cancers, but the efficacy of Met inhibitors is often impeded by resistance. By comparing transcriptional changes at multiple time points after stimulation or inhibition of Met, Lai et al. identified critical mediators of Met-induced proliferation and Met inhibitor resistance in gastric cancer cell lines. Cancer cells or tumors exposed to Met inhibitors had decreased abundance of phosphatases, and this correlated with reactivation of the pro-proliferative MEK-ERK pathway and the emergence of drug resistance. Combining Met and MEK inhibitors was more cytotoxic to gastric cancer cells in culture than was either inhibitor alone, suggesting that this combination strategy may be effective in gastric cancer. The Met receptor tyrosine kinase is activated or genetically amplified in some gastric cancers, but resistance to small-molecule inhibitors of Met often emerges in patients. We found that Met abundance correlated with a proliferation marker in patient gastric tumor sections, and gastric cancer cell lines that have MET amplifications depended on Met for proliferation and anchorage-independent growth in culture. Inhibition of Met induced temporal changes in gene expression in the cell lines, initiated by a rapid decrease in the expression of genes encoding transcription factors, followed by those encoding proteins involved in epithelial-mesenchymal transition, and finally those encoding cell cycle–related proteins. In the gastric cancer cell lines, microarray and chromatin immunoprecipitation analysis revealed considerable overlap between genes regulated in response to Met stimulation and those regulated by signal transducer and activator of transcription 3 (STAT3). The activity of STAT3, extracellular signal–regulated kinase (ERK), and the kinase Akt was decreased by Met inhibition, but only inhibitors of STAT3 were as effective as the Met inhibitor in decreasing tumor cell proliferation in culture and in xenografts, suggesting that STAT3 mediates the pro-proliferative program induced by Met. However, the phosphorylation of ERK increased after prolonged Met inhibition in culture, correlating with decreased abundance of the phosphatases DUSP4 and DUSP6, which inhibit ERK. Combined inhibition of Met and the mitogen-activated protein kinase kinase (MEK)–ERK pathway induced greater cell death in cultured gastric cancer cells than did either inhibitor alone. These findings indicate combination therapies that may counteract resistance to Met inhibitors.

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone

IntroductionBone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases.MethodsTo search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors.ResultsABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5.ConclusionsOur data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.

Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Using transgenic mouse models of breast cancer that ablate Src homology and collagen A (ShcA) expression or oncogene-coupled ShcA signaling, we previously showed that this adaptor is critical for mammary tumor onset and progression. We now provide the first evidence that ShcA regulates mammary tumorigenesis, in part, through its ability to regulate the adaptive immune response. Inactivation of ShcA signaling within tumor cells results in extensive CD4(+) T-cell infiltration and induction of a humoral immune response in mammary tumors. This is associated with a robust CTL response in preneoplastic lesions that are deficient in ShcA signaling. Moreover, mammary tumor progression of ShcA-deficient hyperplasias is accelerated in a T cell-deficient background. We also uncover a clinically relevant correlation between high ShcA expression and low CTL infiltration in human breast cancers. Finally, we define a novel ShcA-regulated immune signature that functions as an independent prognostic marker of survival in human epidermal growth factor receptor 2(+) and basal breast cancers. We reveal a novel role for tumor cell-derived ShcA in the establishment and maintenance of an immunosuppressive state.

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/β1, 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.

Implementing Arithmetic and Other Analytic Operations By Transcriptional Regulation

The transcriptional regulatory machinery of a gene can be viewed as a computational device, with transcription factor concentrations as inputs and expression level as the output. This view begs the question: what kinds of computations are possible? We show that different parameterizations of a simple chemical kinetic model of transcriptional regulation are able to approximate all four standard arithmetic operations: addition, subtraction, multiplication, and division, as well as various equality and inequality operations. This contrasts with other studies that emphasize logical or digital notions of computation in biological networks. We analyze the accuracy and precision of these approximations, showing that they depend on different sets of parameters, and are thus independently tunable. We demonstrate that networks of these “arithmetic” genes can be combined to accomplish yet more complicated computations by designing and simulating a network that detects statistically significant elevations in a time-varying signal. We also consider the much more general problem of approximating analytic functions, showing that this can be achieved by allowing multiple transcription factor binding sites on the promoter. These observations are important for the interpretation of naturally occurring networks and imply new possibilities for the design of synthetic networks.

Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

ABSTRACT ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling.

Abstract A79: Mouse models of breast cancer identify oncogene-associated stroma subtypes

Breast Cancer is a highly heterogeneous disease involving complex crosstalk between tumor cells and their surrounding microenvironment (stroma). Previous work in our lab utilized laser capture microdissection and gene expression analysis to separate Breast Cancer stromal samples into distinct subtypes. Although this and other data demonstrate the importance of the stroma in tumor progression, the cause of these stromal changes are still poorly understood. We hypothesize that changes within stroma are related to individual oncogenic events within the tumor epithelium. Analyzing human data does not allow us to draw causative conclusions due to the presence of compounding factors such as genetic variance and living conditions. In an attempt to circumvent this challenge we used transgenic mouse models of breast cancer in the same strain of mice (FVB) with oncogenes driven by a mammary specific promoter (MMTV). By keeping these variables constant and only varying the oncogene: using either Neu-NDL-2-5, PyVMT or Wnt1 - allowed us to test the aforementioned hypothesis. Class discovery and distinction confirmed that the epithelial and stromal samples from each mouse model were distinct, which validated our hypothesis. Pathway analysis of the genes that distinguish the stroma of the mouse models identifies genes linked to recruitment and activation of different stromal cells. Interestingly our findings corroborate with results from other labs using these mouse models. Citation Format: Sadiq M. Saleh, Paul Savage, Julie Laferriere, Sean Cory, Nicholas Bertos, Margarita Souleimanova, Hong Zhao, Eldad Zacksenhaus, William Muller, Michael Hallett, Morag Park. Mouse models of breast cancer identify oncogene-associated stroma subtypes. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A79. doi:10.1158/1538-7445.CHTME14-A79