Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine-tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling-dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR-mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGFmediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.
Breast cancer incidence is increasing worldwide with more than 600,000 deaths reported in 2018 alone. In current practice treatment options for breast cancer patients consists of surgery, chemotherapy, radiotherapy or targeting of classical markers of breast cancer subtype: estrogen receptor (ER) and HER2. However, these treatments fail to prevent recurrence and metastasis. Improved understanding of breast cancer and metastasis biology will help uncover novel biomarkers and therapeutic opportunities to improve patient stratification and treatment. We will first provide an overview of current methods and models used to study breast cancer biology, focusing on 2D and 3D cell culture, including organoids, and on in vivo models such as the MMTV mouse model and patient-derived xenografts (PDX). Next, genomic, transcriptomic, and proteomic approaches and their integration will be considered in the context of breast cancer susceptibility, breast cancer drivers, and therapeutic response and resistance to treatment. Finally, we will discuss how 'Omics datasets in combination with traditional breast cancer models are useful for generating insights into breast cancer biology, for suggesting individual treatments in precision oncology, and for creating data repositories to undergo further meta-analysis. System biology has the potential to catalyze the next great leap forward in treatment options for breast cancer patients.
SUMMARYIntegration of signaling downstream from individual Receptor Tyrosine Kinases (RTKs) is crucial to fine tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration still remain poorly elucidated. Focusing on distinct Fibroblast Growth Factor Receptors (FGFRs) we generated a detailed picture of recycling-dependent FGF signalling in breast cancer cells by combining quantitative phosphoproteomics and targeted assays. We discovered reciprocal priming between FGFRs and Epidermal Growth Factor Receptor (EGFR) within recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR-mediated cell cycle but not cell invasion. In turn, FGFR signaling primes EGF-mediated outputs. The discovery of reciprocal priming between distict families of RTKs within recycling endosomes will transform our understanding of signalling integration by pointing to recycling endosomes as crucial signalling hubs for orchestrating cellular behaviour. Therefore, targeting reciprocal priming rather than individual receptors may improve personalized therapies in breast and other cancers.
The development of drugs to treat cancer is severely hampered by the inefficiency of translating pre-clinical in vitro and mouse studies into clinical benefit. Over 90% of drugs that progress through pre-clinical studies fail in human trials. Therefore, there is a critical need to improve the accuracy of evaluating pre-clinical drug efficacy through the development of more physiologically relevant human models. This is especially the case for pancreatic cancer, the 4th leading cause of cancer deaths with a 5-year survival rate of <6%, where new therapies are desperately needed. The tumor microenvironment (TME) of PDAC contains numerous cell types including vascular endothelial cells, stellate cells and fibroblasts, as well as a complex extracellular matrix (ECM), all of which contribute to the growth and survival of cancer cells, as well as posing potential targets for therapeutic intervention. Thus, a more complete and nuanced understanding of the PDAC TME is required for the rational development of effective therapies. To address this problem, an in vitro tumor microenvironment system (TMeS) was engineered to incorporate tumor capillary hemodynamics and biological transport with co-cultured human microvascular endothelial cells, along with pancreatic tumor and stellate cells. We demonstrate that significant tumor cell transcriptomic changes occur in the TMeS that correlate with the in vivo xenograft transcriptome, including alterations in cell cycle regulation, oncogene signaling, and metabolism. PDAC tumor cells from two patients were inhibited by the human Cmax dose of gemcitabine to levels paralleling the patients’ clinical responses. Previous work demonstrated that the FAK inhibitor, PF-562,271, worked only on in vivo xenografts and not on 2D in vitro PDAC cultures and here we show that PF-562,271 effectively inhibited PDAC growth in the TMeS indicating in vivo-like drug responses. In sum, the TMeS recapitulates the in vivo xenograft transcriptional program and responds to both established and experimental small molecule inhibitor chemotherapeutics at concentrations that correspond to human therapeutic plasma levels. Therefore, this model provides a unique platform to rigorously test the contributions of the cellular and molecular components of the TME through manipulating individual constituents in a controlled fashion that is not possible in vivo. Moreover, the TMeS can be used for the rapid evaluation of novel precision therapies. Finally, this system is amenable to using patient tumor material directly, enabling the potential use of the TMeS for patient avatars. Citation Format: Daniel G. Gioeli, Chelsi Snow, Michael Simmers, Robert Figler, J. Thomase Parsons, Stephen Hoang, Todd Bauer, Brian Wamhoff. Development of a human pancreatic tumor microenvironment system (TMeS) for evaluation of novel therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4935. doi:10.1158/1538-7445.AM2017-4935
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