Discoidin domain receptor 1 activity drives an aggressive phenotype in gastric carcinoma.

BACKGROUND: Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that utilizes collagen as a ligand, is a key molecule in the progression of solid tumors as it regulates the interaction of cancer cells with the tumor stroma. However, the clinical relevance of DDR1 expression in gastric carcinoma is yet to be investigated. Here, we assessed the role of DDR1 in mediating the aggressive phenotype of gastric carcinoma and its potential as a therapeutic target. METHODS: We conducted DDR1 immunohistochemistry using a tissue microarray of 202 gastric carcinoma specimens. We examined the effect of collagen-induced activation of DDR1 on cell signaling, tumorigenesis, and cell migration in gastric cancer cell lines, and tumor growth in a xenograft animal model of gastric cancer. RESULTS: Our results showed that 50.5% of gastric cancer tissues are positive for DDR1 expression, and positive DDR1 expression was significantly correlated with a poor prognosis (P = 0.015). In a subgroup analysis, DDR1 expression was prognostically meaningful only in patients receiving adjuvant treatment (P = 0.013). We also demonstrated that collagen was able to activate DDR1 and increase the clonogenicity and migration of gastric cancer cells. We observed that a DDR1 inhibitor, 7rh benzamide, suppressed tumor growth in gastric cancer xenografts. CONCLUSIONS: Our findings suggest a key role for DDR1 signaling in mediating the aggressive phenotype of gastric carcinoma. Importantly, inhibition of DDR1 is an attractive strategy for gastric carcinoma therapy.

Recruitment and retention: factors that affect pericyte migration.

Pericytes are critical for vascular morphogenesis and contribute to several pathologies, including cancer development and progression. The mechanisms governing pericyte migration and differentiation are complex and have not been fully established. Current literature suggests that platelet-derived growth factor/platelet-derived growth factor receptor-ß, sphingosine 1-phosphate/endothelial differentiation gene-1, angiopoietin-1/tyrosine kinase with immunoglobulin-like and EGF-like domains 2, angiopoietin-2/tyrosine kinase with immunoglobulin-like and EGF-like domains 2, transforming growth factor ß/activin receptor-like kinase 1, transforming growth factor ß/activin receptor-like kinase 5, Semaphorin-3A/Neuropilin, and matrix metalloproteinase activity regulate the recruitment of pericytes to nascent vessels. Interestingly, many of these pathways are directly affected by secreted protein acidic and rich in cysteine (SPARC). Here, we summarize the function of these factors in pericyte migration and discuss if and how SPARC might influence these activities and thus provide an additional layer of control for the recruitment of vascular support cells. Additionally, the consequences of targeted inhibition of pericytes in tumors and the current understanding of pericyte recruitment in pathological environments are discussed.

SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations.

BACKGROUND: Tumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination. METHODS: M and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer. RESULTS: Comparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases. CONCLUSIONS: The matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer.

Porcupine Inhibition Disrupts Mitochondrial Function and Homeostasis in WNT Ligand-Addicted Pancreatic Cancer.

WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse effects on proliferation, differentiation, survival, and stemness. A subset of PDAC with inactivating mutations in ring finger protein 43 (RNF43) show growth dependency on autocrine WNT ligand signaling and are susceptible to agents that block WNT ligand acylation by Porcupine O-acyltransferase, which is required for proper WNT ligand processing and secretion. For this study, global transcriptomic, proteomic, and metabolomic analyses were performed to explore the therapeutic response of RNF43-mutant PDAC to the Porcupine inhibitor (PORCNi) LGK974. LGK974 disrupted cellular bioenergetics and mitochondrial function through actions that included rapid mitochondrial depolarization, reduced mitochondrial content, and inhibition of oxidative phosphorylation and tricarboxylic acid cycle. LGK974 also broadly altered transcriptional activity, downregulating genes involved in cell cycle, nucleotide metabolism, and ribosomal biogenesis and upregulating genes involved in epithelial-mesenchymal transition, hypoxia, endocytosis, and lysosomes. Autophagy and lysosomal activity were augmented in response to LGK974, which synergistically inhibited tumor cell viability in combination with chloroquine. Autocrine WNT ligand signaling dictates metabolic dependencies in RNF43-mutant PDAC through a combination of transcription dependent and independent effects linked to mitochondrial health and function. Metabolic adaptations to mitochondrial damage and bioenergetic stress represent potential targetable liabilities in combination with PORCNi for the treatment of WNT ligand-addicted PDAC.

WNT Ligand Dependencies in Pancreatic Cancer.

WNT signaling promotes the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) through wide-ranging effects on cellular proliferation, survival, differentiation, stemness, and tumor microenvironment. Of therapeutic interest is a genetically defined subset of PDAC known to have increased WNT/ß-catenin transcriptional activity, growth dependency on WNT ligand signaling, and response to pharmacologic inhibitors of the WNT pathway. Here we review mechanisms underlying WNT ligand addiction in pancreatic tumorigenesis, as well as the potential utility of therapeutic approaches that functionally antagonize WNT ligand secretion or frizzled receptor binding.

Deep proteome profiling of human mammary epithelia at lineage and age resolution.

Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68 years. Data were quality controlled and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Upregulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Age-dependent proteome changes in the breast epithelium identified heretofore unknown potential therapeutic targets for reducing breast cancer susceptibility.

Inhibition of Discoidin Domain Receptor 1 Reduces Collagen-mediated Tumorigenicity in Pancreatic Ductal Adenocarcinoma.

The extracellular matrix (ECM), a principal component of pancreatic ductal adenocarcinoma (PDA), is rich in fibrillar collagens that facilitate tumor cell survival and chemoresistance. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that specifically binds fibrillar collagens and has been implicated in promoting cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. We found that collagen-induced activation of DDR1 stimulated protumorigenic signaling through protein tyrosine kinase 2 (PYK2) and pseudopodium-enriched atypical kinase 1 (PEAK1) in pancreatic cancer cells. Pharmacologic inhibition of DDR1 with an ATP-competitive orally available small-molecule kinase inhibitor (7rh) abrogated collagen-induced DDR1 signaling in pancreatic tumor cells and consequently reduced colony formation and migration. Furthermore, the inhibition of DDR1 with 7rh showed striking efficacy in combination with chemotherapy in orthotopic xenografts and autochthonous pancreatic tumors where it significantly reduced DDR1 activation and downstream signaling, reduced primary tumor burden, and improved chemoresponse. These data demonstrate that targeting collagen signaling in conjunction with conventional cytotoxic chemotherapy has the potential to improve outcome for pancreatic cancer patients. Mol Cancer Ther; 16(11); 2473-85. ©2017 AACR.

Hypoxia Studies with Pimonidazole in vivo.

Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including autophagy (Phan et al., 2013; Hu et al., 2012; Ye et al., 2010). We have recently shown that anti-VEGF therapy-induced hypoxia can result in changes in the extracellular matrix that contribute to the aggressiveness of tumors post therapy (Aguilera et al., 2014). Importantly, therapies that induce hypoxia do not always increase epithelial plasticity and tumor aggressiveness (Ostapoff et al., 2013; Cenik et al., 2013). We have used pimonidazole to evaluate hypoxia in tumors and herein provide a detailed protocol for this useful tool to interrogate the levels of hypoxia in vivo. The utility of the Hypoxyprobe™ (pimonidazole hydrochloride) immunohistochemical analysis approach allows for the assessment of hypoxia in different tissues as well as cell types. Pimonidazole is a 2-nitroimidazole that is reductively activated specifically in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids (Cenik et al., 2013; Arnold et al., 2010; Raleigh and Koch, 1990; Raleigh et al., 1998). Furthermore, the amount of pimonidazole that is detected is directly proportional to the level of hypoxia within tumors.

Collagen signaling enhances tumor progression after anti-VEGF therapy in a murine model of pancreatic ductal adenocarcinoma.

There is growing evidence that antiangiogenic therapy stimulates cancer cell invasion and metastasis. However, the underlying molecular mechanisms responsible for these changes have not been fully defined. Here, we report that anti-VEGF therapy promotes local invasion and metastasis by inducing collagen signaling in cancer cells. We show that chronic VEGF inhibition in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) induces hypoxia, a less differentiated mesenchymal-like tumor cell phenotype, TGF-ß expression, and collagen deposition and signaling. In addition, we show that collagen signaling is critical for protumorigenic activity of TGF-ß in vitro. To further model the impact of collagen signaling in tumors, we evaluated PDA in mice lacking Sparc, a protein that reduces collagen binding to cell surface receptors. Importantly, we show that loss of Sparc increases collagen signaling and tumor progression. Together, these findings suggest that collagen actively promotes PDA spread and that enhanced disease progression associated with anti-VEGF therapy can arise from elevated extracellular matrix-mediated signaling.