Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here, we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types typically seen in response to lung injury, and by striking infidelity amongst transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9 , and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9 -dependent resistance to Natural Killer (NK) cells. Loss of developmental stage-specific constraint in macrometastases triggered by NK cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.

Advanced, metastatic melanomas frequently grow in subcutaneous tissues and portend a poor prognosis. Though subcutaneous tissues are largely composed of adipocytes, the mechanisms by which adipocytes influence melanoma are poorly understood. Using and models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. Additionally, adipocytes directly transfer lipids to melanoma cells, which alters tumor cell metabolism. Adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAF in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. Pharmacologic blockade of FATPs with the small-molecule inhibitor Lipofermata abrogates lipid transport into melanoma cells and reduces melanoma growth and invasion. These data demonstrate that stromal adipocytes can drive melanoma progression through FATP lipid transporters and represent a new target aimed at interrupting adipocyte-melanoma cross-talk. We demonstrate that stromal adipocytes are donors of lipids that mediate melanoma progression. Adipocyte-derived lipids are taken up by FATP proteins that are aberrantly expressed in melanoma. Inhibition of FATPs decreases melanoma lipid uptake, invasion, and growth. We provide a mechanism for how stromal adipocytes drive tumor progression and demonstrate a novel microenvironmental therapeutic target. .

Misexpression of microRNAs (miRNAs) is widespread in human cancers, including in pancreatic cancer. Aberrations of miRNA include overexpression of oncogenic miRs ("Onco-miRs"), or downregulation of so-called tumor suppressor "TSG-miRs". Restitution of TSG-miRs in cancer cells through systemic delivery is a promising avenue for pancreatic cancer therapy. We have synthesized a lipid-based nanoparticle for systemic delivery of miRNA expression vectors to cancer cells ("nanovector"). The plasmid DNA-complexed nanovector is ~100nM in diameter, and demonstrates no apparent histopathological or biochemical evidence of toxicity upon intravenous injection. Two miRNA candidates known to be downregulated in the majority of pancreatic cancers were selected for nanovector delivery: miR-34a, which is a component of the p53 transcriptional network and regulates "cancer stem cell" (CSC) survival, and the miR-143/145 cluster, which together repress the expression of KRAS2, and its downstream effector Ras-responsive element binding protein-1 (RREB1). Systemic intravenous delivery with either miR-34a or miR-143/145 nanovectors inhibited the growth of MiaPaCa-2 subcutaneous xenografts (P<0.01 for miR-34a, P<0.05 for miR-143/145); the effects were even more pronounced in the orthotopic (intra-pancreatic) setting (P<0.0005 for either nanovector), when compared to vehicle or "mock" nanovector delivering an empty plasmid. Tumor growth inhibition was accompanied by increased apoptosis and decreased proliferation. MiRNA restitution was confirmed in treated xenografts by significant upregulation of the corresponding miRNA, and significant decreases in specific miRNA targets (SIRT1, CD44 and aldehyde dehydrogenase for miR34a, and KRAS2 and RREB1 for miR-143/145). The nanovector is a platform with potential broad applicability in systemic miRNA delivery to cancer cells.

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAFV600E;p53−/− fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, μ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (~1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

MicroRNAs (miRNAs) are small non-coding RNAs, which inhibit the stability and/or translation of a mRNA. miRNAs have been found to play a powerful role in various cardiovascular diseases. Recently, we have demonstrated that a microRNA (miR-181c) can be encoded in the nucleus, processed to the mature form in the cytosol, translocated into the mitochondria, and ultimately can regulate mitochondrial gene expression. However the in vivo impact of miR-181c is unknown. Here we report an in-vivo method for administration of miR-181c in rats, which leads to reduced exercise capacity and signs of heart failure, by targeting the 3′-end of mt-COX1 (cytochrome c oxidase subunit 1). We cloned miR-181c and packaged it in lipid-based nanoparticles for systemic delivery. The plasmid DNA complexed nanovector shows no apparent toxicity. We find that the mRNA levels of mitochondrial complex IV genes in the heart, but not any other mitochondrial genes, are significantly altered with miR-181c overexpression, suggesting selective mitochondrial complex IV remodeling due to miR-181c targeting mt-COX1. Isolated heart mitochondrial studies showed significantly altered O2-consumption, ROS production, matrix calcium, and mitochondrial membrane potential in miR-181c-treated animals. For the first time, this study shows that miRNA delivered to the heart in-vivo can lead to cardiac dysfunction by regulating mitochondrial genes.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24+CD44+ and ALDH+ tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for the further investigation of this combination in PDA.

Chromatin state and oncogenic competence Although specific DNA mutations can lead to tumor generation, they are not transforming in all cellular contexts. This may be due to the intrinsic transcriptional program present in the cell of origin. Using zebrafish and human pluripotent stem cell cancer models, Baggiolini et al . report that neural crest cells and melanoblasts (precursors to melanocytes) are susceptible to specific mutation of the BRAF gene, whereas melanocytes are relatively resistant (see the Perspective by Vredevoogd and Peeper). The competent cells display higher levels of chromatin factors such as the protein ATAD2 compared with the less competent ones. ATAD2 forms a complex with the neural crest transcription factor SOX10 and establishes a chromatin state that makes them permissive to BRAF mutagenesis. These data indicate that developmental chromatin programs are a determinant of how cells respond to DNA mutations. —BAP

Summary Mucin 16 (cancer antigen 125) is a cell surface glycoprotein that plays a role in promoting cancer cell growth in ovarian cancer. The aims of this study were to examine mucin 16 expression in a large number of digestive tract adenocarcinomas and precursors and to determine whether mucin 16 up-regulation is correlated with patient outcome. Tissue microarrays were constructed using surgical resection tissues and included pancreatic (115 normal, 29 precursors, 200 pancreatic ductal adenocarcinomas), esophageal (86 normal, 104 precursors, 95 esophageal adenocarcinomas, 35 lymph node metastases), gastric (211 normal, 8 precursors, 119 gastric adenocarcinomas, 62 lymph node metastases), and colorectal (34 normal, 17 precursors, 39 colorectal adenocarcinomas) tissues. Mucin 16 was detected in 81.5%, 69.9%, 41.2%, and 64.1% of the pancreatic ductal adenocarcinomas, esophageal adenocarcinomas, gastric adenocarcinomas, and colorectal adenocarcinomas, respectively. Mucin 16 was seen in a subset of the precursors. On multivariate analysis, moderate/diffuse mucin 16 in pancreatic ductal adenocarcinomas was strongly associated with poor survival (P < .001), independent of other prognosis predictors. A similar trend was observed for esophageal adenocarcinomas (P = .160) and gastric adenocarcinomas (P = .080). Focal mucin 16 in colorectal adenocarcinomas was significantly correlated (P = .044) with a better patient outcome, when compared with mucin 16–negative cases. Using Western blot analysis, we found mucin 16 expression in 3 of 6 pancreatic ductal adenocarcinoma and 1 of 2 esophageal adenocarcinoma cell lines. We conclude that most of the digestive tract adenocarcinomas and a subset of their precursors express mucin 16. Mucin 16 expression is an independent predictor of poor outcome in pancreatic ductal adenocarcinomas and potentially in esophageal adenocarcinomas and gastric adenocarcinomas. We propose that mucin 16 may function as a prognostic marker and therapeutic target in the future.