RelA regulates CXCL1/CXCR2-dependent oncogene-induced senescence in murine Kras-driven pancreatic carcinogenesis

Tumor suppression that is mediated by oncogene-induced senescence (OIS) is considered to function as a safeguard during development of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that regulate OIS in PDAC are poorly understood. Here, we have determined that nuclear RelA reinforces OIS to inhibit carcinogenesis in the Kras mouse model of PDAC. Inactivation of RelA accelerated pancreatic lesion formation in Kras mice by abrogating the senescence-associated secretory phenotype (SASP) gene transcription signature. Using genetic and pharmacological tools, we determined that RelA activation promotes OIS via elevation of the SASP factor CXCL1 (also known as KC), which activates CXCR2, during pancreatic carcinogenesis. In Kras mice, pancreas-specific inactivation of CXCR2 prevented OIS and was correlated with increased tumor proliferation and decreased survival. Moreover, reductions in CXCR2 levels were associated with advanced neoplastic lesions in tissue from human pancreatic specimens. Genetically disabling OIS in Kras mice caused RelA to promote tumor proliferation, suggesting a dual role for RelA signaling in pancreatic carcinogenesis. Taken together, our data suggest a pivotal role for RelA in regulating OIS in preneoplastic lesions and implicate the RelA/CXCL1/CXCR2 axis as an essential mechanism of tumor surveillance in PDAC

Establishment of robust controls for the normalization of miRNA expression in neuroendocrine tumors of the ileum and pancreas.

There is need to determine tissue-specific robust controls for normalization of microRNA expression to avoid false results and misinterpretation. The aim of this study was to evaluate the expression of different small RNAs in neuroendocrine tumors (NETs) and their suitability as normalizers in miRNA real-time PCR experiments. We investigated the expression of the nine small RNAs miR-93, miR-191, SNORD48, SNORD61, SNORD68, SNORD72, SNORD95, SNORD96a, and RNU6-2 in formalin-fixed, paraffin-embedded tissue samples of 25 ileal NETs by real-time PCR determining the most stable controls for expression normalization using four different algorithms. This analysis was expended to ten pancreatic NETs. Finally, five small RNAs were further tested as normalizers for miRNA-133a expression, which is known to be downregulated in metastases of ileal NETs, in ten matched pairs of ileal NETs and their metastases. Ranking of the expression results revealed the following order of stability from high to low: SNORD61 < SNORD95 < SNORD72 < SNORD96a < SNORD68 < miR-191 < miR-93 < RNU6-2 < SNORD48 for ileal NETs and SNORD95 < miR-93 < SNORD96a < SNORD61 < SNORD68 < SNORD72 < RNU6-2 < miR-191 < SNORD48 for pancreatic NETs. The determination of SNORD61 and SNORD95 for ileal NETs and SNORD95 and miR-93 for pancreatic NETs as good normalizers presents a useful tool for experiments involving the analysis of miRNA expression

Lack of CCR7 expression is rate limiting for lymphatic spread of pancreatic ductal adenocarcinoma.

CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21-CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph-node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7-transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph-node metastases than mock-transfected cells. In an analysis using quantitative real-time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate-to-strong immunohistochemical CCR7 expression, found in 58 of 121 well-characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph-node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease-free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor-associated lymph vessels, representing a previously unknown factor of lymphatic spread

Activating mutations in the MAP‐kinase pathway define non‐ossifying fibroma of bone

Non‐ossifying fibroma (NOF), which occasionally results in pathologic fracture, is considered the most common benign and self‐limiting lesion of the growing skeleton. By DNA sequencing we have identified hotspot KRAS, FGFR1 and NF1 mutations in 48 of 59 patients (81.4%) with NOF, at allele frequencies ranging from 0.04 to 0.61. Our findings define NOF as a genetically driven neoplasm caused in most cases by activated MAP‐kinase signalling. Interestingly, this driving force either diminishes over time or at least is not sufficient to prevent autonomous regression and resolution. Beyond its contribution to a better understanding of the molecular pathogenesis of NOF, this study adds another benign lesion to the spectrum of KRAS‐ and MAP‐kinase signalling‐driven tumours

Supplementary Material for: Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains

<p><b><i>Background/Aims:</i></b> Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. <b><i>Methods:</i></b> We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. <b><i>Results:</i></b> Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. <b><i>Conclusion:</i></b> DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18.</p