Chronic Interferon Stimulated Gene Transcription Promotes Oncogene Induced Breast Cancer.

The Mre11 complex (comprising Mre11, Rad50, Nbs1) is integral to the maintenance of genome stability. We previously showed that a hypomorphic Mre11 mutant mouse strain ( Mre11 ATLD1/ATLD1 ) was highly susceptible to oncogene induced breast cancer. Here we used a mammary organoid system to examine which Mre11 dependent responses are tumor suppressive. We found that Mre11 ATLD1/ATLD1 organoids exhibited an elevated interferon stimulated gene (ISG) signature and sustained changes in chromatin accessibility. This Mre11 ATLD1/ATLD1 phenotype depended on DNA binding of a nuclear innate immune sensor, IFI205. Ablation of Ifi205 in Mre11 ATLD1/ATLD1 organoids restored baseline and oncogene-induced chromatin accessibility patterns to those observed in WT . Implantation of Mre11 ATLD1/ATLD1 organoids and activation of oncogene led to aggressive metastatic breast cancer. This outcome was reversed in implanted Ifi205 -/- Mre11 ATLD1/ATLD1 organoids. These data reveal a connection between innate immune signaling and tumor suppression in mammary epithelium. Given the abundance of aberrant DNA structures that arise in the context of genome instability syndromes, the data further suggest that cancer predisposition in those contexts may be partially attributable to tonic innate immune transcriptional programs.

RTEL1 influences the abundance and localization of TERRA RNA.

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.

Epithelial-derived IL-33 promotes intestinal tumorigenesis in Apc Min/+ mice.

Increased expression of Interleukin (IL)-33 has been detected in intestinal samples of patients with ulcerative colitis, a condition associated with increased risk for colon cancer, but its role in the development of colorectal cancer has yet to be fully examined. Here, we investigated the role of epithelial expressed IL-33 during development of intestinal tumors. IL-33 expression was detected in epithelial cells in colorectal cancer specimens and in the Apc Min/+ mice. To better understand the role of epithelial-derived IL-33 in the intestinal tumorigenesis, we generated transgenic mice expressing IL-33 in intestinal epithelial cells (V33 mice). V33 Apc Min/+ mice, resulting from the cross of V33 with Apc Min/+ mice, had increased intestinal tumor burden compared with littermate Apc Min/+ mice. Consistently, Apc Min/+ mice deficient for IL-33 receptor (ST2), had reduced polyp burden. Mechanistically, overexpression of IL-33 promoted expansion of ST2+ regulatory T cells, increased Th2 cytokine milieu, and induced alternatively activated macrophages in the gut. IL-33 promoted marked changes in the expression of antimicrobial peptides, and antibiotic treatment of V33 Apc Min/+ mice abrogated the tumor promoting-effects of IL-33 in the colon. In conclusion, elevated IL-33 signaling increases tumor development in the Apc Min/+ mice.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. METHODS: Hepatocellular carcinoma classification defined in our previous transcriptome meta-analysis (S1, S2 and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumours (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumours (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. RESULTS: Hepatocellular carcinoma subclass-predictive indices were steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-foetoprotein (>400 ng/ml) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with the activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184 and 53 to 30, 43 and 22 for S1, S2 and S3 subclass-targeting therapies respectively. CONCLUSIONS: Hepatocellular carcinoma molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration.

OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100,000/mm(3)), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

Transcriptome profiling of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissue for disease classification.

BACKGROUND: Archived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis. METHODS: Genome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks. RESULTS: RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles. CONCLUSIONS: AS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.

CCR7 deficiency in NOD mice leads to thyroiditis and primary hypothyroidism.

CCR7 is involved in the initiation of immune responses and has been recently implicated in the control of tolerance. To analyze the role of CCR7 in autoimmunity, we backcrossed CCR7(ko/ko) mice (in which ko signifies deficient) onto the autoimmune-prone NOD background. Surprisingly, NODCCR7(ko/ko) mice never developed diabetes, but showed severe inflammation in multiple tissues including thyroid, lung, stomach, intestine, uterus, and testis. NODCCR7(ko/ko) mice had a marked enlargement of the thyroid gland (goiter) that was associated with circulating autoantibodies against thyroglobulin, and development of primary hypothyroidism (decreased levels of serum thyroxin, and augmented levels of thyroid-stimulating hormone in the pituitary gland), features found in Hashimoto's thyroiditis. Cells isolated from diseased thyroids and activated splenocytes from NODCCR7(ko/ko) animals induced goiter in NOD.SCID recipients, demonstrating that autoreactive cells were generated in the absence of CCR7. Moreover, thyroid disease could be accelerated in young NODCCR7(ko/ko) mice by immunization with thyroglobulin. These results demonstrate the complexity in the generation of multiple autoimmune phenotypes in NOD mice and indicate that CCR7 is a key molecule in their development.

Islet expression of M3 uncovers a key role for chemokines in the development and recruitment of diabetogenic cells in NOD mice.

OBJECTIVE: Type 1 diabetes is an autoimmune disease characterized by a local inflammatory reaction in and around islets followed by selective destruction of insulin-secreting beta-cells. We tested the hypothesis that chemokines affect different mechanisms responsible for the development of diabetes in NOD mice. RESEARCH DESIGN AND METHODS: We examined chemokine expression in islets of NOD mice and tested their functional relevance to development of diabetes using transgenic mice expressing the mouse herpesvirus 68-encoded chemokine decoy receptor M3 (NOD-M3 mice) in insulin-secreting beta-cells. RESULTS: Multiple chemokines were expressed in pancreatic islets of NOD mice before development of diabetes. Islet-specific expression of the pan-chemokine inhibitor M3 dramatically reduced leukocyte infiltration and islet destruction and completely blocked development of diabetes in NOD-M3 mice. M3 blocked diabetes by inhibiting the priming of diabetogenic cells in the pancreatic lymph nodes and their recruitment into the islets. This effect was specific to the pancreatic islets because M3 expression did not affect other ongoing autoimmune processes. CONCLUSIONS: These results demonstrate that chemokines mediate afferent and efferent immunity in type 1 diabetes and suggest that broad chemokine blockade may represent a viable strategy to prevent insulitis and islet destruction.

The chemokine binding protein M3 prevents diabetes induced by multiple low doses of streptozotocin.

Multiple injections of low-dose streptozotocin (MLDS) induce lymphocytic insulitis and diabetes in rodents. To test whether the influx of inflammatory cells was associated with changes in the expression of chemokines, we measured the expression of all known chemokine ligands by real-time quantitative PCR in isolated islets. With the exception of CCL20 and CCL19, chemokines were not significantly expressed in islets from wild-type mice before MLDS treatment. Ten days after treatment, the expression of several chemokines, including CXCL9, CCL1, CXCL10, and CCL21, was dramatically up-regulated. The expression of CCL1, CXCL9, and CCL21 protein was confirmed by immunohistochemistry and was mostly associated with the infiltrating cells. The mouse herpesvirus 68-encoded chemokine decoy receptor M3 can broadly engage these chemokines with high affinity. To test whether a blockade of chemokine function would alter the onset or magnitude of insulitis and diabetes, we used transgenic mice expressing M3 in beta cells (rat insulin promoter (RIP)-M3 mice). RIP-M3 mice were normoglycemic and responded normally to glucose challenge but were remarkably resistant to diabetes induced by MLDS. Islets from MLDS-treated RIP-M3 mice had fewer inflammatory cells and expressed lower levels of chemokines than those from MLDS-treated controls. The role of M3 in chemokine blockade during insulitis was further supported by in vitro experiments demonstrating that multiple chemokines up-regulated during islet inflammation are high-affinity M3 ligands that can be simultaneously sequestered. These results implicate chemokines as key mediators of insulitis and suggest that their blockade may represent a novel strategy to prevent insulitis and islet destruction.

The chemokine decoy receptor M3 blocks CC chemokine ligand 2 and CXC chemokine ligand 13 function in vivo.

Chemokines and their receptors play a key role in immune homeostasis regulating leukocyte migration, differentiation, and function. Viruses have acquired and optimized molecules that interact with the chemokine system. These virus-encoded molecules promote cell entry, facilitate dissemination of infected cells, and enable the virus to evade the immune response. One such molecule in the murine gammaherpesvirus 68 genome is the M3 gene, which encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and blocks chemokine signaling in vitro. To test the hypothesis that M3 directly interferes with diverse chemokines in vivo, we examined the interaction of M3 with CCL2 and CXCL13 expressed in the pancreas of transgenic mice. CCL2 expression in the pancreas promoted recruitment of monocytes and dendritic cells; CXCL13 promoted recruitment of B and T lymphocytes. Coexpression of M3 in the pancreas blocked cellular recruitment induced by both CCL2 and CXCL13. These results define M3 as multichemokine blocker and demonstrate its use as a powerful tool to analyze chemokine biology.

Cutting edge: the silent chemokine receptor D6 is required for generating T cell responses that mediate experimental autoimmune encephalomyelitis.

D6, a promiscuous nonsignaling chemokine binding molecule expressed on the lymphatic endothelium, internalizes and degrades CC chemokines, and D6(-/-) mice demonstrated increased cutaneous inflammation following topical phorbol ester or CFA injection. We report that D6(-/-) mice were unexpectedly resistant to the induction of experimental autoimmune encephalomyelitis due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in CFA, D6(-/-) mice showed reduced spinal cord inflammation and demyelination with lower incidence and severity of experimental autoimmune encephalomyelitis attacks as compared with D6(+/+) littermates. In adoptive transfer studies, MOG-primed D6(+/-) T cells equally mediated disease in D6(+/+) or D6(-/-) mice, whereas cells from D6(-/-) mice transferred disease poorly to D6(+/-) recipients. Lymph node cells from MOG-primed D6(-/-) mice showed weak proliferative responses and made reduced IFN-gamma but normal IL-5. CD11c(+) dendritic cells accumulated abnormally in cutaneous immunization sites of D6(-/-) mice. Surprisingly, D6, a "silent" chemokine receptor, supports immune response generation.

A novel model for lymphocytic infiltration of the thyroid gland generated by transgenic expression of the CC chemokine CCL21.

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-alpha but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid.