Penny L. Jeffery

An intestinal epithelial defect conferring ER stress results in inflammation involving both innate and adaptive immunity

We recently characterized Winnie mice carrying a missense mutation in Muc2, leading to severe endoplasmic reticulum stress in intestinal goblet cells and spontaneous colitis. In this study, we characterized the immune responses due to this intestinal epithelial dysfunction. In Winnie, there was a fourfold increase in activated dendritic cells (DCs; CD11c+ major histocompatibility complex (MHC) class IIhi) in the colonic lamina propria accompanied by decreased colonic secretion of an inhibitor of DC activation, thymic stromal lymphopoietin (TSLP). Winnie also displayed a significant increase in mRNA expression of the mucosal TH17 signature genes Il17a, IL17f, Tgfb, and Ccr6, particularly in the distal colon. Winnie mesenteric lymph node leukocytes secreted multiple TH1, TH2, and TH17 cytokines on activation, with a large increase in interleukin-17A (IL-17A) progressively with age. A major source of mucosal IL-17A in Winnie was CD4+ T lymphocytes. Loss of T and B lymphocytes in Rag1-/- × Winnie (RaW) crosses did not prevent spontaneous inflammation but did prevent progression with age in the colon but not the cecum. Adoptive transfer of naive T cells into RaW mice caused more rapid and severe colitis than in Rag1-/-, indicating that the epithelial defect results in an intestinal microenvironment conducive to T-cell activation. Thus, the Winnie primary epithelial defect results in complex multicytokine-mediated colitis involving both innate and adaptive immune components with a prominent IL-23/TH17 response, similar to that of human ulcerative colitis.

IL-10 promotes production of intestinal mucus by suppressing protein misfolding and endoplasmic reticulum stress in goblet cells

BACKGROUND & AIMS Protein misfolding and endoplasmic reticulum (ER) stress have been observed in intestinal secretory cells from patients with inflammatory bowel diseases and induce intestinal inflammation in mice. However, it is not clear how immune factors affect ER stress and therefore disease symptoms. METHODS We analyzed the effects of interleukin (IL)-10 on ER stress in intestinal tissues in wild-type C57BL/6, Winnie, IL-10(-/-), and Winnie × IL-10(+/-) mice. In Winnie mice, misfolding of the intestinal mucin Muc2 initiates ER stress and inflammation. We also analyzed the effects of different inhibitors of IL-10 signaling and the N-glycosylation inhibitor tunicamycin in cultured human LS174T goblet cells. RESULTS Administration of neutralizing antibodies against IL-10 or its receptor (IL-10R1) to Winnie mice rapidly exacerbated ER stress and intestinal inflammation compared with mice given vehicle (controls). Antibodies against IL-10 also increased accumulation of misfolded Muc2 in the ER of goblet cells of Winnie mice and increased T-cell production of inflammatory cytokines. Winnie × IL-10(+/-) mice and IL-10(-/-) mice with a single Winnie allele each developed more severe inflammation than Winnie mice or IL-10(-/-) mice. Administration of tunicamycin to wild-type mice caused intestinal ER stress, which increased when IL-10R1 was blocked. In LS174T cells, induction of ER stress with tunicamycin and misfolding of MUC2 were reduced by administration of IL-10; this reduction required STAT1 and STAT3. In LS174T cells incubated with tunicamycin, IL-10 up-regulated genes involved in MUC2 folding and in ER-associated degradation and maintained correct folding of MUC2, its transport from the ER, and its O-glycosylation and secretion. CONCLUSIONS IL-10 prevents protein misfolding and ER stress by maintaining mucin production in goblet cells and helps the intestine preserve the mucus barrier.

The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis

Background and Aims The MUC13 transmembrane mucin is highly and constitutively expressed in the small and large intestine. Although MUC13 polymorphisms have been associated with human inflammatory bowel diseases and susceptibility to Escherichia coli infection in pigs, the biological functions of MUC13 are unknown. This study aimed to explore whether MUC13 modulates intestinal inflammation. Methods Muc13−/− mice were generated, phenotyped and challenged with the colitis-inducing agent, dextran sodium sulphate (DSS). Colitis was assessed by clinical symptoms and intestinal histopathology. Intestinal epithelial cell apoptosis and proliferation, macrophage infiltration and cytokine production were also quantified. Apoptosis of human LS513 intestinal epithelial cells in response to apoptotic agents, including DSS, was also measured, following knockdown of MUC13 with siRNA. Results Muc13−/− mice were viable, fertile and developed normally, with no spontaneous intestinal pathology except mild focal neutrophilic inflammation in the small and large intestines of old mice. In response to DSS challenge, Muc13−/− mice developed more severe acute colitis, as reflected by increased weight loss, rectal bleeding, diarrhoea and histological colitis scores compared with wild-type mice. Increased numbers of F4/80+ macrophages in inflamed mucosa of Muc13−/− mice were accompanied by increased expression of intestinal IL-1β and TNFα mRNA. Muc13−/− mice had significantly increased intestinal epithelial cell apoptosis within 3 days of DSS exposure. LS513 cells were more susceptible to DSS, actinomycin-D, ultraviolet irradiation and TRAIL-induced apoptosis when MUC13 was knocked down by siRNA. Conclusions These novel findings indicate a protective role for Muc13 in the colonic epithelium by inhibiting toxin-induced apoptosis and have important implications for intestinal infections, inflammatory diseases and the development of intestinal cancer.

Ghrelin in gastrointestinal disease

Enteroendocrine cells of the gastric fundus are the predominant source of ghrelin production, although ghrelin gene transcripts and ghrelin-producing cells have been identified throughout the gastrointestinal tract. Various infectious, inflammatory and malignant disorders of the gastrointestinal system have been shown to alter ghrelin production and secretion and consequently to affect endocrine ghrelin levels and activity. Animal studies have demonstrated that ghrelin and synthetic ghrelin mimetics can reduce the severity of gastric and colonic inflammation and human clinical trials are underway to determine the efficacy of ghrelin in improving motility disorders. This review summarises the impact of gastrointestinal disease on ghrelin synthesis and secretion and the potential use of ghrelin and its mimetics for the treatment of these diseases.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

MUC13 overexpression in renal cell carcinoma plays a central role in tumor progression and drug resistance

Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti‐apoptotic proteins, BCL‐xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi‐kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.

Comparative analysis reveals loss of the appetite-regulating peptide hormone ghrelin in falcons.

Ghrelin and leptin are key peripherally secreted appetite-regulating hormones in vertebrates. Here we consider the ghrelin gene (GHRL) of birds (class Aves), where it has been reported that ghrelin inhibits rather than augments feeding. Thirty-one bird species were compared, revealing that most species harbour a functional copy of GHRL and the coding region for its derived peptides ghrelin and obestatin. We provide evidence for loss of GHRL in saker and peregrine falcons, and this is likely to result from the insertion of an ERVK retrotransposon in intron 0. We hypothesise that the loss of anorexigenic ghrelin is a predatory adaptation that results in increased food-seeking behaviour and feeding in falcons.

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1−/−) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1−/−and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1−/− mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 ± 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 ± 0.12 ng/ml and 2.91 ± 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1−/− HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1−/− HFD-fed mice, but not in NOD/SCID mice. In Rag1−/− HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of long-term Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1−/− mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia.

Gene expression profiling of The Cancer Genome Atlas supports an inverse association between body mass index (BMI) and major oesophageal tumour subtypes

In the last decade the Cancer Genome Atlas (TCGA) program has revealed significant insights into molecular events of dozens of cancers. These data sets are continuously updated, providing an unprecedented resource to the research community. There is now an emerging link between obesity and the development and progression of cancer. In this study we wished to identify genes related to body mass index (BMI) in TCGA datasets. Supporting epidemiological data, our gene expression profiling analyses suggest that oesophageal adenocarcinoma (EAC) can be considered a true obesity-associated cancer subtype, presenting avenues for prevention and treatment.

Abstract 3491: A role for the long non-coding RNAGHRLOSin cancer

Long non-coding RNA (lncRNA) genes are abundant in the human genome, and many are now recognized as oncogenes and/or tumor suppressors. We previously characterized the structure of GHRLOS, an antisense gene on the opposite strand of the multifunctional ghrelin gene (GHRL), however, its expression and function in disease has not been described. To this end, using The Cancer Genome Atlas (TCGA) data set, we revealed that GHRLOS is differentially expressed in a number of cancers. In particular, expression was elevated in endometrial cancer (1.2 - fold; P = 7.1 x 10 -3 Welch9s two-sample t-test; n = 24 vs. n = 175) and prostate cancer (1.2-fold; P = 3.7 x 10 -6 ; n = 52 vs. n = 498) compared to normal tissues. Using qRT-PCR (and commercial cDNA panels) we confirmed that GHRLOS expression was upregulated in endometrial cancer (1.96-fold, P = 0.005 Welch’s two-sample t-test, n = 5 vs. n = 17) and prostate cancer (2.46-fold, P = 0.0045 Welch’s two-sample t-test, n = 5 vs. n = 21) compared to normal tissues. Initial studies, using siRNA designed to silence endogenous GHRLOS expression, significantly reduced cell migration in the PC3 prostate cancer cell line (0.47-fold change, P = 0.042 Kruskal-Wallis test, n = 2) conversely, preliminary data using forced GHRLOS overexpression increased migration and proliferation. Taken together, we show that the long non-coding RNA GHRLOS is differentially expressed in tumor tissue and regulates cell migration and proliferation; possibly by modulating alternative splicing of the overlapping, multifunctional ghrelin gene locus. Targeting GHRLOS could provide a valuable and novel way to target the ghrelin axis in disease. Ongoing studies aim to validate in vitro functional results in complementary mouse xenograft models and identify genes and pathways regulated by this novel lncRNA. Citation Format: Esha T. Shah, Penny Jeffery, Patrick Thomas, Inge Seim, Lisa Chopin. A role for the long non-coding RNA GHRLOS in cancer [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 3491. doi:10.1158/1538-7445.AM2017-3491