A synonymous germline variant in a gene encoding a cell adhesion molecule is associated with cutaneous mast cell tumour development in Labrador and Golden Retrievers

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer. Author summary The combination of various genetic and environmental risk factors makes the understanding of the molecular circuitry behind complex diseases, like cancer, a major challenge. The homogeneous nature of pedigree dog breed genomes makes these dogs ideal for the identification of both simple disease-causing genetic variants and genetic risk factors for complex diseases. Mast cell tumours are the most common type of canine skin cancer, and one of the most common cancers affecting dogs of most breeds. Several breeds, including Labrador Retrievers (which represent one of the most popular dog breeds), have an elevated risk of mast cell tumour development. Here, by using a methodological approach that combined different techniques, we identified a common inherited synonymous variant, that predisposes Labrador Retrievers to mast cell tumour development. Interestingly, we showed that this variant, despite its synonymous nature, appears to have an effect on translation dynamics as it is associated with reduced levels of DSCAM, a cell adhesion molecule. The results presented here reveal dysregulation of cell adhesion to be an important factor in mast cell tumour pathogenesis, and also highlight the important role that synonymous variants can play in complex diseases

Identification of molecular genetic contributants to canine cutaneous mast cell tumour metastasis by global gene expression analysis

<div><p>Cutaneous mast cell tumours are one of the most common canine cancers. Approximately 25% of the tumours metastasise. Activating <i>c-kit</i> mutations are present in about 20% of tumours, but metastases occur in the absence of mutations. Tumour metastasis is associated with significantly diminished survival in spite of adjuvant chemotherapy. Available prognostic tests do not reliably predict whether a tumour will metastasise. In this study we compared the global expression profiles of 20 primary cutaneous mast cell tumours that metastasised with those of 20 primary tumours that did not metastasise. The objective was to identify genes associated with mast cell tumour metastatic progression that may represent targets for therapeutic intervention and biomarkers for prediction of tumour metastasis. Canine Gene 1.1 ST Arrays were employed for genome-wide expression analysis of formalin-fixed, paraffin-embedded biopsies of mast cell tumours borne by dogs that either died due to confirmed mast cell tumour metastasis, or were still alive more than 1000 days post-surgery. Decreased gene expression in the metastasising tumours appears to be associated with a loss of cell polarity, reduced cell-cell and cell-ECM adhesion, and increased cell deformability and motility. Dysregulated gene expression may also promote extracellular matrix and base membrane degradation, suppression of cell cycle arrest and apoptosis, and angiogenesis. Down-regulation of gene expression in the metastasising tumours may be achieved at least in part by small nucleolar RNA-derived RNA and microRNA-effected gene silencing. Employing cross-validation, a linear discriminant analysis-based classifier featuring 19 genes that displayed two-fold differences in expression between metastasising and non-metastasising tumours was estimated to classify metastasising and non-metastasising tumours with accuracies of 90–100% and 70–100%, respectively. The differential expression of 9 of the discriminator genes was confirmed by quantitative reverse transcription-PCR.</p></div

The Complex Link between Apoptosis and Autophagy: a Promising New Role for RB

ABSTRACT Physiological processes, as autophagy, proliferation and apoptosis are affected during carcinogenesis. Restoring cellular sensitivity to apoptotic stimuli, such as the antineoplastic cocktails, has been explored as a strategy to eliminate cancer cells. Autophagy, a physiological process of recycling organelles and macromolecules can be deviated from homeostasis to support cancer cells survival, proliferation, escape from apoptosis, and therapy resistance. The relationship between autophagy and apoptosis is complex and many stimuli can induce both processes. Most chemotherapeutic agents induce autophagy and it is not clear whether and how this chemotherapy-induced autophagy might contribute to resistance to apoptosis. Here, we review current strategies to sensitize cancer cells by interfering with autophagy. Moreover, we discuss a new link between autophagy and apoptosis: the tumor suppressor retinoblastoma protein (RB). Inactivation of RB is one of the earliest and more frequent hallmarks of cancer transformation, known to control cell cycle progression and apoptosis. Therefore, understanding RB functions in controlling cell fate is essential for an effective translation of RB status in cancer samples to the clinical outcome

Immunohistochemical analysis of retinoblastoma and β-catenin as an assistant tool in the differential diagnosis between Crohn's disease and ulcerative colitis.

In about 10-15% of patients with inflammatory bowel diseases (IBD) there is no clear definitive differential diagnosis between Crohn's disease (CD) and ulcerative colitis (UC) and the disease is classified as indeterminate colitis. Since pharmacological and surgical treatments differ in CD and UC, establishing a correct diagnosis is critical. The aim of this work was to access the expression profile of proteins involved in colonic inflammation and cancer in samples from CD and UC. For that, colon samples from 24 CD, 21 UC and 10 control patients were processed for immunohistochemistry using anti-phosphorylated RB at Ser(807/811) and anti-β-catenin. Crypts were blinded, analyzed and counted for phosphorylated RB-positive (phospho-RB) cells or scored for positive β-catenin staining. Western blot was used for confirming immuhistochemical results: RB phosphorylation was significantly greater in colon samples from patients with CD compared with UC (p<0.005). In contrast, the expression of β-catenin was significantly increased in UC compared with CD (p<0.005) samples. Phospho-RB and β-catenin are negatively correlated (CC: -0.573; p = 0.001). A positive phospho-RB test yielded high levels of sensitivity, specificity, negative and positive predictive values, and accuracy for the diagnosis of CD against UC. This work indicates that RB phosphorylation and β-catenin nuclear translocation are differently expressed in CD and UC, and provide novel insights into the pathogenic mechanisms of IBD. In particular, rates of phospho-RB-positive cells in mucosal samples emerge as a promising tool for the differential diagnosis of patients with IBD

Diagnostic capability of phospho-RB and β-catenin immunhistochemical tests in differentiating Crohn's disease from ulcerative colitis.

<p>All values are presented as percentages. Cut-off values were determined by ROC curves, establishing as positive tests: phospho-RB-positive cells ≥24%; and β-catenin score ≤1.4. The positive endpoint was arbitrarily considered for the diagnosis of Crohn's disease. NPV, negative predictive value; PPV, positive predictive value.</p

Immunofluorescence staining for phosphorylated RB is increased in colon biopsies from CD patients.

<p>A. RB phosphorylation was detected in colon biopsies from control (non inflamed; n = 5), CD (n = 7) and UC patients (n = 6) by immunofluorescence staining using phosphorylated RB Ser^807/811 and phosphorylated RB Thr^821/826 antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 50 µm. B. Histogram showing RB fluorescence staining intensity in Ser^807/811 (white bars) and Thr^821/826 (black bars) residues. *p<0.05 when compared with percentage of phosphorylated RB Ser^807/811-positive cells in UC samples and ^##p<0.01 when compared with percentage of phosphorylated RB Thr^821/826 -positive cells in UC samples.</p

β-catenin is overexpressed in colonic samples from UC patients.

<p>A. Colonic biopsies from control (non inflamed), CD and UC patients were stained by immunohistochemistry using anti-β-catenin antibody. Slides were counterstained with methyl green. Scale bar = 50 µm. B. Representative pictures of β-catenin staining scores (0 to 3). C. Graph shows the mean scores of β-catenin expression intensity in control (n = 5), CD (n = 14) and UC (n = 14) samples. *p = 0.019 when compared with β-catenin expression scores in CD samples.</p

Phospho-RB and β-catenin staining pattern helps the differential diagnose of IBDU cases.

<p>A. Low phospho-RB ser^807/811and high β-catenin staining in a 50-year old woman patient lately diagnosed with UC (Patient A). B. High phospho-RB ser^807/811and low β-catenin staining in a 28-year old woman lately diagnosed with CD (Patient B). Slides were counterstained with methyl green. Scale bar = 50 µm.</p