Background: N6-methyladenosine (m⁶A) is the most common internal RNA modification and is involved in regulation of RNA and protein expression. AlkB family member 5 (ALKBH5) is a m⁶A demethylase. Given the important role of m⁶A in biological mechanisms, m⁶A and its regulators, have been implicated in many disease processes, including cancer. However, the contribution of ALKBH5 to invasive breast cancer (BC) remains poorly understood. The aim of this study was to evaluate the clinicopathological value of ALKBH5 in BC. Methods: Publicly available data were used to investigate ALKBH5 mRNA alterations, prognostic significance, and association with clinical parameters at the genomic and transcriptomic level. Differentially expressed genes (DEGs) and enriched pathways with low or high ALKBH5 expression were investigated. Immunohistochemistry (IHC) was used to assess ALKBH5 protein expression in a large well-characterised BC series (n = 1327) to determine the clinical significance and association of ALKBH5 expression. Results: Reduced ALKBH5 mRNA expression was significantly associated with poor prognosis and unfavourable clinical parameters. ALKBH5 gene harboured few mutations and/or copy number alternations, but low ALKBH5 mRNA expression was seen. Patients with low ALKBH5 mRNA expression had a number of differentially expressed genes and enriched pathways, including the cytokine-cytokine receptor interaction pathway. Low ALKBH5 protein expression was significantly associated with unfavourable clinical parameters associated with tumour progression including larger tumour size and worse Nottingham Prognostic Index group. Conclusion: This study implicates ALKBH5 in BC and highlights the need for further functional studies to decipher the role of ALKBH5 and RNA m⁶A methylation in BC progression.

Osteosarcoma (OSA) is the most common type of primary bone malignancy in people and dogs. Our previous molecular comparisons of canine OSA against healthy bone resulted in the identification of differentially expressed protein-expressing genes (forkhead box protein O4 (FOXO4), interferon regulatory factor 8 (IRF8), and lymphoid enhancer binding factor 1 (LEF1)). Immunohistochemistry (IHC) and H-scoring provided semi-quantitative assessment of nuclear and cytoplasmic staining alongside qualitative data to contextualise staining (n = 26 patients). FOXO4 was expressed predominantly in the cytoplasm with significantly lower nuclear H-scores. IRF8 H-scores ranged from 0 to 3 throughout the cohort in the nucleus and cytoplasm. LEF1 was expressed in all patients with significantly lower cytoplasmic staining compared to nuclear. No sex or anatomical location differences were observed. While reduced levels of FOXO4 might indicate malignancy, the weak or absent protein expression limits its primary use as diagnostic tumour marker. IRF8 and LEF1 have more potential for prognostic and diagnostic uses and facilitate further understanding of their roles within their respective molecular pathways, including Wnt/beta-catenin/LEF1 signalling and differential regulation of tumour suppressor genes. Deeper understanding of the mechanisms involved in OSA are essential contributions towards the development of novel diagnostic, prognostic, and treatment options in human and veterinary medicine contexts.

Background: Osteosarcoma (OSA) is the most common type of bone cancer in canines. Novel therapies are required to prevent the growth, survival, and metastatic progression of this cancer, to increase life expectancy of patients. Immunohistochemical (IHC) studies and RNA sequencing help us gain a deeper understanding into the molecular mechanisms of the disease. Methods: We previously compared canine OSA tissues with patient matched non-tumour tissues, revealing 442 overexpressed genes within the samples. The present research used IHC staining for four of these genes in OSA tissues: G protein-coupled receptor 64 (GPR64), TOX High Mobility Group Box Family Member 3 (TOX3), Matrix Metallopeptidase 12 (MMP-12), and Forkhead Box F1 (FOXF1). H-scoring was performed to quantitatively assess protein expression and qualitatively contextualise staining locations. Additional analyses addressed whether gender or anatomical location of lesions (axial or appendicular tumours) affected protein expression. cBioPortal was employed to analyse expression and genetic alterations in patients. Results: GPR64, TOX3, MMP-12, and FOXF1 showed high mRNA expression and genetic alterations in people with OSA. GPR64, TOX3, MMP-12, and FOXF1 were all expressed in canine OSA with novel findings regarding cellular expression. Additionally, differential sex expression was revealed for GPR64 and TOX3. Potential biomarkers or therapeutic targets were identified. Conclusions: These studies, and subsequent analysis, have provided insights into the molecular mechanisms associated with OSA progression and revealed potential biomarkers for diagnostic and prognostic purposes. A deeper understanding of genetic and protein interactions will support and progress novel pathways towards diagnostic, prognostic, and treatment interventions for OSA in both veterinary and human medicine.

Osteosarcoma (OSA) is the most prevalent bone malignancy in people and dogs. Current survival rates show the need for advances in novel therapies to help overcome the growth, survival and metastatic progression of the cancer. Canine models are often used to advance prognostic and treatment opportunities for OSA due to the similarities in the disease between species. This study focusses on the genetic and molecular similarities of OSA between human and canine specimens. Differentially expressed genes (DEGs) were compared and identified in canine and human OSA tumours, revealing 86 common genes, 36 having high and 50 having low expression. Further immunohistochemical analysis of the corresponding proteins of three identified DEGs (ASPN, STK3, BAMBI) allowed for the visualisation of protein expression in canine OSA tissues (n = 19). Overall nuclear and cytoplasmic H-scores were generated, and nuclear and cytoplasmic scores in males and females and in different anatomical locations (axial versus appendicular) were also investigated, presenting unique opportunities to understand the expression in this cancer type. This study contributes to a deeper knowledge of genetic pathways changes and identifies avenues for the diagnosis, prognosis and treatment of OSA in people and dogs, whilst encompassing the One Health concept in medicine.

Breast cancer (BCa) is one of the most commonly diagnosed malignancies worldwide and is clinically heterogenous. BCa is classified into distinct histopathological and molecular subtypes that inform diagnosis, treatment, and prognosis. Although therapeutic advances, particularly targeted therapies, have improved outcomes, metastatic BCa remains an unmet clinical need. In addition, treatment options remain limited especially for triple negative BCa (TNBC). There is an urgent need to develop novel approaches that can prevent, delay, or reverse disease progression in these patients. The roles of genetic and epigenetic alterations in BCa are well established. Emerging evidence highlights the dysregulation of epitranscriptomic mechanisms involving covalent RNA modifications as a contributing factor in BCa pathogenesis. Notably, recent evidence supports functional crosstalk between epigenetic and epitranscriptomic processes with potential clinical and therapeutic relevance. This review explores key epitranscriptomic RNA modifications, m⁶A, m⁶A_m, m⁵C, m⁷G, and m¹A in the context of BCa. The functional consequences of the epitranscriptomic regulators ("writers," "erasers", and "readers") are discussed alongside the accumulating evidence of their contribution to cancer development and progression. This review considers how RNA modifications and their regulators might serve as biomarkers or therapeutic targets and offers new directions for translational research and clinical intervention in BCa.

Osteosarcoma (OSA) is a metastatic bone tumour. In dogs, it predominantly affects older animals and large and giant breeds. The cancer has poor survival rates due to most patients developing dissemination to secondary sites prior to an initial diagnosis. Therefore, novel therapeutic approaches are required to help overcome the growth and metastatic progression of the disease and increase survival rates. Immunohistochemical staining and histological scoring (H-scoring) are often used for diagnostic and prognostic purposes in human medicine. Unfortunately, these techniques have not yet been fully established and implemented within the veterinary field, but ongoing investigations and developments of potential biomarkers are expanding. Four proteins (ELOVL2, LIPG, RPL4, and RPSA) whose genes showed higher expression in canine OSA underwent immunohistochemical investigations. Immunohistochemistry (IHC) and consequent H-score analysis were performed to investigate nuclear and/or cytoplasmic protein expression encoded by the four canine overexpressed genes (ELOVL2 (n = 22), LIPG (n = 18), RPL4 (n = 18), and RPSA (n = 19)). Statistical analyses of the nuclear/cytoplasmic staining scores of the whole cohort, as well as sex (male versus female) and anatomical location (axial and soft tissue versus appendicular) differences, were conducted. This study revealed novel findings regarding the subcellular expression of the proteins corresponding to the overexpressed genes of interest (ELOVL2, LIPG, RPL4, and RPSA) in Rottweiler OSA, including the identification of significant sex-associated differences for LIPG nuclear staining. These candidate biomarkers could be utilised in future investigations to validate their potential as diagnostic, prognostic, and treatment avenues for canine OSA.