Background: Osteosarcoma (OS) is a highly aggressive bone malignancy with limited treatment options and poor prognosis. This exploratory study aimed to identify molecular subtypes of early-stage, treatment-naive OS to guide precise therapeutic strategies. Methods: We analyzed RNA-seq data obtained from tumor tissues from 102 OS patients using a non-negative matrix factorization algorithm (NMF) to classify the tumors into three subtypes: S1, S2, and S3. Differential gene expression was evaluated using DESeq2, followed by functional enrichment analysis with clusterProfiler and CancerHallmarks. The tumor microenvironment was assessed through ESTIMATE and CIBERSORT, and drug sensitivity was predicted using OncoPredict. SAOS-2 and MG63 cells, representing the S1 subtype, were used in the viability essays to determine the effect of hesperidin, a natural phenolic compound noted for its anti-cancer potential, alone and in combination with doxorubicin and 5-fluorouracil. Results: This study revealed three OS subtypes: S1 was enriched in cell cycle regulation, vesicular transport, and RNA metabolism while S2 and S3 were enriched in pathways related to extracellular matrix organization and protein translation, respectively. S1 displayed high tumor purity, significant chemoresistance, and overexpression of KIF20 A, correlating with poor prognosis. AURKB, a hesperidin target, was implicated in S1 pathogenesis. In vitro, hesperidin significantly reduced the viability of SAOS-2 and MG63 cells and enhanced doxorubicin efficacy. Conclusions: Our findings support the molecular subclassification of OS, emphasizing subtype-specific mechanisms of tumor progression and chemoresistance, with hesperidin offering potential as a therapeutic adjunct for high-risk OS patients.

In contrast to humans, zebrafish have an outstanding ability to regenerate injured heart through a highly orchestrated process involving all cardiac structures. To replace the lost myocardium, resident cardiomyocytes (CMs) dedifferentiate and proliferate, invading the injured area. The response of the myocardium is preceded by the activation of the epicardium and endocardium, which form active scaffolds to provide mechanical and paracrine support to guide regeneration. New CMs use protrusions to migrate and invade fibrotic injured tissue, replacing it with functional myocardium. Here, we investigated the expression profile of the stress-responsive ankrd1a gene in different cardiac structures, at key time points during regeneration, aiming to gain insight into its precise roles during zebrafish heart regeneration. In the TgBAC(ankrd1a:EGFP) reporter line, transgene upregulation was restricted to the myocardium, initiated as early as 15 h post-cryoinjury, and consistently marked CMs bordering the injury or scar area during regeneration. Transcriptome profiling and immunostaining revealed a potential role of ankrd1a in regulating CMs' dedifferentiation, as well as changes in the expression of genes associated with antigen presentation and extracellular matrix composition in the ankrd1a mutant. Our results indicate that ankrd1a is dispensable for ventricular regeneration after cryoinjury, while it may serve as a marker and fine-tuner in the healing process of injured cardiac muscle.

Background: Osteosarcoma (OS) is a highly aggressive primary bone malignancy, for which treatment and prognosis remain unchanged for three decades. This study aimed to delineate molecular subtypes of early-stage, treatment-naive OS to improve molecular stratification and guide precise therapeutic strategies. Methods: The top 10% of the most variable genes of 102 OS tumour tissues were used in NMF to classify the tumours into three subtypes, S1, S2, and S3. Differential expression analysis was carried out using DESeq2 and functional enrichment analysis using clusterProfiler and CancerHallmarks. Tumor microenvironment composition was estimated through ESTIMATE and CIBERSORT, and drug sensitivity prediction using OncoPredict. NMF was also applied to RNA-seq data from 19 osteosarcoma cell lines and DEGs between the groups were correlated with the DEGs from patients' subtypes. SAOS-2, representing S1, was used in the viability assays to determine the effects of hesperidin, alone or in combination with doxorubicin and 5-fluorouracil. Results: S1 was enriched in cell cycle regulation, vesicular transport, and RNA metabolism with high tumor purity and high resistance to chemotherapeutics. S2 and S3 were enriched in extracellular matrix organization and protein translation pathways, respectively. KIF20A was significantly overexpressed gene indicating a poor prognosis of S1. AURKB, a hesperidin target, was found as an important gene for the pathogenesis of S1. Hesperidin significantly reduced SAOS-2 cell viability and enhanced the efficacy of doxorubicin. Conclusions: Our results offer a strong molecular basis for OS subclassification, highlighting subtype-specific mechanisms of tumour progression and chemoresistance. Our findings support the prospect of customized therapeutic approaches, with hesperidin being a promising co-adjuvant to improve the prognosis of high-risk OS patients.

This study identifies ankrd1a as a novel modulator of skeletal muscle repair in adult zebrafish. Revealing its dual role in newly forming and intact myofibers near injury, the work highlights ankrd1a’s function in fine-tuning myogenic differentiation during repair. We position ankrd1a as both a marker of myofibers that repopulate wounded tissue and a player in coordination of adaptive responses essential for effective muscle tissue recovery.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to Western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30>RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may be lethal for RMS cells and opens a window for therapeutic targeting.