Molecular Oncology最新文献

Liquid biopsies enable noninvasive tumor profiling and longitudinal disease monitoring. Their analytical performance is strongly influenced by pre-analytical factors, yet direct comparisons between cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (evDNA) remain scarce. We prospectively evaluated four pre-analytical variables: processing delay, storage temperature, tube type, and plasma input volume, on cfDNA and evDNA from cancer patient plasma (n = 244) using ddPCR, Qubit, and TapeStation. Key findings were validated in archived plasma samples (n = 723). In the prospective cohort, cfDNA concentrations increased after 24 h and evDNA after 48 h at room temperature, while retrospective analysis revealed earlier changes (cfDNA: 6 h; evDNA: 24 h). Storage conditions influenced both analytes, as short-term refrigeration (4 °C) better preserved DNA quality than -80 °C freezing, while extracted DNA remained stable at -80 °C. Acid citrate dextrose (ACD) and K₂EDTA tubes performed similarly under prompt processing. Higher plasma volumes improved evDNA, but not cfDNA, for mutation detection. evDNA demonstrates greater resilience than cfDNA under suboptimal conditions. Standardized workflows and prompt processing are essential to ensure reliable mutation detection in clinical liquid biopsy applications.

Myelodysplastic neoplasms (MDS) are heterogeneous malignancies originating in hematopoietic stem cells. In this explorative study, we carried out ultra-deep total RNA sequencing on FACS-sorted CD34+ bone marrow cells from 71 patients and eight healthy age-matched controls. We investigated the expression of circular RNAs (circRNAs), a group of noncoding RNAs produced by back-splicing of nonadjacent splice sites. Key findings were further explored in an independent cohort of 118 patients with MDS and ring sideroblasts. circRNA abundance was higher in the disease groups than in controls, and different spliceosome mutations were associated with distinct circRNA expression patterns. Expression of the proliferation-related gene MKI67 was negatively correlated with circRNA abundance. High circRNA abundance was associated with a significantly increased risk of disease progression at 3 years. The majority of the 38 circRNAs that were significantly upregulated in MDS demonstrated highly correlated expression, and many were associated with risk of leukemic progression. Furthermore, we confirmed the specificity of circZEB1 expression to cases with SF3B1 mutations. We conclude that aberrant circRNA expression is found in MDS and displays associations with disease characteristics and patient outcomes.

High-grade serous ovarian cancer (HGSOC) is the most prevalent and lethal subtype of epithelial ovarian cancer (EOC), characterised by extensive peritoneal metastasis. The intermediate filament keratin 19 (KRT19) has been linked to tumour progression and chemoresistance in various cancers. However, its role varies across tumour types and remains unclear for HGSOC. We evaluated KRT19 protein expression in 199 HGSOC patients and correlated findings with clinical outcomes. In vitro, we assessed the effects of KRT19 on tumour-associated mechanisms, including proliferation, migration, adhesion, and spheroid formation. A xenograft mouse model was used to assess tumour burden in vivo. Publicly available datasets enabled in silico validation. KRT19 was significantly overexpressed in HGSOC, and high expression was associated with reduced overall survival. In vivo, KRT19-overexpression increased peritoneal tumour burden. In vitro and ex vivo, KRT19 induced a hybrid epithelial phenotype through enhanced epithelial-mesenchymal plasticity (EMP), promoting adhesion, migration, and spheroid integrity, thereby potentially supporting metastatic processes. Further, KRT19 could contribute to paclitaxel resistance. Altogether, KRT19 represents a potential independent prognostic marker and therapeutic target to inhibit metastatic dissemination.

The nucleoside analogue cytarabine (ara-C) is part of standard treatment against acute myeloid leukaemia (AML). The efficacy of this therapy is dependent upon accumulation of the active triphosphate metabolite ara-CTP, which mis-incorporates into genomic DNA, triggering cell death. The deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) SAMHD1 can hydrolyse ara-CTP and thereby convert the active metabolite back to its inactive prodrug form. This constitutes a barrier to treatment efficacy and thus strategies to target SAMHD1 are warranted. SAMHD1 activity is allosterically regulated by nucleotides, which are synthesised in cells via distinct pathways. We screened a collection of drugs targeting nucleotide biosynthetic enzymes and identified that inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH), responsible for catalysing the rate-limiting step in guanine nucleotide biosynthesis, sensitises AML cell lines to ara-C in a SAMHD1-dependent manner. We show that approved drugs inhibiting IMPDH-mycophenolic acid and ribavirin-imbalance deoxyribonucleoside triphosphate pools and increase ara-C efficacy in SAMHD1-proficient, but not deficient, leukaemic cells. Altogether, we provide insight into SAMHD1 regulation in leukaemic cells and show how this process can be exploited by approved drugs to improve ara-C therapy.

Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis as early-stage asymptomaticity leads to late-stage diagnoses. Strategies to detect PDAC earlier or identify high-risk individuals are therefore paramount. Here, we report results from genetically engineered mice and PDAC patients that identify serum proteins associated with pancreatic intraepithelial neoplasms (PanINs), the most common PDAC precursor, and early-stage PDAC. Initially, we screened previously described PanIN-abundant mice, harbouring pancreatic and duodenal homeobox 1 (Pdx1)-Cre, Lox-STOP-Lox-Kras^G12D/+ and floxed alleles of essential autophagy genes autophagy-related 7 (Atg7) or autophagy-related 5 (Atg5). Sera from these mice were assessed by proteomics and hits were compared to those in Lox-STOP-Lox-Kras^G12D/+ Lox-STOP-Lox-Trp53^R172H/+ Pdx1-Cre (KPC) mice, which closely recapitulate human disease, and early-stage (I-II) PDAC patients. Levels of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) were significantly elevated in all three screens, with complement C5, complement factors B and H (CFB/CFH), and monocyte differentiation antigen CD14 increased in KPC mice and PDAC patients; and all were significantly increased co-ordinately in PDAC according to disease stage. Serum levels of C5, CFH and CD14 together constitute a novel panel for identifying PanINs and early-stage PDAC with confidence, and when combined with additional screening, could help increase survival from this dismal disease.

Recurrent ovarian cancer (OC) remains a major cause of mortality due to chemoresistance and metastasis. Epigenetic aberrations, particularly dysregulated microRNA (miRNA) expression, contribute to disease progression and represent a promising therapeutic target. Here, we identify the miR-214-3p/miR-199a-5p cluster as a stage-associated, tumor-suppressive network that is lost in recurrent and chemoresistant OC but can be elevated using engineered small extracellular vesicles enriched with this miRNA cluster (m214-sEVs). Using a clinically relevant mouse model that recapitulates spontaneous OC relapse following platinum-based chemotherapy, we show that m214-sEVs are internalized by OC cells and niche fibroblasts, leading to increased intracellular levels of this cluster and suppression of key chemoresistance-associated pathways, including through downregulation of Toll-like receptor 4 (TLR4), β-catenin, and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein YKT6. m214-sEV treatment reprograms secondary tumor-derived sEVs toward a less prometastatic cargo profile and decreases carboplatin resistance and cell migration. Enforced YKT6 overexpression abrogates these effects, establishing YKT6 as a key downstream effector. Collectively, these findings support engineered sEVs as a translatable strategy to overcome chemoresistance and disrupt pro-tumorigenic EV signaling in recurrent OC.

Epithelial-to-mesenchymal transition (EMT), driven by cues from the tumor microenvironment, is a critical initiator of metastatic progression. In breast cancer patients, elevated expression of cartilage oligomeric matrix protein (COMP) is associated with shorter survival and increased metastatic risk. Here, we investigated the role of COMP in regulating EMT in breast cancer. Breast cancer cells treated with recombinant COMP or engineered to overexpress COMP exhibited a marked decrease in the epithelial marker CDH1 and an increase in mesenchymal markers such as VIM and VCAN. Consistent with these in vitro findings, COMP-expressing xenograft tumor tissues showed enhanced EMT characteristics. Functionally, COMP promoted increased migration and invasion of breast cancer cells in both autocrine and paracrine manners, dependent on its thrombospondin (TSP) and C-terminal domains. We further identified protein TMEPAI (encoded by gene PMEPA1) as a functional COMP-binding partner that mediates COMP-induced EMT, primarily through interaction with the TSP domain of COMP. Mechanistically, COMP shifted transforming growth factor beta (TGFβ) signaling from canonical phosphorylated mothers against decapentaplegic homolog 2/3 (pSMAD2/3) activation toward pSMAD1/5, likely through its interaction with PMEPA1. This study suggests the COMP-PMEPA1 axis as a new driver of EMT in breast cancer models.

Receptor-interacting protein kinase 4 (RIPK4) has been implicated in the progression of numerous tumours. In nonmelanoma skin cancer, RIPK4 plays a suppressor role, whereas in melanoma, it functions as an oncogene that modulates key signalling pathways involved in melanoma cell survival and expansion. Increased RIPK4 levels in metastatic melanoma biopsies prompted us to investigate the consequences of RIPK4 loss for the invasive and metastatic phenotype of melanoma cells. Using an integrated approach involving clinical samples, in vivo xenograft models, transcriptomic analysis and 3D functional assays, we show that RIPK4 deletion significantly reduces pulmonary metastasis formation. This reflects its role in late-stage metastatic events, such as extravasation and colonization, particularly since this phenotype correlates with extensive transcriptional reprogramming of adhesion- and motility-related genes in melanoma cells, as evidenced by next-generation sequencing and functional validation in spheroid and collagen-based models. Despite exhibiting features of a partial shift towards an amoeboid phenotype such as membrane blebbing and increased MLC2 phosphorylation, RIPK4 knockout cells display impaired motility and invasion. Re-expression of RIPK4 restores mesenchymal morphology and migratory capacity. Together, our results establish RIPK4 as a critical regulator of melanoma invasion and metastasis. Nonetheless, they also demonstrate that the loss of RIPK4 function activates compensatory phenotypic shifts in melanoma cells that fail to fully rescue their invasive potential.

Targeted drug therapy is very important for the treatment of triple-negative breast cancer (TNBC), and the development of carrier systems to deliver apoptosis-inducing proteins such as TRAIL to cells is important in cancer therapy. In this study, a nanosystem formulation (TRAIL-PEG-Apt-PLGA) encapsulating TNBC-targeted aptamer-bound-TRAIL protein was performed and the efficacy of this system was evaluated in a mouse tumor model. The characterization of TRAIL-PEG-Apt-PLGA was confirmed by FTIR, NTA and SEM microscopy. The efficacy of TRAIL-PEG-Apt-PLGA was evaluated by in vitro release assays and interactions with TNBC cells (MDA-MB-231) and healthy breast cells (MCF-10A). TRAIL-PEG-Apt-PLGA was administered intravenously to NOD/SCID gamma mouse breast tumors and evaluated in vivo. Pharmacokinetics, bioavailability testing, histological staining (DR4/DR5, TUNEL, HE staining) and molecular alterations with PCR array were evaluated in tumor tissues. TRAIL-PEG-Apt-PLGA induced apoptosis in both in vivo and in vitro studies. It was found that it regulated cellular responses along with apoptotic mechanisms in cells without developing resistance in suppressing tumor growth by making changes on Atf2, Casp8, Bcl2 and Irf5 genes and proteins. As a result, the biotechnological drug potential of TRAIL was discovered in an aptamer-bound nanosystem for the treatment of triple-negative breast cancer and innovative applications for clinical use.

Cervical cancer remains a leading cause of mortality among women, particularly in low- and middle-income countries. Despite distinct prognoses and clinical outcomes between its main histological subtypes, squamous cell carcinoma (SCC) and adenocarcinoma (ADC), current treatment regimens remain largely similar, creating an urgent need for targeted therapeutic strategies based on molecular distinctions. To address this gap, the long noncoding RNA IGFL2-AS1 was investigated as a potential prognostic biomarker and therapeutic target in cervical adenocarcinoma. A translational approach was employed that integrated patient transcriptome data, in silico analysis from public databases, and in vitro validation. Using CRISPR/dCas9 technology, IGFL2-AS1 expression was modulated in HeLa (ADC) and SiHa (SCC) cell lines to assess its impact on cellular characteristics associated with tumorigenesis. In silico analysis revealed that IGFL2-AS1 expression was significantly reduced in ADC compared to SCC, and its low expression was consistently linked to poorer ADC prognosis and decreased patient survival. Notably, overexpression of IGFL2-AS1 in HeLa cells significantly reduced cell proliferation, migration, clonogenic survival, and enhanced sensitivity to cisplatin and doxorubicin. Conversely, IGFL2-AS1 repression in SiHa cells yielded no significant phenotypic changes, suggesting a context-dependent mechanism. IGFL2-AS1 is identified as a histological subtype-specific prognostic biomarker and promising therapeutic target for cervical adenocarcinoma.