Molecular Oncology最新文献

Combining chemotherapy with chemosensitizing agents is a common strategy to enhance anticancer efficacy while mitigating treatment-related side effects. This study investigated the potential of dammarenediol II (DM2), a ginsenoside precursor, to enhance the anticancer effects of etoposide by downregulating O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) and modulating the Akt signaling pathway in HepG2 human liver cancer cells. The effect of DM2 on O-GlcNAcylation regulation was analyzed using Pharmaco-Net, an artificial intelligence-driven drug screening platform and further validated using O-GlcNAc transferase (OGT) activity assay. DM2 cotreatment enhanced etoposide's anticancer efficacy, which was quantitatively evaluated by viability, Annexin V binding, membrane integrity, and caspase-3/7 activity assays in HepG2 cells. Results showed that DM2 reduced O-GlcNAc levels by directly interacting with OGT, as confirmed through Pharmaco-Net. Cotreatment with 40 μm DM2 and 20 μm etoposide produced synergistic anticancer effects, lowering etoposide's IC₅₀ for cell viability by 2.29-fold and its EC₅₀ for caspase-3/7 activity by 3.64-fold. Mechanistically, DM2 dose-dependently suppressed Akt/GSK3β/mTOR signaling. Using the Akt activator SC79, additional experiments confirmed that Akt signaling acts downstream of O-GlcNAcylation regulated by etoposide and DM2. These effects were also observed in multiple human liver cancer cell lines, as well as in A549 lung and Caco-2 colorectal cancer cells. This supports the broader anticancer and Akt-inhibitory potential of DM2. This study is the first to demonstrate that DM2 enhances anticancer synergy by suppressing O-GlcNAcylation and Akt signaling, highlighting its potential as a novel chemotherapy adjuvant.

Plecstatin (PST) is a potent anticancer agent in preclinical models, yet its precise mechanism of action and molecular specificity regarding its main targets, plectin and outer dense fiber protein 2 (ODF2), remain incompletely understood. Here, we dissected PST's mode of action using knockouts of plectin (PLEC) and ODF2 in SNU-475 hepatocellular carcinoma (HCC) cells. PST suppressed anchorage-independent growth and impaired 2D and 3D migration in a dose-dependent manner in both wild-type and ODF2-deficient cells, but not in PLEC-deficient cells, establishing plectin as the principal effector of PST's antitumor activity. Proteomic and functional analyses revealed that PST primarily disrupts cytoskeletal remodeling through plectin, while selectively affecting ciliogenesis-related pathways linked to ODF2 loss. Deletion of either protein attenuated PST-induced Ser51 phosphorylation of eIF2α, ATF4/GADD34 induction, and cytochrome c release, indicating cooperative involvement in integrated stress response (ISR). Correlative analysis of patient datasets confirmed associations between PLEC/ODF2 expression and ISR-related gene signatures, supporting the clinical relevance of this pathway. Together, these findings identify plectin as a key target of PST in disrupting cytoskeletal integrity and establish plectin/ODF2 axis in PST-driven stress adaptation in HCC.

The limiting dilution assay (LDA) is a key method to quantify clonogenic cells with self-renewing capacity in vitro, crucial for preclinical cancer research and therapy response assessment. It estimates the frequency of individual clonogenic, stem-like cells within a population based on their ability to form colonies with ≥50 cells at limiting cell numbers. Standard LDA analysis relies on linear, single-hit Poisson models, yet clonogenic growth under single-cell conditions often involves cooperative or competitive dynamics, violating this linearity assumption. Here, we present a modeling framework incorporating non-linear population dynamics into LDA analysis and introduce LDAcoop, an R-based tool for universal quantification of clonogenic cells in LDA formats. Across multiple cancer cell types, we benchmarked LDA against the colony formation assay (CFA) and show that LDA outperforms CFA, especially for patient-derived organoids, suspension cultures, and higher throughput applications. This renders the LDA format particularly suitable for larger-scale pharmacogenomic screening and drug sensitivity testing in complex models. Our results establish LDA and LDAcoop as versatile, scalable tools for robust quantification of clonogenic growth, supporting preclinical drug development and molecular precision oncology research.

Complete surgical resection is essential for oropharyngeal squamous cell carcinoma (OPSCC) therapy, underscoring the need for improved intraoperative margin assessment. To advance in vivo diagnostics for OPSCC, Nanosecond infrared laser (NIRL) tissue sampling combined with shotgun lipidomic analysis reveals lipidome differences between OPSCC tissue and adjacent healthy tissue. In this study, ablations were performed on tonsil squamous cell carcinoma in 28 samples from 11 patients using an established chamber setup, and a subset of six samples from three patients with a custom-made laser fiber-coupled applicator, designed for handheld use. Welch's t-test results (p = 0.05, two-fold change) revealed a similar OPSCC lipid profile in seven out of 11 patients. Potential tumor lipid markers were identified as consistently and significantly increased, despite the biological heterogeneity of the samples, underscoring their potential diagnostic value. Tissue ablation with fiber-coupled applicator was successful and the lipidomic analysis was consistent with the chamber setup. While limited to off-line analysis, our approach highlights the potential of fiber-based laser sampling as a rapid and minimally invasive method to obtain tissue material for advanced molecular profiling in surgical and endoscopic settings.

Single circulating tumor cell (sCTC) analysis enables the determination of predominant CTC phenotypes and genotypes. We previously demonstrated the feasibility of sCTC detection and genomic characterization in high-grade serous ovarian cancer (HGSOC) by combining immune-magnetic enrichment and image-based sorting, followed by whole-genome amplification (WGA) and next-generation sequencing-based copy number alteration analysis (CNA). Here we aimed to improve our workflow by incorporating HGSOC-specific markers, folate receptor alpha (FRα), and markers to identify epithelial (cytokeratin) and mesenchymal (vimentin) phenotypes for the phenotypic as well as genotypic analysis of sCTCs over the course of treatment in 42 HGSOC patients. We detected a significant reduction of FRα-positive cells (P = 0.0205) and an expansion of cells with a high nuclear staining and no target antigen expression (P = 0.002). Before treatment, sCTCs showed an enrichment in CNAs of Chromosomes 2, 7, and 12, while CNA dynamics of sCTCs suggested a potential selection of distinct CNAs specific to the homologous recombination pathway. sCTCs revealed persistent CNAs in the CDK4 and emerging ones in the ALK oncogene. Notably, primary tumors revealed considerable fractions of shared genomic aberrations.

Copy number variations (CNVs) play a crucial role in cancer diagnostics and prognostics, potentially impacting treatment decisions. Ultra-low-pass whole-genome sequencing (ULP-WGS) has emerged as a promising alternative to array-based methods for CNV detection, especially in formalin-fixed paraffin-embedded (FFPE) samples. However, sequencing biases and sample heterogeneity necessitate the optimization of CNV detection tools for FFPE sample-derived data. This study evaluates three open-source CNV callers (CNVpytor, ichorCNA, and WisecondorX) using ULP-WGS and compares their performance against a single nucleotide polymorphism (SNP) array. Our results demonstrate that under optimal experimental conditions, ichorCNA and WisecondorX achieved equal detection of true positive results, with reduced false positive results compared to the SNP array. The SNP array detection pattern differed somewhat from that of the CNV callers, while ichorCNA and WisecondorX had the most comparable detection pattern. We highlight the importance of (pre-)analytical parameters such as neoplastic cell content, sequencing coverage, and bin size selection on CNV detection accuracy. Our findings support the adoption of ULP-WGS-based CNV detection as a robust alternative to SNP arrays, with WisecondorX emerging as the most suitable tool for clinical implementation.

Receptor tyrosine kinase ERBB4 (HER4) is frequently mutated in human cancer, and ERBB4 mutations have been identified in patients relapsing on targeted therapy. Here, we addressed the functional consequences of recurrent cancer-associated ERBB4 mutations that are located at regions important for receptor activation and/or are paralogous to known oncogenic hotspot mutations in other ERBB genes. Eleven out of 18 analyzed mutations were transforming in cell models, thus suggesting oncogenic potential for more than half of the recurrent ERBB4 mutations. More detailed analyses of the most potent mutations, S303F, E452K, and L798R, showed that they are activating, can co-operate with other ERBB receptors and are sensitive to clinically available second-generation pan-ERBB inhibitors neratinib, afatinib, and dacomitinib. Furthermore, the S303F mutation, together with a previously identified activating ERBB4 mutation, E715K, promoted resistance to third-generation EGFR inhibitor osimertinib in EGFR-mutant lung cancer model in vitro and in vivo. Together, these results are expected to facilitate clinical interpretation of the most recurrent cancer-associated ERBB4 mutations. The findings provide rationale for testing the efficacy of clinically used pan-ERBB inhibitors in patients harboring driver ERBB4 mutations both in the treatment-naïve setting, and upon development of resistance to targeted agents.

Monomethyl auristatin E (MMAE) is used as the cytotoxic payload for enfortumab vedotin (EV) in the treatment of locally advanced and metastatic bladder cancer (BC). However, the development of resistance to MMAE in BC is a therapeutic problem. To explore the mechanism of resistance to MMAE in BC, we established MMAE-resistant BC cells (MR-BCs). RNA sequencing analysis showed that the expression of dipeptidyl peptidase 4 (DPP4, also called CD26) increased significantly in MR-BCs compared with parental BC cells. Knock down of DPP4 expression using small interfering RNA inhibited the viability of MR-BCs. In addition, the DPP4 inhibitor sitagliptin suppressed the proliferation, migration, and invasion of BC cells, and cotreatment with MMAE effectively induced cell apoptosis, arrested cells in the G₂M phase of the cell cycle, increased reactive oxygen species production by inhibiting the AKT pathway, and significantly inhibited the in vivo growth of MMAE-resistant cells. This study provides insights into the use of DPP4 inhibitors as a treatment strategy for MMAE-resistant BC.

Metabolic dysfunction-associated steatohepatitis (MASH) is emerging as a major driver of hepatocellular carcinoma (HCC). Crouchet et al. identify PRDX2 as a key regulator linking oxidative stress, metabolic imbalance, and oncogenic signaling. Across multiple in vivo and in vitro models, PRDX2 inhibition restores metabolic homeostasis, reduces tumor initiation, and selectively impairs HCC cell survival. These findings highlight PRDX2 as a promising biomarker and hepatocyte-directed target for chemoprevention, emphasizing the importance of the interplay between metabolism and liver cancer development.