Triple-negative breast cancer (TNBC) remains highly aggressive and refractory to conventional treatments, underscoring the need for novel combination strategies. Here, we employed 2D and 3D in vitro models, transcriptomic profiling, and in vivo xenograft studies to evaluate the anticancer efficacy of cannabinoids combined with the terpene β-caryophyllene (BC) in resistant TNBC models. Among the tested cannabinoids, cannabichromene (CBC) exhibited the greatest potency, and its combination with BC at sub-toxic concentrations significantly reduced IC50 values, enhanced cytotoxicity in spheroids, and suppressed colony formation and migration. The combination treatment induced pronounced G1 cell cycle arrest and increased apoptotic cell death. Western blot analyses revealed downregulation of PARP, Survivin, mTOR, Vimentin, Glypican-5, and PD-L1, while RNA sequencing demonstrated suppression of proliferative and migratory signaling pathways alongside activation of apoptosis, autophagy, and ferroptosis-related pathways. In vivo, CBC + BC significantly inhibited tumor growth in MDA-MB-231 xenografts, outperforming single-agent treatments. Collectively, these findings demonstrate that BC synergistically enhances cannabinoid activity, yielding superior antiproliferative and anti-migratory effects, and highlight this combination as a promising therapeutic strategy for resistant TNBC.

Male urological cancers, including clear cell renal cell carcinoma (ccRCC), bladder cancer (BC), and prostate cancer (PCa), are characterized by extensive heterogeneity and complex ecosystems, yet the underlying mechanisms remain incompletely understood. In this study, scRNA-seq, scATAC-seq and spatial transcriptomics data are integrated to systematically characterize the features of the tumor microenvironment (TME). We identify tumor cell subclones and elucidate the impact of chromosomal abnormalities on their characteristic functions. We further identify transcription factor regulatory networks by analyzing tumor cell differentiation trajectories. Importantly, we integrate DNA methylation and SNP information to deeply dissect the tumor cell differentiation process, revealing the multilayer regulatory mechanisms of tumor-related genes. Additionally, we reveal the evolution of cellular states across ecotypes to provide a more comprehensive characterization of TME. Finally, we screened potential therapeutic agents targeting the molecular mechanisms underlying tumor cell differentiation (Amivantamab in ccRCC, Levothyroxine in BC, Ouabain in PCa) and signature genes of ecotype. In conclusion, our work establishes a comprehensive framework for tumor assessment and informs the development of precision therapeutic strategies.

Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers for cancer risk assessment, yet prospective evidence for breast cancer (BC) remains limited. We conducted a nested case-control study within a prospective cohort to examine whether pre-diagnostic circulating miRNAs are associated with subsequent BC risk and to explore their potential relevance in prospective population-based settings. Baseline serum from 160 women (80 incident BC cases; 80 matched controls) was analyzed, with a median time to diagnosis of 8.9 years. Eight candidate miRNAs were quantified by droplet digital PCR (ddPCR) and normalized to miR-484. Group differences were evaluated by non-parametric tests, and odds ratios for BC were estimated using logistic regression models adjusted for established risk factors, with Bonferroni correction for multiple testing. Cases and controls were comparable at baseline. Among the candidates, lower circulating miR-181 levels showed a suggestive inverse association with BC risk in fully adjusted models, while lower Let7 levels showed only a non-significant, hypothesis-generating inverse trend that did not survive Bonferroni correction. No other miRNA displayed clear associations with BC risk. These findings, while preliminary, support further large-scale prospective investigations specifically designed to assess predictive performance and external validation. employing standardized pre-analytical and analytical protocols, repeated sampling, and independent replication/external validation to clarify the etiologic relevance and potential risk-prediction value of circulating miRNAs for BC.

Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer deaths, with resistance to targeted therapies posing a major clinical challenge. Drug-tolerant persister (DTP) cells are key contributors to resistance, and targeting them offers new strategies to enhance existing treatments. MicroRNAs (miRNAs), particularly the tumour-suppressive miR-15/107 family, offer promise due to their ability to target multiple oncogenic pathways. This study evaluated a synthetic consensus miRNA mimic, conmiR-15/107, in NSCLC cell line models. Dose-response assays showed robust, dose-dependent growth inhibition in both EGFR-mutant (PC9) and KRAS-mutant (H358 and A549) lung adenocarcinoma cells, but not in the human bronchial epithelial cell line BEAS-2B. When combined with EGFR inhibitors (osimertinib and gefitinib) in PC9 cells, the mimics showed a higher rate of growth inhibition compared with the controls and reduced IC50 values. Similarly, conmiR-15/107 enhanced growth inhibition by the KRAS inhibitors sotorasib and adagrasib in H358 cells. RT-qPCR confirmed downregulation of conmiR-15/107 targets, including MEK1, BCL2 and BRCA1, suggesting a multi-target mechanism of action. Long-term assays showed that the mimics reduced the survival and delayed the proliferation of DTPs in osimertinib-treated PC9 cells as well as sotorasib-treated H358 cells. These findings support conmiR-15/107 as a potential adjunct to targeted therapy, capable of enhancing treatment efficacy and delaying resistance in lung adenocarcinoma.

Soft tissue sarcomas are rare malignant mesenchymal tumors for which accurate diagnosis, prognostic stratification, and therapeutic decision-making remain challenging. Although histopathology and immunohistochemistry are essential diagnostic tools, they frequently fail to capture the molecular complexity underlying tumor aggressiveness and treatment resistance. In this study, we evaluated the utility of RNA-based next-generation sequencing for the molecular characterization of STS and for elucidating transcriptomic mechanisms associated with aggressive tumor behavior. An observational cohort of 24 patients with histologically confirmed soft tissue sarcomas was analyzed, using adipose and skeletal muscle tissue as controls. RNA was extracted from tumor samples, libraries were prepared with a targeted pan-cancer panel, and sequencing was performed on the Illumina platform, followed by bioinformatic analysis using DRAGEN pipelines and DESeq2. RNA-NGS identified a predominance of single-nucleotide polymorphisms and significant differential gene expression, with overexpression of proliferation-related genes (TOP2A, MKI67, BUB1B), extracellular matrix and microenvironment-associated genes (COL11A1, SPP1), and developmental regulators (HOXD13, MELK). Subgroup analysis revealed a distinct transcriptomic profile in leiomyosarcoma, while gene fusion analysis detected clinically relevant alterations. These findings demonstrate that RNA-NGS provides biologically and clinically meaningful insights into the molecular landscape of soft tissue sarcomas and supports its integration into precision medicine-oriented diagnostic workflows.

Dedifferentiation-the acquisition of an early developmental state-is a hallmark of cancer. However, the underlying mechanisms that lead to cancer-associated dedifferentiation are not fully understood. Transposable elements (TEs) are becoming increasingly recognised as important regulators of development and disease. The recruitment of TE sequences has played an important role in placental evolution, and TE-derived genes play critical roles in placental development. Although important biological differences exist between tumours and the placenta, the placenta shares certain features with tumours, including the capacity to invade surrounding tissue and modulate the maternal immune response. In this regard, TEs have been implicated in cancer development, and are documented to contribute to oncogenesis through multiple different mechanisms. Moreover, cancers reacquire an epigenetic landscape, which is reflective of early development, and which corresponds to increased phenotypic plasticity, including facilitating the activation of early developmental genes. Many cancers can repurpose developmental genes, including TE-associated genes, which may contribute to pathways involved in invasion and metastasis. Determining whether TE activation is a consequence of broader epigenetic reprogramming or actively contributes to dedifferentiation will be important for understanding cancer biology and may facilitate improvements in cancer diagnosis and treatment.

The efficacy of immunotherapy in colorectal cancer (CRC) has long been considered to be closely associated with microsatellite instability (MSI) status. Patients with microsatellite stable (MSS) tumors typically exhibit poor responses to PD-1/PD-L1 inhibitors and a poor prognosis, often being categorized as immunologically 'cold' tumors. However, some MSS patients can still achieve favorable therapeutic responses, sometimes even surpassing those of certain MSI patients. Immune-cold and immune-hot tumor phenotypes are largely determined by the abundance, clonal expansion, and functional states of tumor-infiltrating T cells. This suggests that immunotherapy responses are driven by dynamic remodeling of T-cell clonality rather than by MSI status alone. To elucidate the underlying T cell clonal dynamics, integrated single-cell transcriptome (scRNA-seq) and T cell receptor sequencing (scTCR-seq) data analyses from 43 blood and tissue samples of MSI and MSS colorectal cancer patients before and after anti-PD-1 therapy were performed. Using our developed TCR reconstruction pipeline (TORBiT), we systematically analyzed the clonal architecture of the TCR repertoire, inter-tissue migration, and its association with T-cell functional state transitions. From a TCR clonal kinetic perspective, we revealed two distinct modes of immune Coercion that may further affect the immune response: a "high-fluctuation, deep-exhaustion" pattern in MSI tumors and a "high-baseline, strong-suppression" pattern in MSS tumors. These findings provide a novel theoretical foundation and research perspective for understanding the responsiveness and resistance mechanisms to immune checkpoint inhibitors.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs initially identified in germline cells as genome guardians that silence transposable elements. Recent studies have expanded this view, revealing that piRNAs and PIWI proteins are broadly expressed in somatic tissues and participate in epigenetic and post-transcriptional gene regulation. This review systematically summarizes piRNA biogenesis and molecular mechanisms, with a focus on their functional diversification from germline to somatic cells. We detail piRNA dysregulation and its association with various human diseases, including cancer, cardiovascular disorders, neurodegenerative diseases, immune dysfunction, and reproductive disorders. By integrating recent findings, this review provides a comprehensive overview of piRNA-mediated regulatory networks and highlights their potential as novel biomarkers and therapeutic targets.

Glial brain tumours, including astrocytoma IDH (Isocitrate Dehydrogenase) mutant and glioblastoma IDH wild-type, are highly malignant brain tumours with poor clinical outcomes. Genomic instability, encompassing microsatellite (MIN) and chromosomal instability (CIN), drives tumour heterogeneity and evolution. In this study, genomic instability was analysed in 85 patients using AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) by comparing tumour and normal tissue (blood) DNA profiles of the same patient. Both types of alterations were present in all analysed samples, contributing almost equally to the total level of genomic instability. The dominant pattern of genomic instability in our cohort was low overall instability, predominantly manifesting as low-degree microsatellite instability. A general decrease in genomic instability was observed with increasing tumour grade. Glioblastoma IDH wild-type was more prevalent in older patients, whereas astrocytoma IDH mutant predominated in younger individuals. Notably, low genomic instability (both MIN and CIN) was associated with poorer survival in patients over 50 years of age. Females, compared to males, exhibited higher MIN in grade 2 tumours and elevated CIN in grade 4 tumours. Our results confirm that genomic instability contributes to tumour progression, MIN being the pivotal factor, and could serve as a prognostic biomarker in malignant gliomas.

Triple-negative breast cancer (TNBC) lacks effective targeted therapies, and the mechanisms by which developmental endothelial locus-1 (EDIL3/Del-1) promotes TNBC remain incompletely defined. We profiled Del-1 and AMPK subunits in TNBC cell lines by RT-PCR and immunoblotting, performed functional assays in CRISPR/Cas9 Del-1 knockout and AMPKβ-manipulated cells, and evaluated AMPKβ in early-stage TNBC tumors using tissue microarrays (TMA) (immunohistochemistry; n = 100) and AMPKβ2 mRNA quantification. Del-1 and AMPKβ were enriched in TNBC cells, most prominently in the mesenchymal stem-like subtype, whereas AMPKα levels were relatively stable. Increased Del-1 and activated AMPKβ enhanced proliferation and invasion, while Del-1 deletion reduced AMPKβ expression and suppressed tumor-promoting phenotypes. Mechanistically, Del-1 increased AMPKβ phosphorylation at serine 108, and a phospho-mimetic AMPKβ mutant further amplified oncogenic effects. In the pilot TMA study, high AMPKβ protein expression showed a trend toward poorer DFS in Kaplan-Meier analysis, while multivariate analysis identified high AMPKβ protein expression as an independent factor associated with poorer DFS in patients with early TNBC. These data support AMPKβ as a key mediator of Del-1-driven signaling and suggest AMPKβ could be a therapeutic target in aggressive TNBC subsets.

Pituitary neuroendocrine tumors (PitNETs) are usually benign intracranial neoplasms that may exhibit invasion of the cavernous sinus, complicating surgery and increasing the risk of recurrence. This study aimed to investigate membranous E-cadherin (mE-cad) expression across PitNET subtypes and transcription factor (TF) lineages, including Pit-1 (pituitary-specific positive transcription factor 1), SF-1 (Steroidogenic Factor 1), and TPIT (T-box pituitary transcription factor), and its association with tumor invasiveness in sixty-nine patients. mE-cad expression was evaluated as the percentage of positive cells (0%, 1-10%, >10%) and by immunoreactive score (IRS). Staining intensity was scored as: 0, no staining; 1, weak; 2, moderate; 3, strong. The proportion of positive cells was scored as: 0, none; 1, <10%; 2, 10-50%; 3, 51-80%; 4, >80%. Mean mE-cad expression was 5.2% in gonadotroph, 3.2% in corticotroph, 0.5% in lactotroph, and 17.5% in plurihormonal PitNETs. By TF lineage, the mean expression was 5.3% for Pit-1, 3.2% for TPIT, and 5.1% for SF-1. Low mE-cad expression (IRS 1-2) was associated with higher odds of cavernous sinus invasion compared with IRS 3-6 (adjusted OR = 6.0, 95% CI 1.08-33.4, p = 0.04), independent of tumor volume (adjusted OR = 4.0, 95% CI 1.50-10.7, p = 0.01). After restricting the analysis to the gonadotroph PitNET group, tumors with an IRS of 1-2 showed significantly higher invasiveness compared with those with an IRS of 3-6 (p = 0.012). These findings suggest that mE-cad may serve as a biomarker of PitNET invasiveness, with expression varying according to TF lineage and tumor subtype.

CRISPR screens are a powerful functional genomics approach for identifying genes that confer sensitivity and resistance to anti-cancer therapies. CFI-402257 (luvixasertib, 2257) is a small molecule inhibitor of threonine tyrosine kinase (TTK), a promising therapeutic target in genomically unstable cancers due to its critical role in establishing the spindle assembly checkpoint (SAC) during mitosis. To inform its ongoing development and evaluation in clinical trials, we sought to use CRISPR activation (i.e., gain of function) screens to identify cellular mechanisms of resistance to 2257 in models of triple-negative breast cancer (TNBC). In vitro screens conducted in two TNBC cell lines nominated ABCG2 as the top resistance-conferring gene in both models. Validation studies assessing clonogenic survival and apoptosis confirmed that ABCG2 overexpression enhanced TNBC resistance to 2257 in vitro, while knockdown enhanced sensitivity. These findings suggest that 2257 is a substrate of ABCG2's drug efflux activity. However, overexpression of ABCG2 failed to confer resistance to 2257 in TNBC xenografts grown in mice and treated with a moderately active dose and schedule. Our results highlight the potential impact of drug transporters in in vitro CRISPR screens and the importance of confirming the relevance of drug response mechanisms identified in cultured cells using in vivo models that recapitulate drug pharmacokinetics and pharmacodynamics.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy with especially high incidence in Middle Eastern populations. While classical hereditary syndromes explain a minority of cases, the broader germline landscape of non-syndromic PTC remains unclear. Whole-exome sequencing was performed on 245 unselected Saudi PTC patients to identify germline pathogenic or likely pathogenic variants (PVs/LPVs) in cancer predisposition genes. Clinical and molecular characteristics, and family history were integrated to assess phenotypic correlations. Eleven patients (4.5%) harbored germline PVs/LPVs in cancer susceptibility genes including STK11, TP53, BRCA1, BRCA2, FANCA, SLX4, RAD50, MSH6, POLD1 and NF1. Four patients (36.4%) carried PVs/LPVs in canonical FA pathway genes; this increased to five patients (45.5%) when RAD50 was included. Two unrelated patients harbored the same STK11 variant (p.R304Q) without classical Peutz-Jeghers syndrome features. A TP53 hotspot mutation (p.R175H) was identified in a patient with a personal history of gastric cancer, a malignancy associated with Li-Fraumeni syndrome. Notably, the BRCA1 PV detected matches a known Saudi founder mutation in hereditary breast cancer, now observed in PTC. Most germline positive cases lacked syndromic manifestations, underscoring limitations of phenotype or family history-driven genetic testing strategies. These findings suggest that a small subset of non-syndromic PTC cases may carry germline PVs/LPVs in cancer predisposition genes, highlighting the need for broader genetic screening frameworks. Unbiased whole-exome analysis in unselected cohorts can uncover under-recognized genetic risk and guide screening strategies to address the unique hereditary landscape of thyroid cancer in underrepresented populations.

Leucine-rich repeat-containing 8A (LRRC8A; also known as SWELL1), the essential subunit of volume-regulated anion channels (VRACs), is amplified in multiple malignancies and has been implicated in tumor progression and therapeutic resistance. Three-dimensional (3D) cancer spheroids have been well-established as in vitro models that recapitulate characteristics of tumor stemness and intrinsic drug resistance. In the present study, spheroid formation in human breast cancer cell lines, YMB-1 and MDA-MB-468, conferred resistance to multiple anticancer drugs, including doxorubicin (DOX), gemcitabine (GEM), and 5-fluorouracil (5-FU), thereby mimicking the characteristic properties of breast cancer stem-like cells. LRRC8A expression was upregulated in 3D spheroids compared with adherent 2D monolayers, and its pharmacological inhibition induced membrane hyperpolarization accompanied by intracellular Cl- accumulation. Inhibition of LRRC8A significantly sensitized spheroids to DOX, GEM, and 5-FU. Spheroid formation increased the expression of multidrug resistance-related protein 3 (MRP3) and the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4), whereas LRRC8A inhibition suppressed their expression. The transcriptional upregulation of MRP3 and CYP3A4 was mediated through the NRF2-CEBPB/D transcriptional axis. Collectively, these findings suggest that LRRC8A inhibition may represent a therapeutic strategy to overcome chemoresistance by repressing MRP3 and/or CYP3A4 expression in breast cancer stem cells.

Oligoprogressive disease (OPD) in non-small-cell lung cancer (NSCLC) is a clinical entity with peculiar behavior and treatment. OPD patients, during systemic therapy, may receive local ablative treatment (LAT) with survival benefit. The importance of OPD and the role of LAT has been comprehensively assessed in the setting of EGFR mutant and ALK-rearranged NSCLC during tyrosine kinase inhibitor (TKI) treatment, but it is still almost unexplored in the context of NSCLC harboring actionable oncogenic drivers other than EGFR and ALK. The aim of our review is to collect and discuss the available data about standard treatment in this latter setting, with special consideration given to the role of LAT in case of OPD in systemic treatment. Through a comprehensive PubMed and ClinicalTrials.gov search, we identified the available data and ongoing clinical trials addressing these aims. To date, only limited evidence supports the use of LAT in OPD involving NSCLC driven by these molecular alterations, mainly deriving from case reports and retrospective series. This highlights an unmet clinical need that warrants systematic and multicentric data collection to generate more robust evidence.

There is no approved drug therapy for schwannomas associated with NF2-related schwannomatosis (NF2-SWN). Neither life-saving surgical resection or radiation are curative and can compound the debilitating neurological effects of the schwannomas. We previously identified fimepinostat, a dual histone deacetylase (HDAC)/phosphoinositide-3 kinase (PI3K) inhibitor, as a promising drug candidate with pro-apoptotic effects on NF2-related schwannomas. This preclinical study used the pharmaceutical formulation of fimepinostat to confirm its efficacy in schwannomas and identify pro-apoptotic signaling pathways. Fimepinostat was tested in human schwannoma model cells, patient-derived primary vestibular and non-vestibular schwannoma cells, and in a sciatic nerve allograft model. The signaling pathways leading to caspase-3-dependent apoptosis were elucidated using immune assays, flow cytometry, imaging, proteome, and acetylome analysis. Acute exposure to fimepinostat led to p21-dependent cell cycle inhibition, upregulation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL R2), and downregulation of tumor necrosis factor receptor 1 (TNFR1), Yes-associated protein (YAP), and inhibitors of apoptosis. Moreover, fimepinostat downregulated cytokine and chemokine secretion increased by merlin loss in schwannoma cells. Fimepinostat is a promising new drug intervention for NF2-SWN patients with the potential to promote tumor regression.

In glioblastoma, the strong immunosuppression of the tumor immune microenvironment fosters tumor aggressiveness and decreases the effectiveness of therapeutic interventions, including immunotherapies. An intricate network of connections among tumor cells, stroma and infiltrating immune cells sustains immunosuppression. Lectins are immunoregulatory glycan-binding receptors contributing to immunosuppression. Their targeting is proposed as an appealing strategy for anti-cancer therapy. In this work, network-based approaches were exploited to identify a lectin profile that could dissect the complexity of tumor-immunity interactions in glioblastoma. Differential co-expression analysis, employing TCGA, CGGA and GTEx databases (145, 133 and 255 samples, respectively), identified a cluster of novel C-type lectins, with ASGR2 and CLEC12A as principal hubs. Furthermore, TIMER2.0 analysis revealed that their expression was significantly associated with immunosuppressive cells. ASGR2 and CLEC12A expression was also validated by cytofluorimetric analysis on both tumor and liquid biopsies from 20 glioblastoma patients. We report that ASGR2 and CLEC12A C-type lectins are associated with tumor-infiltrating immunosuppressive myeloid subsets and discriminate patients' poor prognosis. These results suggest that C-type lectins may contribute to the immunosuppressive network sustained by infiltrating myeloid immune cells in GB, resulting in exploitable targets for therapeutic interventions.

RNA editing is a critical post-transcriptional modification that contributes to transcriptomic and proteomic diversity. The most common A-to-I (recognized as G) RNA editing enzymes are adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2, respectively), which mediate alterations across all regions of mRNA molecules. However, a systematic cross-tissue view of RNA editing and its molecular correlates is still lacking. Here, we developed a rapid method for ADAR editing assessment based on 24 frequently edited positions in coding regions, which enables faster estimation of RNA editing levels than previous methods. We applied this metric to assess RNA editing in normal and cancerous lung and colorectal tissues. We analyzed RNA and whole exome sequencing profiles of experimental 172 colorectal and 144 lung cancer samples, and literature 646 colorectal and 1037 lung cancer samples. We also examined two types of control tissues: tumor-matched normal tissues (51 colorectal and 108 lung samples) and healthy tissues (6 colorectal and 7 lung samples). Overall ADAR-mediated RNA editing levels were ~2.9- and ~4.7-fold higher in healthy controls than in colorectal and lung cancers, respectively. In addition to their well-known association with immune cells, we identified positive correlations of ADAR editing with 740 molecular pathways including those responsible for extracellular matrix organization, RAS-MAPK axis and G2/M phase cell cycle arrest, and negative-with 139 pathways responsible for DNA repair, apoptosis, expression of transposable elements, and other factors.

Chronic lymphocytic leukemia (CLL) is uncommon in Asia, and longitudinal genomic data from Asian cohorts are limited. We conducted serial whole-exome sequencing (WES) in a multicenter Korean cohort of newly diagnosed, elderly CLL treated with chlorambucil-obinutuzumab to evaluate mutational heterogeneity and clonal hematopoiesis of indeterminate potential (CHIP) during treatment and follow-up. Tumor-only variants were filtered, restricted to nonsynonymous or loss-of-function coding/splice-site mutations, and summarized as a binary patient-by-gene matrix for principal component analysis (PCA), trajectory analysis, and k-means clustering. CHIP was defined as ≥1 qualifying mutation in a prespecified CHIP gene set. Baseline PCA was more compact in patients with complete response at end of treatment, whereas partial response or progressive disease cases were more dispersed. PCA trajectories were compact and directionally consistent in complete responders, more dispersed in partial responders, and highly heterogeneous without a dominant direction in progressive disease. Clustering identified dispersed and compact clusters, and CHIP-associated mutations were enriched in the dispersed cluster (55.6% vs. 8.3%, Fisher's exact p = 0.0086). In paired samples collected 3-5 months after end of treatment, CHIP status changed in some patients. Serial WES may provide complementary information to treatment response, although these observations require confirmation in larger cohorts.

Deuterium abundance has been proposed as a modulator of cellular metabolism; however, its influence on cancer-associated gene expression networks remains incompletely characterized. We analyzed A549 lung adenocarcinoma cells cultured across four deuterium concentrations (40, 80, 150, and 300 ppm) using NanoString nCounter profiling. Expression data were processed through multistep filtering, symbolic trajectory encoding, and density-based spatial clustering (DBSCAN) to identify extreme expression responders, and Gaussian mixture modeling (GMM-6) to resolve coordinated gene-expression modules. DBSCAN identified 11 outlier genes under deuterium depletion, including reduced expression of multidrug-resistance-associated ABCB1 (-42% at 80 ppm), proliferative signaling component FGFR4 (-19%), and transcriptional amplifier MYCN (-24%). In contrast, enrichment at 300 ppm produced a broad increase in oncogenic expression (mean +44%), with marked elevation of inflammation-related (IL6, TGFBR2) and invasion-associated (MMP9) genes. GMM-6 clustering of the remaining core network resolved six functional modules, indicating that depletion preferentially reduces expression of genes associated with plasticity-related programs (Cluster 5: TGFB1, S100A4), while basal survival-associated genes (Cluster 6: BIRC5, RET) remain comparatively stable. Together, these results indicate that deuterium concentration acts as a bidirectional modulator of gene expression programs in the A549 model, with enrichment broadly elevating oncogenic expression and moderate depletion associated with selective downregulation of genes linked to resistance, signaling, and invasive behavior. Significance: Deuterium depletion is associated with reduced expression of genes involved in multidrug resistance, growth-factor signaling, and transcriptional amplification, revealing deuterium-responsive transcriptional vulnerabilities within the A549 lung adenocarcinoma model.