Cancer Gene Therapy

While recent therapeutic advances have extended the survival of patients with non-small cell lung cancer (NSCLC), overcoming metastatic progression in the CNS remains a significant challenge. Some patients with NSCLC may require concurrent management of CNS and extracranial metastases, while others develop isolated brain metastasis or leptomeningeal disease. These heterogenous clinical outcomes are difficult to predict and diagnose for early intervention with current surveillance modalities. Herein, we comprehensively analyzed gene mutations, copy number variations, and DNA methylation of NSCLC brain metastasis tissue collected at the time of craniotomy, combined with ctDNA sequencing of paired plasma and CSF liquid biopsies. We confirmed a high concordance between the molecular features of brain metastasis tissue with ctDNA from CSF which were largely distinct from ctDNA alterations in paired plasma samples. Plasma ctDNA tumor fraction and ctDNA hypermethylation were most significantly associated with extracranial metastasis and overall survival. Alternatively, we identified specific hypermethylated DNA loci in brain metastasis tissue and CSF ctDNA as significant correlates of brain metastasis progression and risk of leptomeningeal disease. Our findings support the utility of integrating ctDNA testing from CSF and plasma, while revealing distinct epigenetic features and biomarkers of brain metastasis or leptomeningeal disease.

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

TP53 (also called TRP53 or p53) is a critical tumour suppressor that prevents cancer development by inducing a transcriptional program which can lead to diverse cellular responses, most prominently, cell proliferation arrest/senescence with survival of cells or cell death by apoptosis. Why distinct cell types undergo different outcomes after p53 activation remains unclear. Using integrated RNA-sequencing, proteomic and functional analyses across a diverse range of murine primary cell types, we demonstrate that cell fate is governed by the balance between pro-survival BCL-2 and pro-apoptotic BH3-only proteins. Cells resistant to apoptosis displays a higher starting ratio of pro-survival BCL-2 to pro-apoptotic BH3-only proteins, along with transcriptional upregulation of the pro-survival gene Bcl2l1, encoding BCL-XL. This control of cell fate is also seen in human wild-type p53 cancer cell lines. These findings reveal the mechanism for understanding p53-driven cell fate decisions, suggest therapeutic strategies to shift p53-induced cell proliferation arrest/senescence toward apoptotic cell death and allowed generation of an RNAseq data-based predictor of outcome for cancer cells after p53 activation.

Abstract Background: The lack of effective biomarkers and therapeutic targets to overcome radioresistance in cervical cancer remains a major clinical challenge. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase pivotal in immune and inflammatory signaling, has been implicated in various malignancies. However, its role in radioresistance in cervical cancer remains unclear. Methods: TRAF6 expression was evaluated in cervical cancer tissues from 162 patients who underwent postoperative radiotherapy at our institution and in 304 cases from the TCGA-CESC cohort. The prognostic significance of TRAF6 was assessed using Kaplan-Meier and Cox regression analyses. A nomogram integrating TRAF6 expression with clinicopathological factors was constructed to predict overall survival (OS) and progression-free survival (PFS). The functional role of TRAF6 in malignant phenotypes and radiosensitivity was investigated using shRNA-mediated knockdown in HeLa and C33A cervical cancer cells. Immune cell infiltration patterns associated with TRAF6 expression were analyzed using ssGSEA and xCELL algorithms based on TCGA data. Results: TRAF6 expression was significantly elevated in cervical cancer tissues compared with adjacent normal tissues (70.99% vs. control, P < 0.001) and was higher in radioresistant than in radiosensitive patients (P < 0.001). High TRAF6 expression was associated with shorter OS (HR = 18.73, P = 0.004) and PFS (HR = 8.44, P < 0.001) and was identified as an independent risk factor for radiotherapy resistance (OR = 8.44, P < 0.001). The TRAF6-integrated nomogram demonstrated good predictive accuracy for OS (C-index = 0.7351) and PFS (C-index = 0.7444). TRAF6 knockdown in cervical cancer cells significantly suppressed proliferation, migration, and invasion, while substantially enhancing radiosensitivity of tumor cells. Functional enrichment analysis revealed that TRAF6-related genes were enriched in autophagy, mitophagy, and HPV infection pathways. Immune cell infiltration analysis showed that TRAF6 expression correlated with distinct immune cell profiles, characterized by enrichment of activated dendritic cells, M1 macrophages, and regulatory T cells, alongside depletion of cytotoxic effectors such as CD8+ T cells and {gamma}{delta} T cells. Conclusions: TRAF6 could be a prognostic biomarker associated with poor outcomes and indicator of radiotherapy resistance in cervical cancer, TRAF6 represents a potential therapeutic target for overcoming radioresistance in cervical cancer.

Young-onset lung cancer is enriched for never-smoking and oncogene-driven tumors, yet its inherited genetic basis remains poorly defined. We performed germline whole-genome sequencing in 251 young-onset lung cancer cases (median age 37), which we jointly analyzed with never-smoking cases (n=196; median age 68) and cancer-free controls (n=1,883). We identified enrichments of rare deleterious coding variants across 55 cancer-related gene sets, including EGFR/ERBB2 signaling and genes implicated by prior lung cancer GWAS. Exome-wide analyses of rare coding variants affirmed TP53 as a penetrant lung cancer predisposition gene (odds ratio [OR]=36.1, p=1.02x10-7) and discovered two novel exome-wide significant tumor subtype-dependent associations: IREB2 in cases with fusion-driven tumors (p=1.39x10-6) and SMAD6 in fusion-negative tumors (p=2.05x10-6). Structural variants contributed distinct risk, with enrichment in constrained, lung-expressed genes (OR=5.79, p=5.8x10-5) and very large germline deletions being markedly enriched in cases with fusion-driven tumors. Polygenic risk scores for lung cancer were inversely correlated with rare variant burden, consistent with additive risk from rare and common variants. Collectively, these findings delineate a complex germline architecture underlying susceptibility and molecular subtype in young-onset lung cancer.

Lung cancer is a highly heterogeneous disease and remains the leading cause of cancer-related mortality worldwide. While mouse models and patient-derived organoids have advanced our understanding of lung cancer, key interactions within the tumor microenvironment (TME) remain poorly characterized. We developed microtumor models from lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC) using mouse and patient samples, including surgical resections and rapid autopsy specimens. Microtumors preserve structural, cellular, and molecular features of the native TME, enabling mechanistic studies of tumor progression ex vivo. Multi-omics analyses of LUAD microtumors revealed progression-associated changes, including increased epithelial-to-mesenchymal transition (EMT) and metabolic reprogramming toward fatty acid synthesis. Pharmacologic inhibition of fatty acid synthesis through ACC1/2 reduced proliferation in patient-derived microtumors, identifying a targetable vulnerability. This platform provides a robust system for studying tumor progression, therapeutic response, and resistance mechanisms in lung cancer, including culturing postmortem specimens that are not accessible in current models.

Peripheral whole-blood gene expression profiling offers a minimally invasive route to lung cancer detection, but high-dimensional transcriptomic data are prone to optimistic bias when preprocessing and model selection are not properly separated from performance evaluation. We applied L1-penalised (LASSO) logistic regression to 303 peripheral whole-blood microarray profiles (123 lung cancer cases and 180 healthy controls; Gene Expression Omnibus accession GSE252168; Illumina HumanHT-12 v4) within a leakage-free nested cross-validation framework (5 outer and 3 inner folds), in which all data-dependent steps (imputation, univariate feature screening by ANOVA F-test with k = 500, and standardisation) were confined strictly to training partitions. Statistical significance was assessed by permutation testing (B = 100), and feature selection stability was quantified across outer folds. LASSO was compared with ridge logistic regression, linear support vector machines, and random forest under the same framework. The LASSO model identified a sparse 29-probe signature with a pooled out-of-fold area under the ROC curve (AUC) of 0.990 (nested estimate 0.989 +/- 0.015), accuracy 97.4%, sensitivity 94.3%, and specificity 99.4% at a 0.50 threshold; permutation testing confirmed significance (p = 0.0099). Six probes, including CDC42, U2AF1, and RPS15A, were selected in all five outer folds, forming a stable core, and all classifiers exceeded AUC 0.987, indicating a strong, algorithm-independent signal. A leakage-free nested cross-validation framework enables unbiased performance estimation and reproducible feature selection in blood-based lung cancer classification. The 29-probe panel is an internally validated candidate requiring prospective, multicentre external validation before clinical use.

Background & Aims: Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality, with most patients presenting at advanced stages requiring systemic therapy. Despite promising outcomes with immune checkpoint inhibitors (ICI), responses remain variable due to an immunosuppressive tumor microenvironment. Y90 radioembolization offers potential immune priming, but only a subset of patients benefit. Here, we apply spatial multi-omics to delineate baseline and treatment-induced immune features and identify predictive signatures of progressive disease (PD) for early detection of patients unlikely to benefit from therapy. Approach & Results: Paired baseline (Day 0) and on-treatment (Day 35) biopsies were obtained from 33 patients, following Y90 radioembolization (Day 14) and nivolumab. Multiplex immunohistochemistry (mIHC) was used for cell-cell interaction analysis. A subset was further profiled using Visium (n=13) for tissue category-specific analysis and NanoString GeoMx DSP (n=12) for cell type-resolved transcriptomic and pathway analyses. Global spatial transcriptomics analysis revealed minimal baseline immune activity in PD, indicating an intrinsically immune-deficient TME. Despite treatment-induced activation, PD exhibited reduced CD8+ T cell abundance and limited reinvigoration of exhausted subsets, and persistent LAG-3-associated exhaustion. DSP showed downregulation of antigen presentation and T cell activation pathways. Macrophage profiling revealed enrichment of CD38+ phenotypes, contrasting CXCL9-CXCR3-associated responses in responders. Furthermore, a 72-gene PD signature was identified and validated in TCGA, associating with poorer survival. Conclusions: Integrated spatial multi-omics reveals that PD in HCC is associated with an immune-deficient TME, characterized by LAG-3-associated CD8+ exhaustion and immunosuppressive macrophages. A 72-gene signature enables early identification and supports alternative therapeutic strategies.

Deregulation of sphingolipid (SL) metabolism is well-established across many cancers, yet the underlying mechanisms that drive changes in SLs are poorly understood. We previously identified dihydroceramide desaturase 1 (DES1) as a downstream target of HER2 and implicated DES1 as a driver of anchorage-independent survival in breast cancer. In this study, we expand on these results to establish the oncogenic PI3K pathway as a driver of post-translational DES1 activity following cell detachment from the extracellular matrix. PI3K activation of DES1 required glucose uptake and metabolism through both glycolysis and the pentose phosphate pathway. However, it did not require glucose flux into the TCA cycle and was independent of antioxidant capacity of the cell. Moreover, Instead, results identify GAPDH - a point of convergence between glycolysis and PPP - as important for oncogene-driven DES1 activity. Overall, this study defines a novel pathway of DES1 regulation and establishes DES1 as a point of crosstalk between glucose and SL metabolic pathways.

Scaffold integrity is essential for the activity of proteins that function through protein-protein interactions rather than catalytic output. RAF1 exemplifies this duality: although it is a bona fide kinase and a core component of the MAPK cascade, its tumor-promoting role is largely kinase-independent, relying instead on scaffold-mediated suppression of apoptosis. Genetic Raf1 ablation in KRAS-driven lung adenocarcinoma mouse models induces tumor regression without systemic toxicity, making it an attractive candidate for targeted protein degradation. Chemogenetic systems like the dTAG platform are widely used for preclinical target validation. Here, we generated a dTAG-RAF1 mouse model and showed that pharmacological degradation is efficient and systemically well tolerated, but fails to reproduce the tumor regression observed upon genetic Raf1 ablation. Mechanistically, the N-terminal FKBP12F36V tag (dTAG) perturbs the RAF1 interactome, including scaffold associations with apoptotic regulators, thereby blunting the phenotypic consequences of its degradation. These results establish scaffold integrity as a determinant of chemogenetic system fidelity and argue that degradation tools must be validated at the functional level, not only for target elimination, before assessing their therapeutic relevance.

SMAD4 is a central transcriptional effector of the TGF-{beta} signaling pathway and a frequently inactivated tumor suppressor gene in various cancers. Missense mutations in its MH2 domain are among the most prevalent somatic alterations in colorectal cancer (CRC). These mutations are associated with disease progression and poor prognosis, yet their precise mechanistic consequences have remained incompletely characterized. Here, we show that CRC-derived SMAD4 MH2 hotspot mutations (D351H, S357P, R361C, and R361H) selectively impair co-activator recruitment without disrupting chromatin occupancy. RNA-seq profiling demonstrated broad suppression of TGF-{beta} target gene expression across all mutants. Notably, the mutations confer distinct degrees of TGF-{beta} pathway unresponsiveness: R361H is completely refractory to TGF-{beta} stimulation, whereas R361C and S357P retain partial transcriptional responsiveness suggesting allele-specific differences in the severity of co-activator interface disruption. Genome-wide chromatin binding analysis by greenCUT&RUN confirmed that all mutants maintain wild-type-like genomic occupancy, as expected given that the MH1 DNA-binding domain is intact in each case. Proximity-dependent biotinylation mass spectrometry in COLO205 cells revealed that all four mutants exhibit markedly reduced interactions with the CREBBP/EP300 histone acetyltransferase complex and BRD4 relative to wild-type SMAD4 identifying disrupted co-activator engagement. Collectively, our findings establish that SMAD4 MH2 mutations impair TGF-{beta}-induced transcription by selectively reducing CREBBP/EP300 recruitment, which provides a molecular mechanism for the loss-of-function SMAD4 phenotype in CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/735541v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@14f542eorg.highwire.dtl.DTLVardef@11fd220org.highwire.dtl.DTLVardef@1c3aa1org.highwire.dtl.DTLVardef@14d5a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Melanoma, one of the most metastatic and multidrug resistant cancer, is the first leading cause of death from skin cancer. This complex disease requires identification of additional cooperating events that contribute to progression, invasion and metastasis to reinforce therapeutics. RhoGTPases play key roles in cancer development and metastasis. Rhophilin-2 (RHPN2), a Rho effector, is amplified in various human cancers and its role in melanoma remains unexplored. Here, we combined knock-down experiments in human melanoma cells, with knock-out and overexpression experiments in zebrafish to uncover the roles of RHPN2 in melanoma development. We show that in human melanoma cells RHPN2 contributes to growth, and to clonogenic, migratory and invasive properties of the cells. Using NRASQ61L and BRAFV600E zebrafish models, we provide the first in vivo evidence that Rhpn2 promotes melanoma onset and development. Histological analysis of the Rhpn2 deficient tumors showed decreased cellular density and absence of primary cilia structures at the invasive tumor/stroma borders. Transcriptomic profiling of the Rhpn2-KO melanoma revealed increased expression of the IFN1-responsive genes and modulation of genes involved in lipid metabolism and cilia function. Together these findings position RHPN2 as a modulator of melanoma, offering new perspectives in considering it as a target to impair the development of the tumor.

TMEM127 is an adaptor protein that bridges substrates to E3 ubiquitin ligases of the HECT family. Among its interacting partners is the major histocompatibility class I (MHC-I), a critical component of the antigen presentation pathway and the adaptive immune response. MHC-I is ubiquitinated and fated for lysosome-mediated degradation by the WWP2 E3 ligase in a complex that involves TMEM127 and a second adaptor protein, SUSD6. However, the interacting dynamics among complex components remains to be determined, a key knowledge gap towards the development of pharmacological modulators. Here, using in vitro and in vivo models, we report that TMEM127-WWP2 interaction stabilizes the MHC-I degradation complex and reveals an asymmetric role of the two adaptor proteins. Specifically, we find that TMEM127 regulates WWP2 catalytic activity, abundance and localization through its canonical PY motif interaction with the WW domain of WWP2 with contribution of a TMEM127 endocytic motif, providing a mechanism to restrain complex activity. Further, we validate the impact of TMEM127 dosage in the endogenous complex assembly and regulation. Our results nominate TMEM127 as a critical member of the MHC-I degradation complex and highlight the TMEM127-WWP2 interaction as a target for augmenting MHC-I-mediated antigen presentation, a long sought goal in cancer immunology.

Structural AbstractO_ST_ABSBackgroundC_ST_ABSTriple-negative breast cancer (TNBC) represents a major clinical challenge due to its aggressiveness, heterogeneity and limited availability of effective targeted therapy. We investigated whether LRP-1, a multifunctional cell-surface endocytic and signaling receptor, contributes to TNBC progression. MethodsUsing CRISPR-Cas9, LRP-1-deficient murine 4T1 and human HS578-T TNBC cells were used. Functional consequences were assessed through migration, invasion, and 3D spheroid assays, imaging of focal adhesions and actin organization, atomic force microscopy, and plasmin activity assays. Global molecular reprogramming was analyzed by label-free quantitative proteomics and secretomics. LRP-1-deficient or proficient 4T1 cells were implanted orthotopically in immunocompetent mice; tumor progression was monitored longitudinally while peritumoral collagen architecture and immune microenvironment composition were characterized by second harmonic generation imaging and immunohistochemistry. ResultsWe show that LRP-1 loss reduces TNBC aggressiveness, as reflected by decreased migration and invasive capacity, reduced spheroid evasion, and significant morphological changes in focal adhesion and actin structure. LRP-1-deficient cells became stiffer and showed lower LOXL-4 levels, while pericellular proteolytic activity remained unchanged, suggesting other proteases mechanism. Multi-omic analysis revealed alterations in extracellular matrix (ECM), epithelial-mesenchymal transition, and inflammatory pathways. In vivo, LRP-1-deficiency reduced tumor progression and peritumoral collagen deposition, while increasing CD8+ T and Natural Killer cell infiltration, together with a cytokine profiling compatible with a more immune-permissive microenvironment. ConclusionsLRP-1 act as a key contributor in TNBC progression through matrix remodeling, mechano-adaptation, and immune exclusion. Positioning it as a candidate biomarker for TNBC patients who are likely to benefit from stroma-targeting therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/732906v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b595c2org.highwire.dtl.DTLVardef@7b208aorg.highwire.dtl.DTLVardef@1956e54org.highwire.dtl.DTLVardef@17e55d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The intensification of neoadjuvant therapy for early triple-negative breast cancer (eTNBC) - through the addition of carboplatin to standard chemotherapy and the incorporation of pembrolizumab - has markedly improved prognosis in recent years. However, this escalation carries a substantial risk of toxicity, and not all patients require the full regimen to achieve benefit. Realizing individualized treatment strategies will therefore depend on prognostic and predictive biomarkers that can forecast treatment response and long-term outcome. In the present study, we interrogated public gene expression datasets to develop transcriptomic signatures predicting response to neoadjuvant treatment and risk of recurrence. To validate these signatures, we used the Multiplex8+ platform for spatially informed comprehensive transcriptomic profiling in a real-world, multicenter, retrospective cohort of 590 patients diagnosed with eTNBC and treated with neoadjuvant chemotherapy with or without immunotherapy. The diagnostic Multiplex8+ test uses H&E and multiplexed RNA-FISH to guide the selection of specific tumor areas for the whole transcriptome sequencing and signature analysis. In the real-world cohort, the Multiplex8+ signatures were associated with both response and prognosis, remaining highly significant in multivariable models that included clinical parameters. The signatures were complementary to established biomarkers such as stromal tumor-infiltrating lymphocytes. These findings warrant prospective integration of the signatures into risk-stratified clinical trials to support future de-escalation and escalation strategies, enabling a better balance of efficacy, toxicity, cost, and drug availability.

Intracellular iron homeostasis is increasingly recognized as a regulator of cancer cell behavior, but how iron distribution influences extracellular matrix (ECM) organization and invasion remains poorly understood. Here, we show that loss of divalent metal transporter 1 (DMT1/SLC11A2) disrupts intracellular iron homeostasis and promotes cancer cell invasion through an iron-ER-ECM axis. In MDA-MB-231 cells, DMT1 knockout (KO) reduced total iron content but increased the labile iron pool (LIP) in both 2D and 3D culture models, indicating altered intracellular iron distribution. Across transcriptomic and phenotypic readouts, DMT1-dependent effects were more evident in 3D than in 2D models, with DMT1 KO inducing endoplasmic reticulum (ER) stress and impaired collagen/ECM organization. Functionally, the DMT1-loss phenotype was marked by reduced 2D motility, whereas in 3D spheroid models DMT1 KO cells displayed enhanced invasive outgrowth in both Matrigel and collagen matrices. Iron chelation further modulated this phenotype in a DMT1-dependent manner. Pharmacologic induction of ER stress phenocopied the loose spheroid architecture and invasive behavior, supporting ER stress as a mechanistic link between altered iron handling and ECM destabilization. Together, these findings identify intracellular iron distribution, rather than total iron abundance alone, as a determinant of ECM integrity and context-dependent cancer cell invasion. Significance StatementOur study identifies an iron-ER-ECM axis through which intracellular iron homeostasis regulates cancer cell invasion. Total cellular iron content alone is insufficient to predict invasive behavior without considering how iron is distributed within the cell. By preserving intracellular iron homeostasis and ER function, DMT1 supports collagen synthesis and maintains ECM integrity. In contrast, DMT1 loss disrupts these processes, promoting formation of loosely aggregated spheroids and enhanced invasion in 3D tumor models despite reduced total iron levels. These findings challenge the assumption that lowering bulk iron uniformly suppresses invasive phenotypes and instead highlight intracellular iron trafficking as a potential therapeutic target for limiting cancer cell invasion.

Cytotoxic T lymphocytes (CTLs) play a central role in antitumor immunity; however, metabolic reprogramming within the tumor microenvironment often compromises their effector function, making metabolic targeting crucial for the improvement of T cell function. Folate-dependent purine synthesis, a core pathway sustaining the nucleotide pool, is highly activated in tumors, yet its role in regulating tumor immune sensitivity remains unclear. Here, by establishing a co-culture system of melanoma cells and human T Cell Receptor (TCR)-engineered T cells, we systematically evaluated the effects of folate-dependent purine synthesis inhibitors on tumor cell response to CD8+ T cell cytotoxicity. We found that inhibition of key enzymes such as methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and glycinamide ribonucleotide transformylase (GART) markedly enhanced tumor cell sensitivity to T cell killing, an effect also observed with exogenous nucleoside supplementation. Mechanistically, inhibition of folate-dependent purine synthesis suppresses glycolysis by downregulating critical glycolytic enzymes, thereby reducing lactate production. Reduction in lactate further weakens lactylation and stability of the immune checkpoint protein PD-L1. In parallel, impaired purine synthesis disrupts uridine metabolism, blocks ribose salvage, and distally influences glycolysis. Collectively, our study identified the folate-dependent purine synthesis-glycolysis axis as key regulator of tumor immune response and highlights metabolic targeting as a promising strategy to improve cancer immunotherapy.

TAOK3 is a lesser-studied MAPK family serine/threonine kinase our group has shown to be targeted by HPV integration, suggesting a potential role in driving invasive cervical cancer (ICC). Here, we profiled TAOK3 expression in patient tumors, metastases, and cervical cancer models and localized TAOK3 within a tumor epithelial subpopulation by integrating two single-cell RNA-seq datasets. Functional consequences of TAOK3 loss were assessed with siRNA and CRISPRi in cell lines and 3D spheroids. In vivo effects were evaluated in intracervical xenografts with species-specific RNA-seq to resolve tumor versus microenvironmental responses. TAOK3 mRNA/protein were elevated in primary and metastatic ICC and primarily localized to a keratin-positive epithelial subset (T3epi) enriched for cadherin/S100 binding, vesicle/endocytic pathways, and leading-edge programs. TAOK3 silencing reprogrammed transcriptomes and proteomes toward reduced WNT/cell-cycle and motility signaling, altered endocytosis and cytoskeleton organization, and reshaped phospho-networks linked to chromatin remodeling and ERBB2-ERBB3/cytoskeletal kinase activity. Functionally, TAOK3 inhibition prolonged G2/M, suppressed invasion, and enhanced sensitivity to low dose paclitaxel. Prolonged inactivation induced methuosis-like cell death with extracellular ATP release. In xenografts, TAOK3 knockdown reduced tumor burden, downregulated KRT14--a leader cell marker--within the human tumor compartment, and enriched microenvironmental pathways for immune activation, with a specific decrease in CD206+ M2 macrophages. TAOK3 delineates an invasion-competent epithelial state in ICC and coordinates cell-cycle control, cytoskeleton-membrane dynamics, and tumor-immune crosstalk. Genetic or pharmacologic TAOK3 inhibition constrains tumor growth, potentiates paclitaxel, and remodels the microenvironment toward anti-tumor immunity, supporting TAOK3 as a potential therapeutic target and biomarker in ICC. Statement of SignificanceTAOK3 marks an invasion-competent epithelial subpopulation in cervical cancer. TAOK3 inhibition slows tumor growth, enhances chemoresponse, and reduces M2 macrophages, revealing TAOK3 as a potential therapeutic target and biomarker for patient stratification.

Canine hemangiosarcoma (HSA) is an aggressive endothelial malignancy with limited therapeutic options, and its progression is closely associated with vascular architecture, stromal remodeling, and inflammatory cell recruitment. Sulfoquinovosylacylpropanediol (SQAP) is a sulfoquinovosyl lipid radiosensitizer reported to affect angiogenic and tumor-microenvironmental pathways, but its effects in canine HSA are unknown. Here, we evaluated SQAP in canine HSA cell lines and patient-derived xenograft (PDX) models. SQAP showed minimal direct cytotoxicity against HSA cell lines in vitro, whereas it significantly suppressed tumor growth in three canine HSA PDX models. Transcriptome analysis of SQAP-treated HSA PDX tumors detected more SQAP-responsive genes in mouse host-derived cells than in canine tumor cells. Gene-set enrichment analysis of the mouse host-derived fraction showed positive enrichment of angiogenesis, hypoxia, and stromal remodeling-related gene sets after SQAP treatment. Subsequent tissue analysis showed that SQAP reduced host-derived CD31-positive vascular area and increased -smooth muscle actin coverage of remaining vessels in two of the three PDX models, while altering macrophage-associated marker profiles in a model-dependent manner. These findings indicate that SQAP suppresses canine HSA PDX growth primarily through vascular and macrophage-associated remodeling of the tumor microenvironment rather than direct tumor-cell cytotoxicity.

BackgroundCancer-associated cachexia is characterized by progressive loss of skeletal muscle and adipose tissue driven by systemic inflammation and metabolic dysregulation. AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, but its role in cachexia and its therapeutic potential remains incompletely defined. We investigated AMPK signaling during cachexia and whether pharmacological AMPK activation alone or combined with ghrelin could ameliorate disease manifestations. MethodsCachexia was induced in male C57BL/6 mice by Lewis lung carcinoma (LLC) implantation. Additional models included fibrosarcoma (CHX and MN/MCA1) and chronic lymphocytic choriomeningitis virus (LCMV) infection. AMPK was activated using AICAR and BC1618 (AB), alone or combined with ghrelin (AB+G). Metabolic, inflammatory, and functional outcomes were assessed in hypothalamus, skeletal muscle, adipose tissue, and serum. ResultsLLC-bearing mice developed cachexia characterized by reduced body weight, lean and fat mass, hypophagia, and elevated circulating IL-6 and corticosterone. Cachectic LLC mice displayed increased Il6 and Il1{beta} expression in hypothalamus, skeletal muscle, and white adipose tissue (WAT). Furthermore, AMPK activation failed to increase in hypothalamus or peripheral tissues despite profound energy deficit. A similar defect in AMPK responsiveness was observed in CHX and LCMV models, indicating a conserved feature of cachexia. AB treatment in LLC mice reduced circulating IL-6 and corticosterone levels and decreased skeletal muscle atrogene expression and IL-6/STAT3 signaling, partially preserving muscle mass, fiber size, and grip strength. However, food intake remained low, and WAT was largely unresponsive, maintaining elevated Il6 expression and tissue loss. Ghrelin alone increased food intake in LLC mice but did not ameliorate the cachectic phenotype. In contrast, AB+G restored food intake and prevented loss of lean and fat mass. LLC AB+G mice exhibited reduced hypothalamic Il6 and serotonin transporter (Slc6a4) expression, normalized adipocyte morphology and serum leptin levels, decreased adipose Il6 and Atgl expression and reduced WAT sympathetic innervation. AB+G further lowered circulating corticosterone levels, and provided greater protection against muscle wasting, with increased Pgc1 expression and improved muscle function. Neither intervention affected tumor growth or tumor inflammatory gene expression. ConclusionsCancer cachexia is associated with a central and peripheral failure to appropriately activate AMPK signaling in response to the energetic stress imposed by cachexia. Combined AMPK activation and ghrelin administration exerted complementary effects on energy homeostasis, inflammation, and tissue wasting, resulting in greater protection against cachexia than either intervention alone. These findings support combined AMPK-ghrelin targeting as a promising therapeutic strategy for cancer cachexia.