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Chemoresistance is a major obstacle in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. Here we identify hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance by developing chemoresistant TNBC tumors in vivo and characterizing their transcriptomes by RNA-sequencing. Inhibiting LOX reduces collagen cross-linking and fibronectin assembly, increases drug penetration, and downregulates ITGA5/FN1 expression, resulting in inhibition of FAK/Src signaling, induction of apoptosis and re-sensitization to chemotherapy. Similarly, inhibiting FAK/Src results in chemosensitization. These effects are observed in 3D-cultured cell lines, tumor organoids, chemoresistant xenografts, syngeneic tumors and PDX models. Re-expressing the hypoxia-repressed miR-142-3p, which targets HIF1A , LOX and ITGA5 , causes further suppression of the HIF-1α/LOX/ITGA5/FN1 axis. Notably, higher LOX, ITGA5, or FN1, or lower miR-142-3p levels are associated with shorter survival in chemotherapy-treated TNBC patients. These results provide strong pre-clinical rationale for developing and testing LOX inhibitors to overcome chemoresistance in TNBC patients. The development of chemoresistance is a major hurdle in triple negative breast cancer (TNBC). Here, the authors show that lysyl oxidase (LOX) is overexpressed in chemoresistant TNBCs, and when inhibited reduces collagen cross-linking, fibronectin fibril assembly, and downstream integrin signalling, overcoming resistance.

Deregulation of chemokine CCL8 expression is common in various malignancies and other pathologies and plays a causative role in disease progression. However, despite CCL8's acknowledged role in pathology, inhibition of its activity does not represent a strategy of choice for cancer management. This is because its function overlaps with that of the other structurally related chemokines, and its activity is mediated by more than one receptor. To overcome this limitation, we hypothesized that ablation of CCL8 cellular targets, as opposed to disruption of CCL8 activity, may be more advantageous. Therefore, we developed DTCCL8, a chimeric cytotoxic peptide that delivers diphtheria toxin into cells expressing CCL8 receptors which are overexpressed in both the cells of tumor microenvironment and the cancer cells. The specificity of this peptide was confirmed in vitro by testing the cytotoxic activity of breast cancer cells overexpressing CCR5, a major CCL8 receptor, and by a neutralizing anti‐CCL8 antibody we developed. In vivo, DTCCL8 transiently reduced lymphocytes in blood, and its anticancer activity was confirmed in mouse breast cancers triggered by the polyoma middle T oncogene. These findings suggest that DTCCL8 can be used as a prototype for the development of a novel class of breast cancer therapeutics, targeting chemokine targets instead of inhibiting their activity. These cytotoxic peptides may also be useful for managing cancers and other immune system‐associated pathologies. DTCCL8 is a recombinant fusion toxin that targets cancer cells expressing chemokine receptors. By combining diphtheria toxin with CCL8, DTCCL8 binds to multiple receptors on tumor cells and induces selective cytotoxicity. This strategy enables receptor‐mediated targeting of cancer and may support the development of chemokine‐guided therapeutics.

Detection and identification of blood, semen and saliva stains, the most common body fluids encountered at a crime scene, are very important aspects of forensic science today. This study targets the development of a nondestructive, confirmatory method for body fluid identification based on Raman spectroscopy coupled with advanced statistical analysis. Dry traces of blood, semen and saliva obtained from multiple donors were probed using a confocal Raman microscope with a 785-nm excitation wavelength under controlled laboratory conditions. Results demonstrated the capability of Raman spectroscopy to identify an unknown substance to be semen, blood or saliva with high confidence.

Raman spectroscopy has demonstrated remarkable capabilities in identifying blood in controlled laboratory conditions. However, substrate interference presents a significant challenge toward characterizing body fluid traces with Raman spectroscopy at a crime scene. Here, several possible solutions are explored, including the selection of laser excitation, isolating the signal of blood using spectral subtraction and using a favorable substrate for collection which minimizes interference. Simulated blood stain evidence was prepared and analyzed using a Raman microscope with variable laser capabilities. It is shown that the best approach for detecting blood depends on the nature of the substrate and the type of interference encountered.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Stress granule formation is a type of liquid–liquid phase separation in the cytoplasm, leading to RNA–protein condensates that are associated with various cellular stress responses and implicated in numerous pathologies, including cancer, neurodegeneration, inflammation, and cellular senescence. One of the key components of mammalian stress granules is the DEAD-box RNA helicase DDX3, which unwinds RNA in an ATP-dependent manner. DDX3 is involved in multiple steps of RNA metabolism, facilitating gene transcription, splicing, and nuclear export and regulating cytoplasmic translation. In this study, we investigate the role of the RNA helicase DDX3’s enzymatic activity in shaping the RNA content of ribonucleoprotein (RNP) condensates formed during arsenite-induced stress by inhibiting DDX3 activity with RK-33, a small molecule previously shown to be effective in cancer clinical studies. Using the human osteosarcoma U2OS cell line, we purified the RNP granule fraction and performed RNA sequencing to assess changes in the RNA pool. Our results reveal that RK-33 treatment alters the composition of non-coding RNAs within the RNP granule fraction. We observed a DDX3-dependent increase in circular RNA (circRNA) content and alterations in the granule-associated intronic RNAs, suggesting a novel role for DDX3 in regulating the cytoplasmic redistribution of non-coding RNAs.

Conventional confirmatory biochemical tests used in the forensic analysis of body fluid traces found at a crime scene are destructive and not universal. Recently, we reported on the application of near-infrared (NIR) Raman microspectroscopy for non-destructive confirmatory identification of pure blood, saliva, semen, vaginal fluid and sweat. Here we expand the method to include dry mixtures of semen and blood. A classification algorithm was developed for differentiating pure body fluids and their mixtures. The classification methodology is based on an effective combination of Support Vector Machine (SVM) regression (data selection) and SVM Discriminant Analysis of preprocessed experimental Raman spectra collected using an automatic mapping of the sample. This extensive cross-validation of the obtained results demonstrated that the detection limit of the minor contributor is as low as a few percent. The developed methodology can be further expanded to any binary mixture of complex solutions, including but not limited to mixtures of other body fluids.

The unfolded protein response (UPR) is involved in the pathogenesis of metabolic disorders, yet whether variations in the UPR among individuals influence the propensity for metabolic disease remains unexplored. Using outbred deer mice as a model, we show that the intensity of UPR in fibroblasts isolated early in life predicts the extent of body weight gain after high-fat diet (HFD) administration. Contrary to those with intense UPR, animals with moderate UPR in fibroblasts and therefore displaying compromised stress resolution did not gain body weight but developed inflammation, especially in the skin, after HFD administration. Fibroblasts emerged as potent modifiers of this differential responsiveness to HFD, as indicated by the comparison of the UPR profiles of fibroblasts responding to fatty acids in vitro, by correlation analyses between UPR and proinflammatory cytokine-associated transcriptomes, and by BiP (also known as HSPA5) immunolocalization in skin lesions from animals receiving HFD. These results suggest that the UPR operates as a modifier of an individual's propensity for body weight gain in a manner that, at least in part, involves the regulation of an inflammatory response by skin fibroblasts. This article has an associated First Person interview with the first author of the paper.

HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 μM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 μM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1β. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.

Evaluation of gene co‐regulation is a powerful approach for revealing regulatory associations between genes and predicting biological function, especially in genetically diverse samples. Here, we applied this strategy to identify transcripts that are co‐regulated with unfolded protein response (UPR) genes in cultured fibroblasts from outbred deer mice. Our analyses showed that the transcriptome associated with RASSF1, a tumor suppressor involved in cell cycle regulation and not previously linked to UPR, is highly correlated with the transcriptome of several UPR‐related genes, such as BiP/GRP78, DNAJB9, GRP94, ATF4, DNAJC3, and CHOP/DDIT3. Conversely, gene ontology analyses for genes co‐regulated with RASSF1 predicted a previously unreported involvement in UPR‐associated apoptosis. Bioinformatic analyses indicated the presence of ATF4‐binding sites in the RASSF1 promoter, which were shown to be operational using chromatin immunoprecipitation. Reporter assays revealed that the RASSF1 promoter is responsive to ATF4, while ablation of RASSF1 mitigated the expression of the ATF4 effector BBC3 and abrogated tunicamycin‐induced apoptosis. Collectively, these results implicate RASSF1 in the regulation of endoplasmic reticulum stress‐associated apoptosis downstream of ATF4. They also illustrate the power of gene coordination analysis in predicting biological functions and revealing regulatory associations between genes. RASSF1 is a tumor suppressor involved in cell cycle regulation and not previously linked to unfolded protein response (UPR). By evaluating gene coordination in genetically diverse samples, we found RASSF1 is highly correlated with transcriptomes of several UPR‐related genes. Our further investigations identified RASSF1 as a target of ATF4 in the regulation of endoplasmic reticulum (ER) stress‐associated apoptosis.