Gasdermin C (GSDMC), predominantly expressed in the gut, belongs to the Gasdermin (Gsdm) protein family, which only becomes functional after the protease cleavage-mediated release of the N-terminus from their self-inhibited C-terminus. Previous studies have identified caspase 8 as the protease responsible for cleaving human GSDMC in specific cancer cell types, leading to pyroptosis, a form of programmed inflammatory cell death. However, the direct protease that cleaves intestinal GSDMC is unknown. Here, we showed that the protease Cathepsin S (CTSS) is the direct protease for GSDMC. Intriguingly, unlike other Gasdermin N-terminal fragments (GSDMs^N-ter), the CTSS-processed GSDMC^N-ter was found to be ineffective in promoting pyroptosis. This ineffectiveness is attributed to an incomplete lipid interaction motif, which limits its ability to disrupt cellular membranes—a hallmark of pyroptosis. Instead, CTSS-processed GSDMC^N-ter localized to and penetrated RAB7⁺ vesicles, which are considered the marker for late endosomes. Consequently, targeting of RAB7⁺ vesicles resulted in the release of endocytic cargo, resulting in enhanced protein production during plasmid transfection and an improved transfectability of cell lines. Furthermore, Rab7 associates with lipid droplets and regulates lysosome recruitment to lipid droplets. We found that CTSS-generated GSDMC^N-ter resulted in impaired lipid droplet breakdown and turnover under starvation conditions. This newly described biological function expands the role of Gsdm beyond pyroptosis and highlight a novel role for Gsdm proteins in modulating Rab7+ vesicles, such as late endosome/lipid droplet functions.

Introduction:
Lung cancer (LC) is a major cause of death among cancer patients worldwide. About 30-55% of LC patients suffer from brain metastasis (BM), with lung adenocarcinoma (LUAD) being one of the main tumor types that develop BM. Consequently, researchers have been increasingly conducting studies related to BM-LUAD in recent years. However, due to the unique characteristics of BM tumors, obtaining genuine BM cells remains challenging. Therefore, researchers often use nude mouse models to establish BM cell lines. Previous methods have several shortcomings, such as high costs, lengthy timeframes, the need for specialized imaging equipment, and difficulties in capturing early features of BM occurrence. To address these issues, we employed an improved method and successfully obtained various BM-LUAD cell lines that exhibit different stage features of BM.

Methods:
By performing low-cell-number (500-4000 cells) circulating intracranial injections in nude mice, we obtained the H1975-BM1, BM2, and BM3 cell lines. We then conducted RNA-Seq and utilized various bioinformatics analyses to reveal the transcriptomic characteristics of the different stages of BM cell lines. Subsequently, we validated our findings through experiments including Transwell assays, CCK8, cell adhesion, and nude mice subcutaneous tumor implantation.

Results:
In the H1975-BM1 cell line, the tumor cells are more focused on proliferation and migration. In contrast, in the H1975-BM3 cell line, the function of the tumor cells shifts more toward cell adhesion and secretion, particularly the secretion of various cytokines related to immune cell recruitment. Subcutaneous tumor implantation also confirmed this finding, as H1975-BM3 exhibited a larger tumor volume and formed a bigger secretion cystic cavity.

Conclusions:
We successfully obtained high-confidence BM-LUAD cell lines through an improved new method, revealing distinct characteristics between the different stage cell lines of BM-LUAD.

Introduction: Oesophageal cancer is the eleventh most diagnosed malignancy and the seventh most common cause of cancer-associated mortality globally. Studies have shown that cancer stem cells (CSCs) are the main drivers for resistance in oesophageal cancer. Photodynamic therapy (PDT), a light-based treatment strategy, has demonstrated efficiency against several cancers. However, the therapeutic effect of aluminium phthalocyanine chloride tetra sulfonate (AlPcS₄Cl)-mediated PDT in inducing oxidative stress and potentiating apoptotic activity in oesophageal CSCs is limited. This study examined the effects of AlPcS₄Cl in promoting oxidative stress and apoptotic cell death in HKESC-1 human oesophageal CSCs.

Method: The CSCs were isolated from HKESC-1 cells and grouped into control and treatment groups. The CSC treatment group was exposed to AlPcS₄Cl-PDT at 5J/cm². Twenty-four hours after PDT, the anticarcinogenic actions of AlPcS₄Cl on oesophageal CSCs in inhibiting cell growth and promoting oxidative stress and cell death were evaluated. Cell viability was assessed using a viability assay; a cellular ROS assay was used to determine the induction of oxidative stress, rhodamine-123 flow cytometry analysis was used for mitochondria membrane potential, Annexin V-FITC/PI double-staining flow cytometry analysis for cell death mechanism, and Caspase-Glo 3/7 fluorometric assay for caspase activities. All treatment and control cells were conducted in biological and technical replicates. GraphPad Prism (v5) was used to collate the results and perform statistical analysis. The treatment groups were compared relative to the control cells. One-way ANOVA was used, and a p-value of 0.05 indicated statistical significance.

Results: Findings from our study showed that AlPcS₄Cl-PDT with 5 J/cm² irradiation significantly inhibited cell growth, induced oxidative stress via increased intracellular ROS production, altered the integrity of mitochondria membrane potential, and induced caspase 3/7-mediated apoptosis in oesophageal CSCs.

Conclusion: This study demonstrates the promising use of AlPcS₄Cl-PDT in eradicating oesophageal CSCs and improving prognosis.

Introduction. The G protein-coupled estrogen receptor (GPER) is known for its ability to mediate rapid estrogen signaling in diverse normal and malignant cell contexts, including breast cancer (BC). Of note, the role of the GPER in promoting pro-tumorigenic traits in triple-negative breast cancer (TNBC) has been suggested. In this study, we sought to provide novel insights into the transcriptionally guided biological behavior of TNBC cells lacking GPER expression.

Methods. GPER knock-out (KO) MDA-MB-231 TNBC cells were obtained using the CRISPR/Cas9 genome editing technology. RNA sequencing (RNA-seq) and Gene Ontology (GO) enrichment analyses served to assess the differentially expressed genes (DEGs) between the GPER KO and wild-type (WT) cells and their biological role. Chromatin immunoprecipitation assays, real-time PCR, immunoblots, immunofluorescence, ELISA, and flow cytometric experiments, as well as the use of RNA interference techniques, allowed us to uncover the molecular routes implicated in the biological features triggered by GPER silencing in the TNBC cells. Survival analyses were performed on TNBC patients ffrom the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset.

Results. The RNA-seq data revealed that GPER silencing in MDA-MB-231 cells induces a pro-apoptotic gene signature. Accordingly, the biological assays demonstrated that GPER KO cells exhibit enhanced mitochondria-dependent apoptotic death with respect to these levels in WT cells. Mechanistically, we found that reduced cAMP levels in GPER KO cells may trigger the activation of the pro-apoptotic JNK/c-Jun/p53/Noxa pathway. In accordance with these results, the bioinformatics analyses on the TNBC patients revealed that high NOXA gene expression levels are associated with better outcomes.

Conclusions. Collectively, our findings unveil the role of the GPER in sustaining anti-apoptotic signals in TNBC cells, thus suggesting this receptor as a potential valuable therapeutic target for preventing the progression of this malignancy.

Cachexia is a syndrome characterized by significant weight loss and is a major complication in cancer development. Despite its importance, there are no effective treatments available. Recently, resistance training has been suggested as a non-pharmacological therapy to prevent muscle atrophy and functional decline in preclinical models. The C26 tumor-bearing mouse model is commonly used, showing progressive muscle and fat loss along with systemic inflammation. However, the impact of these changes on the cerebellum, which is crucial for motor function and balance, remains unclear. In this study, we combined the C26 tumor model with an endurance protocol to investigate cerebellar alterations in sedentary (SED T+) and trained (TR T+) tumor groups compared to sedentary (SED T-) and trained (TR T-) non-tumor groups. The tumor groups were sacrificed at the onset of cachexia to assess the impact of proactive endurance training. After confirming cachexia onset through body weight and strength loss, histological analysis revealed that the cerebellar area was similar across groups. However, a significant reduction in the Purkinje cell layer (PCL) thickness was observed in SED T+ mice. Since PCs are the sole efferent neurons in the cerebellum, we examined their condition using histological analysis, immunohistochemistry, and immunofluorescence by ZIC4 labelling. The PC body size decreased in the tumor groups, while their number was reduced only in TR T+ mice. Interestingly, the SED T+ group exhibited the highest number of PC with abnormal morphology. Additionally, to assess the effects on cerebellar vascularity, we found that tumor groups had an increased number of vessels in the white matter. Altogether, these results suggest that in the C26 tumor mouse model, the cerebellum is affected by PCL hypotrophy with smaller and abnormal PC. Endurance training only partially counteracts these alterations. Further studies are needed to clarify Purkinje cell aberrations due to their crucial role in motor function.

Introduction Cancer cells exploit aerobic glycolysis, also known as the Warburg effect, which consists of the fermentation of pyruvate into lactate, even in the presence of oxygen and functioning mitochondria. This metabolic rewiring fuels the uncontrolled growth of the tumor mass and promotes cancer progression. Glycolytic reprogramming occurs not exclusively in cancer cells but also in the stroma, especially in cancer-associated fibroblasts (CAFs), a process that has been named the “reverse Warburg effect”. Autophagy, an evolutionary conserved catabolic process devoted to macromolecular turnover, is often dysregulated in cancer and plays a "double-edged sword" role in regulating the tumor microenvironment.

Methods To mimic the tumor microenvironment in vitro, we employed IL-6, a pro-inflammatory cytokine abundant in ovarian cancer patients, and we set up an indirect co-culture method by cultivating fibroblasts with the conditioning medium of ovarian cancer cells.

Results Our work aimed to dissect how the glycolysis/autophagy interplay affects ovarian cancer cell motility and the phenoconversion of CAFs. Our data show that IL-6 induces cancer cell migration only in cases of active glycolysis. On the other hand, nutraceutical resveratrol (RV) inhibits glucose uptake and metabolism while promoting autophagy, collectively hampering cancer cell motility. Moreover, our bioinformatic interrogation of TCGA data shows that patients with a low expression of glycolytic markers and displaying active autophagy exhibit favorable clinical outcomes. Next, we show that the glycolysis-dependent inhibition of autophagy promotes CAF activation. The conditioning medium of the ovarian cancer cells induces the glycolytic reprogramming that is required for CAF activation. Notably, glycolysis inhibition using 2-deoxy-D-glucose (2DG) or RV induces autophagy, which reprograms the CAFs into quiescent fibroblasts.

Conclusions Taken together, our data support the use of glycolysis inhibitors and/or autophagy inducers as an adjuvant strategy to improve the therapy success rate and limit the risk of metastasis.

Neuroblastoma (NB) is a pediatric malignancy originating from the neural crest cells of the sympathetic nervous system, and it often progresses aggressively due to the influence of epidermal growth factor (EGF). Our earlier research demonstrated that cathepsin D (CD) disrupts EGF-driven proliferation in NB cells cultured in 2D by inhibiting the EGFR/MAPK signaling pathway. While this highlights CD's regulatory function in tumor growth, its potential role in metastasis remains unexplored. In order to reproduce tumor heterogeneity, we engineered neuroblastoma (NB) clones with either silenced or overexpressed CD expression and co-cultured the clones in different proportions. We then analyzed the growth of the mixed populations in 2D (adherent) and 3D (suspension) culture conditions to mimic the stages of the metastatic process in response to EGF stimulation. We unexpectedly observed an opposite behavior in 2D and 3D cells with different levels of CD. In particular, cells overexpressing CD demonstrated a greater adaptability to growth in suspension cultures. In contrast, cells with suppressed CD expression exhibited enhanced growth under adherent conditions in two-dimensional environments. Different CD levels lead to different behaviors even during the transition from 3D to 2D: cells that overexpress CD demonstrated an increase in N-cadherin levels, while cells with silenced CD showed a greater propensity to revert to a mesenchymal-to-epithelial-like phenotype, as evidenced by the elevated expression of E-cadherin. The contrasting roles of CD in driving cancer cell growth under 2D and 3D conditions indicate that clonal evolution may favor the emergence of subpopulations with distinct CD levels, optimizing their adaptability to various metastatic niches. This highlights the potential epigenetic regulation of CD as a mechanism supporting survival and proliferation in both substrate-adherent and neurosphere-forming NB cells. Consequently, disrupting the epigenetic control of CD could represent a promising therapeutic approach to hinder NB progression and metastasis.

Background: The tumor microenvironment plays a pivotal role in shaping tumor aggressiveness and driving disease progression. In this context, the identification of gene signatures characterizing cancer-associated fibroblasts (CAFs) obtained from the two most frequently diagnosed cancers worldwide, such as breast and prostate tumors, may improve outcome prediction and therapeutic strategies for patients.

Methods: The transcriptomes of CAFs isolated from breast and prostate cancer biopsies were analyzed using RNA sequencing. Data from The Cancer Genome Atlas (TCGA) were used to compare the gene expression profiles of CAFs with those of breast and prostate cancer patients. The clusterProfiler package was employed to perform pathway enrichment analysis, while the gene signature associated withprostate CAFs was identified by applying K-means clustering. Kaplan--Meier curves and log-rank tests were used to assess the prognostic significance of the signature in prostate cancer patients. A decision-tree classification approach validated the clustering results and the prognostic relevance of the gene signature.

Results: The comparisons of either breast and prostate CAFs transcriptomes or the gene expression landscapes of breast and prostate cancer patients allowed us to construct a gene signature counting 11 genes (IL13RA2, GDF7, IL33, CXCL1, TNFRSF19, CXCL6, LIFR, CXCL5, IL7, TSLP, and TNFSF15) with clinical implications in prostate cancer. Notably, the aforementioned genes are implicated in immune-related transduction pathways. Thereafter, clustering and classification analyses revealed that prostate cancer patients exhibiting low expression levels of these 11 genes are characterized by a worse prognosis with a high prediction accuracy.

Conclusions: The prostate CAFs-related gene signature identified here might serve as a prognostic indicator and might offer a valuable set of biomarkers for improving the management of prostate cancer patients.

Age is the main risk factor for late-onset Alzheimer’s disease (LOAD) and all processes in normal aging also occur in LOAD pathology. While some factors appear to be more specific to LOAD and may be genetically determined, others are acquired through the aging process. Because bioenergetic metabolism is among the most fundamental features of cell functions, changes thereof have profound effects on every aspect of aging. In both fibroblasts and induced pluripotent stem cell (iPSC)-derived immature and mature brain cells of subjects with LOAD, we observed bioenergetic substrate deficiencies, including reductions in the redox agent nicotinamide adenine dinucleotide (NAD) or glucose uptake and metabolism, and alterations of associated bioenergetic-dependent cell functions. These data suggest that dysfunctional bioenergetics is an inherent cell-specific and cell-autonomous risk factor in LOAD. Together with findings from the brains of normally aging individuals or patients with LOAD, our data support the view that in LOAD, inherent cell-metabolic dysfunctions may occur in development and early life, altering the trajectory of the normal aging process in youth and middle-age, and predisposing neuropathological events that lead to symptomatic features of LOAD later in life. Our studies seek to further identify and characterize the cellular mechanisms underlying LOAD-associated bioenergetic abnormalities. The results of these studies may reveal targets and methods for treating individuals at risk for LOAD before illness develops or delaying disease onset and progression later in life.

BACKGROUND

Melanoma ranks among the most lethal cancers worldwide. Advanced-stage melanoma is treated with various therapeutic strategies, often accompanied by significant side effects. Recent advancements in targeted therapies, particularly those aimed at receptor tyrosine kinases and immune checkpoints, have substantially improved overall survival (OS) and long-term disease control. However, resistance mechanisms frequently develop, leading to disease progression. Consequently, the need for alternative therapeutic approaches for advanced melanoma remains pressing. The transient receptor potential melastatin-8 (TRPM8) channel has emerged as a promising molecular target implicated in the migration and proliferation of malignant cells. However, its specific role in melanoma progression remains unclear.

AIM

This study aims to investigate the effects of novel TRPM8 modulators on androgen-induced migration, invasiveness, and spheroid growth in melanoma cells.

METHODS

Melanoma cell lines with varying malignancy degrees were treated with or without androgens in the presence or absence of newly synthesized TRPM8 modulators. Wound scratch and Boyden’s chambers analysis were performed to evaluate cell migration and invasion. The most effective compounds were further tested in melanoma 3D spheroid models.

RESULTS

TRPM8 modulators effectively inhibited the androgen-induced migration and invasion of melanoma cells and reduced the growth of melanoma cell-derived spheroids.

CONCLUSIONS

These preclinical findings highlight TRPM8 as a promising therapeutic target in melanoma, offering potential for innovative treatment strategies to overcome limitations in current therapies.