BackgroundTriple-negative breast cancer (TNBC) frequently develops resistance to chemotherapy. Cancer-supporting roles of the endogenous gaseous mediator hydrogen sulfide (H2S) have been identified. We investigated whether endogenous H2S, produced by 3-mercaptopyruvate sulfurtransferase (3-MST), mediates chemoresistance in TNBC and elucidated the underlying mechanisms involved.MethodsA 3-MST inhibitor (HMPSNE) was used along with different chemotherapeutic drugs to determine whether 3-MST affects TNBC cell (MDA-MB-231) chemoresistance. H2S production was measured via AzMC fluorescence. H2S-synthesizing and H2S-degrading enzymes were quantified via Western blotting together with downstream signaling molecules involved in the PI3K/Akt/mTOR pathway. Cell viability, colony formation and migration assays were performed. qRT‒PCR and flow cytometry were conducted to assess the expression of the cancer stem cell marker CD44.ResultsHMPSNE enhanced the cytotoxic, anticlonogenic and antimigratory effects of doxorubicin on MDA-MB-231 cells. Doxorubicin increased H2S-synthesizing enzymes, whereas HMPSNE resulted in their downregulation, especially cystathionine beta-synthase (CBS) and 3-MST. A similar trend was observed for H2S-metabolizing enzymes, particularly thiosulfate sulfurtransferase (TST). A significant increase in CD44 was revealed upon doxorubicin treatment; 3-MST slightly affected this response. With respect to the PI3K/AKT/mTOR pathway, HMPSNE did not significantly modulate the effect of doxorubicin.ConclusionThese findings suggest that TNBC chemoresistance is linked to the 3-MST/H2S pathway. Pharmacological inhibition of 3-MST by HMPSNE enhances the chemotherapeutic effect of doxorubicin on TNBC. Some of these effects may be related to the regulation of CD44 but are unlikely to be mediated via the PI3K/AKT/mTOR pathway. Therefore, pharmacological inhibition of 3-MST may serve as a promising target for further investigations to increase the sensitivity of TNBC cells to doxorubicin-based therapies.

High mobility group box protein 1 (HMGB1) is a ubiquitous protein that has multiple functions in the nucleus and cytosol. Following release into the extracellular space, HMGB1 acts as a mediator of inflammation. HMGB1 is overexpressed in many cancers and has both oncogenic and antitumoral effects. Here we investigated the role of HMGB1 expression by cancer cells in the antitumor immune response. Subcutaneous tumors were induced in mice by injection of wild-type or HMGB1-low engineered syngeneic cancer cells. Tumor growth and immune infiltration were assessed, and gene expression was analysed in whole tumors and isolated tumor cells by RNA sequencing analysis. Downregulation of cancer cell-intrinsic HMGB1 prevented tumor growth and reprogrammed the tumor microenvironment into a more inflamed phenotype. Tumor growth was not impacted in immunodeficient mice or in mice deficient in CD8+ T cells, demonstrating that the impact of HMGB1 downregulation on tumor progression was immune-dependent and mediated by CD8+ T cells. Transcriptional profiling showed that HMGB1low tumors were enriched in immune pathways such as antigen presentation, T-cell receptor and cytokine signalling. Immune activation was robust and detectable in both tumor tissue and secondary lymphoid organs of mice bearing HMGB1low tumors, but this did not facilitate immune infiltration of wild-type tumors that had been co-injected in the contralateral flank. We propose that tumor cell-intrinsic HMGB1 plays a critical role in modulating the local tumor microenvironment and in limiting anticancer immune responses. Thus, targeting cancer cell-intrinsic HMGB1 may represent a novel approach to improve the outcome of cancer immunotherapy.

Hydrogen sulfide (H2S) is a redox-active gasotransmitter implicated in tumor progression and immune regulation. The enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is a key contributor to endogenous H2S and polysulfide production, but its role in tumor-immune interactions remains poorly defined. Here, we show that 3-MST is the most abundantly expressed H2S-synthesizing enzyme in human renal cell carcinoma cells (RCC) and that high 3-MST expression correlates with reduced patient survival. Pharmacological inhibition of 3-MST lowered intracellular H2S levels in Renca renal carcinoma cells, suppressed proliferation, induced apoptosis, disrupted cellular metabolism, and increased expression of immune-related genes and proteins. In immune cells, partial inhibition of 3-MST promoted T cell activation, as evidenced by increased CD69 expression on CD3+, CD4+, and CD8+ T cells. In contrast, complete inhibition of 3-MST, achieved by high concentrations of the inhibitor, modestly reduced CD8+ T cell proliferation. Functionally, 3-MST inhibition potentiated antigen-specific CD8+ T cell-mediated killing of tumor cells, an effect further amplified by PD-L1 blockade. These results establish 3-MST as a redox-sensitive metabolic driver of tumor growth and immune evasion in RCC and demonstrate that its inhibition can boost antitumor immune responses, offering a potential avenue for combination immunotherapy.

Multiple myeloma is a plasma cell malignancy with a poor prognosis despite the recent development of new therapeutic options. Histone deacetylase 6 (HDAC6) is overexpressed in multiple myeloma cells and may be involved in the acquisition of resistance to conventional anti-proteasome treatments. In addition to displaying a deacetylase catalytic activity, HDAC6 plays an essential role in the regulation of autophagy and cell death by recognizing ubiquitinated motifs from misfolded proteins through its C-terminal ZnF-UBP binding domain. These defective proteins are sent to the aggresome to facilitate their degradation by autophagy. Here, we explore the role of the ZnF-UBP binding domain of HDAC6 in the function of multiple myeloma cells. A non-functional ZnF-UBP domain containing a 2-residue mutation in the binding site was designed, and the absence of ubiquitin binding was confirmed in a cell-free assay. Molecular docking simulations and electrostatic calculations revealed a significant decrease in the electrostatic potential of the mutated peptide, which is crucial for the stability of the complex with ubiquitin. A multiple myeloma cell line containing the non-functional ZnF-UBP domain was then engineered. Although the deacetylase activity of HDAC6 was maintained in these cells, they showed reduced cell growth, impaired aggresome formation, and a dysregulated gene expression profile that was more pronounced than cells entirely deficient in HDAC6. These results indicate that a non-functional ZnF-UBP binding domain impacts the function of multiple myeloma cells. Based on these findings, a series of quinazolinylpropanoic acid derivatives was synthesized to explore the inhibitory activity of small molecules to this domain. We propose that ZnF-UBP binding domain inhibitors should be further evaluated as potential therapeutic agents in multiple myeloma.

Although obesity is a major risk factor for cancer, it may also improve the response to cancer therapy. Here we investigated the impact of obesity on the efficacy of immune checkpoint inhibitors (ICI). In male mice, obesity promoted tumor growth but enhanced the response to ICI. This was associated with higher expression of immune-related genes within the tumor and enhanced infiltration of tumor-specific CD8+ T cells. Further, obesity in mice was associated with higher estrogen levels and enrichment of estrogen response genes in the tumor, and anti-programmed cell death 1 (anti-PD-1) efficacy was reduced upon administration of the aromatase inhibitor letrozole, which blocks the production of estrogens. Mechanistically, adipocyte-derived estrogens increased antigen presentation by dendritic cells and tumor-specific CD8+ T cell cytotoxicity. Last, overweight and obese men with melanoma responded better to ICI, with high estrogen levels being associated with improved response and survival. Our results suggest that estrogens may serve as a predictive factor of response to ICI in men with melanoma.

The chemokine CCL22 is constitutively expressed at high levels in lymphoid organs, where it controls immunity by promoting contacts between dendritic cells (DC) and regulatory T cells (Treg). However, its regulation and impact in the context of pattern recognition receptor (PRR) stimulation and microbial infection are unknown. Here we show that CCL22 levels in lymphoid organs of mice were strongly suppressed upon stimulation with TLR agonists. In vitro, activation of Toll-like receptors (TLR), RIG-I like helicases (RLH) and stimulator of interferon genes (STING) resulted in a potent downregulation of CCL22. Mechanistically, the suppression of DC-derived CCL22 secretion was exerted by inflammatory cytokines such as IFN-α, IFN-γ and IL-10 released upon TLR activation by B and T cells. Decreased CCL22 synthesis correlated with reduced frequencies of cellular clustering between Treg and DC in co-cultures. CCL22 suppression was also observed upon microbial infection, since CCL22 levels were significantly reduced in lymphoid organs of mice upon injection of Salmonella typhimurium. As a clinical correlate, CCL22 serum concentrations were decreased in patients with sepsis compared to controls. Taken together, we demonstrate a strong and long-lasting suppression of CCL22 as a consequence of innate immune activation. In the context of microbial infection, transient reduction of CCL22 reduces Treg-DC interactions and may thereby represent a mechanism to weaken Treg function in order to enable an effective immune response and pathogen clearance.

Obesity increases cancer risk, yet obese patients often respond better to cancer immunotherapy. In our study, we uncovered why: high estrogen levels, especially in obese men, boost the immune response triggered by immunotherapy. This reveals a key role for estrogens in improving treatment effectiveness.

Chronic kidney disease (CKD) is a growing public health crisis, affecting over 10% of the global population and significantly increasing mortality and morbidity. Irrespective of its underlying cause, tubulointerstitial fibrosis (TIF) is a hallmark of CKD progression, with myofibroblasts being the primary effectors of renal fibrosis. Here, we show that 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a critical driver of pathogenic myofibroblast differentiation and fibrosis in CKD. Using genetic deletion and pharmacological inhibition of 11beta-HSD1 in mouse models, we demonstrate a marked reduction in TIF severity and improved renal function, linked to the suppression of a regulatory myofibroblast (Reg-MF) subpopulation. Single-cell and spatial transcriptomics data reveal that 11beta-HSD1 is essential for the activation and expansion of Reg-MFs, which is conserved across species and predicts worse outcomes in CKD patients and kidney allograft recipients. These findings establish a direct link between 11beta-HSD1 activity and renal fibrogenesis, highlighting its role during the transition from pericytes to pathogenic Reg-MFs. Our results support 11beta-HSD1 inhibition as a promising therapeutic strategy to mitigate CKD progression, offering both mechanistic insights and translational potential for improving patient outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Gasotransmitters play crucial roles in regulating many physiological processes, including cell signaling, cellular proliferation, angiogenesis, mitochondrial function, antioxidant production, nervous system functions and immune responses. Hydrogen sulfide (H2S) is the most recently identified gasotransmitter, which is characterized by its biphasic behavior. At low concentrations, H2S promotes cellular bioenergetics, whereas at high concentrations, it can exert cytotoxic effects. Cystathionine β-synthetase (CBS), cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST), and cysteinyl-tRNA synthetase 2 (CARS2) are pivotal players in H2S biosynthesis in mammalian cells and tissues. The focus of this review is the regulation of the various pathways involved in H2S metabolism in various forms of cancer. Key enzymes in this process include the sulfide oxidation unit (SOU), which includes sulfide:quinone oxidoreductase (SQOR), human ethylmalonic encephalopathy protein 1 (hETHE1), rhodanese, sulfite oxidase (SUOX/SO), and cytochrome c oxidase (CcO) enzymes. Furthermore, the potential role of H2S methylation processes mediated by thiol S-methyltransferase (TMT) and thioether S-methyltransferase (TEMT) is outlined in cancer biology, with potential opportunities for targeting them for clinical translation. In order to understand the role of H2S in oncogenesis and tumor progression, one must appreciate the intricate interplay between H2S-synthesizing and H2S-catabolizing enzymes.

Recently, myriad studies have defined the versatile abilities of gasotransmitters and their synthesizing enzymes to play a "Maestro" role in orchestrating several oncological and non-oncological circuits and, thus, nominated them as possible therapeutic targets. Although a significant amount of work has been conducted on the role of nitric oxide (NO) and carbon monoxide (CO) and their inter-relationship in the field of oncology, research about hydrogen sulfide (H2S) remains in its infancy. Recently, non-coding RNAs (ncRNAs) have been reported to play a dominating role in the regulation of the endogenous machinery system of H2S in several pathological contexts. A growing list of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are leading the way as upstream regulators for H2S biosynthesis in different mammalian cells during the development and progression of human diseases; therefore, their targeting can be of great therapeutic benefit. In the current review, the authors shed the light onto the biosynthetic pathways of H2S and their regulation by miRNAs and lncRNAs in various oncological and non-oncological disorders.

Inflammation plays a pivotal role in cancer development, with chronic inflammation promoting tumor progression and treatment resistance, whereas acute inflammatory responses contribute to protective anti-tumor immunity. Gasdermin D (GSDMD) mediates the release of pro-inflammatory cytokines such as IL-1β. While the release of IL-1β is directly linked to the progression of several types of cancers, the role of GSDMD in cancer is less clear. In this study, we show that GSDMD expression is upregulated in human breast, kidney, liver, and prostate cancer. Higher GSDMD expression correlated with increased survival in primary breast invasive carcinoma (BRCA), but not in liver hepatocellular carcinoma (LIHC). In BRCA, but not in LIHC, high GSDMD expression correlated with a myeloid cell signature associated with improved prognosis. To further investigate the role of GSDMD in anticancer immunity, we induced breast cancer and hepatoma tumors in GSDMD-deficient mice. Contrary to our expectations, GSDMD deficiency had no effect on tumor growth, immune cell infiltration, or cytokine expression in the tumor microenvironment, except for Cxcl10 upregulation in hepatoma tumors. In vitro and in vivo innate immune activation with TLR ligands, that prime inflammatory responses, revealed no significant difference between GSDMD-deficient and wild-type mice. These results suggest that the impact of GSDMD on anticancer immunity is dependent on the tumor type. They underscore the complex role of inflammatory pathways in cancer, emphasizing the need for further exploration into the multifaceted effects of GSDMD in various tumor microenvironments. As several pharmacological modulators of GSDMD are available, this may lead to novel strategies for combination therapy in cancer.

Abstract Background Hydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells. Methods BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays. Results miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins. Conclusion This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

In humans, cystathionine-β-synthase (CBS), cystathionine gamma-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) biosynthesize Hydrogen Sulphide (H2S). The expression of these enzymes was found to be significantly upregulated in breast cancer (BC). H2S causes various alterations in cellular metabolism and plays a major role in metabolic reprogramming in several malignant tumors. At low concentrations H2S stimulates cellular bioenergetics and increases ATP production. It induces persulfidation of different transcription factors such as PGC1α and thus, promotes the mitochondrial biogenesis.

Hydrogen sulfide (H2S) is an endogenous gaseous mediator implicated in breast cancer (BC) progression. The 3 principle enzymes responsible for H2S production in mammals are cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST), whose overexpression was correlated with poor prognosis in BC patients. Besides, H2Swas linked to the suppression of tumor immune microenvironment in BC. Our group recently reported that upon single or dual inhibition of any of H2S-synthesizing enzymes, the other untargeted enzyme(s) is/are upregulated/activated as a compensatory mechanism for maintaining the level of H2Sin cancer cells. Thus, the aim of this work is to find a pan-repressor of all 3 enzymes to skip the above compensatory/escape mechanism and to investigate its impact on oncogenic and immunogenic profiles of BC cells. BC female patients (n=20) were recruited. In-silico analysis was used to identify microRNAs (miRNAs) that target CBS, CSE, and 3MST. MDA-MB-231 TNBC cells were cultured and transfected by oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. Western blot analysis was performed. H2S levels were measured by AzMc. BC hallmarks were assessed by MTT, transwell migration, and clonogenic assays. MiRNA-193a was validated to regulate the expression of H2S enzymes by 8 different bioinformatics software, and was found to be significantly downregulated in BC tissues. Further, miR-193a was negatively correlated with CBS, CSE and 3MST levels in BC patients. Ectopic expression of miR-193a resulted in a marked repression of CBS, CSE, and 3MST expression, inducing a significant decrease in cellular H2S production. in TNBC cells overexpressing miRNA-193a, not only the cancer hallmarks were markedly suppressed but also the immune-suppressor proteins Galectin (GAL) 3 and GAL 9 were decreased significantly. This study identifies miRNA-193a as a pan-repressor of the 3 principle H2S-synthesizing enzymes in BC, bypassing the compensatory behavior observed at single or dual inhibition of H2S enzymes. Thus, miR-193a emerges as a novel tumor suppressor and immunomodulatory miRNA in TNBC.

Supplementary Figure 1 from Systemic Cancer Therapy with a Small Molecule Agonist of Toll-like Receptor 7 Can Be Improved by Circumventing TLR Tolerance

Supplementary Figure 1-Treatment with CpG causes a reduction of Treg within the CD4 T cell population and an increase of CD8+ tumor-infiltrating cells.