Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

Identification of myoferlin as a mitochondria-associated membranes component required for calcium signaling in PDAC cell lines.

BACKGROUND: Pancreatic ductal adenocarcinoma is an aggressive cancer type with one of the lowest survival rates due to late diagnosis and the absence of effective treatments. A better understanding of PDAC biology will help researchers to discover the Achilles' heel of cancer cells. In that regard, our research team investigated the function of an emerging oncoprotein known as myoferlin. Myoferlin is overexpressed in PDAC and its silencing/targeting has been shown to affect cancer cell proliferation, migration, mitochondrial dynamics and metabolism. Nevertheless, our comprehension of myoferlin functions in cells remains limited. In this study, we aimed to understand the molecular mechanism linking myoferlin silencing to mitochondrial dynamics. METHODS: Experiments were performed on two pancreas cancer cell lines, Panc-1 and MiaPaCa-2. Myoferlin localization on mitochondria was evaluated by immunofluorescence, proximity ligation assay, and cell fractionation. The presence of myoferlin in mitochondria-associated membranes was assessed by cell fractionation and its function in mitochondrial calcium transfer was evaluated using calcium flow experiments, proximity ligation assays, co-immunoprecipitation, and timelapse fluorescence microscopy in living cells. RESULTS: Myoferlin localization on mitochondria was investigated. Our results suggest that myoferlin is unlikely to be located on mitochondria. Instead, we identified myoferlin as a new component of mitochondria-associated membranes. Its silencing significantly reduces the mitochondrial calcium level upon stimulation, probably through myoferlin interaction with the inositol 1,4,5-triphosphate receptors 3. CONCLUSIONS: For the first time, myoferlin was specifically demonstrated to be located in mitochondria-associated membranes where it participates to calcium flow. We hypothesized that this function explains our previous results on mitochondrial dynamics. This study improves our comprehension of myoferlin localization and function in cancer biology.

The transcription factor c-Jun inhibits RBM39 to reprogram pre-mRNA splicing during genotoxic stress.

Genotoxic agents, that are used in cancer therapy, elicit the reprogramming of the transcriptome of cancer cells. These changes reflect the cellular response to stress and underlie some of the mechanisms leading to drug resistance. Here, we profiled genome-wide changes in pre-mRNA splicing induced by cisplatin in breast cancer cells. Among the set of cisplatin-induced alternative splicing events we focused on COASY, a gene encoding a mitochondrial enzyme involved in coenzyme A biosynthesis. Treatment with cisplatin induces the production of a short isoform of COASY lacking exons 4 and 5, whose depletion impedes mitochondrial function and decreases sensitivity to cisplatin. We identified RBM39 as a major effector of the cisplatin-induced effect on COASY splicing. RBM39 also controls a genome-wide set of alternative splicing events partially overlapping with the cisplatin-mediated ones. Unexpectedly, inactivation of RBM39 in response to cisplatin involves its interaction with the AP-1 family transcription factor c-Jun that prevents RBM39 binding to pre-mRNA. Our findings therefore uncover a novel cisplatin-induced interaction between a splicing regulator and a transcription factor that has a global impact on alternative splicing and contributes to drug resistance.

Treatment algorithm and prognostic factors for patients with stage I-III carcinoma of the anal canal: a 20-year multicenter study.

Despite a growing incidence in developed countries and a recent improved understanding of its pathogenesis, anal cancer management has not evolved over the past decades and drug combination used as first-line regimen still largely depends on clinician preferences. Aiming at paving the way for precision medicine, a large cohort of 372 HIV-negative patients diagnosed over a 20-year time period with locally advanced anal carcinoma was collected and carefully characterized at the clinical, demographic, histopathologic, immunologic, and virologic levels. Both the prognostic relevance of each clinicopathological parameter and the efficacy of different concurrent chemoradiation strategies were determined. Overall, the incidence of anal cancer peaked during the sixth decade (mean: 63.4) and females outnumbered males (ratio: 2.51). After completion of treatment, 95 (25.5%) patients experienced progression of persistent disease or local/distant recurrence and 102 (27.4%) died during the follow-up period (median: 53.8 months). Importantly, uni-multivariate analyses indicated that both negative HPV/p16ink4a status and aberrant p53 expression were far better predictors for reduced progression-free survival than traditional risk factors such as tumor size and nodal status. As for overall survival, the significant influences of age at diagnosis, p16ink4a status, cTNM classification as well as both CD3+ and CD4+ T-cell infiltrations within tumor microenvironment were highlighted. Cisplatin-based chemoradiotherapy was superior to both radiotherapy alone and other concurrent chemoradiation therapies in the treatment of HPV-positive tumors. Regarding their HPV-uninfected counterparts, frequent relapses were observed, whatever the treatment regimen administered. Taken together, our findings reveal that current anal cancer management and treatment have reached their limits. A dualistic classification according to HPV/p53 status should be considered with implications for therapy personalization and optimization.

Methylglyoxal, a glycolysis metabolite, triggers metastasis through MEK/ERK/SMAD1 pathway activation in breast cancer.

BACKGROUND: Elevated aerobic glycolysis rate is a biochemical alteration associated with malignant transformation and cancer progression. This metabolic shift unavoidably generates methylglyoxal (MG), a potent inducer of dicarbonyl stress through the formation of advanced glycation end products (AGEs). We have previously shown that the silencing of glyoxalase 1 (GLO1), the main MG detoxifying enzyme, generates endogenous dicarbonyl stress resulting in enhanced growth and metastasis in vivo. However, the molecular mechanisms through which MG stress promotes metastasis development remain to be unveiled. METHODS: In this study, we used RNA sequencing analysis to investigate gene-expression profiling of GLO1-depleted breast cancer cells and we validated the regulated expression of selected genes of interest by RT-qPCR. Using in vitro and in vivo assays, we demonstrated the acquisition of a pro-metastatic phenotype related to dicarbonyl stress in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cellular models. Hyperactivation of MEK/ERK/SMAD1 pathway was evidenced using western blotting upon endogenous MG stress and exogenous MG treatment conditions. MEK and SMAD1 regulation of MG pro-metastatic signature genes in breast cancer cells was demonstrated by RT-qPCR. RESULTS: High-throughput transcriptome profiling of GLO1-depleted breast cancer cells highlighted a pro-metastatic signature that establishes novel connections between MG dicarbonyl stress, extracellular matrix (ECM) remodeling by neoplastic cells and enhanced cell migration. Mechanistically, we showed that these metastasis-related processes are functionally linked to MEK/ERK/SMAD1 cascade activation in breast cancer cells. We showed that sustained MEK/ERK activation in GLO1-depleted cells notably occurred through the down-regulation of the expression of dual specificity phosphatases in MG-stressed breast cancer cells. The use of carnosine and aminoguanidine, two potent MG scavengers, reversed MG stress effects in in vitro and in vivo experimental settings. CONCLUSIONS: These results uncover for the first time the key role of MG dicarbonyl stress in the induction of ECM remodeling and the activation of migratory signaling pathways, both in favor of enhanced metastatic dissemination of breast cancer cells. Importantly, the efficient inhibition of mitogen-activated protein kinase (MAPK) signaling using MG scavengers further emphasizes the need to investigate their therapeutic potential across different malignancies.

Transforming growth factor beta-induced, an extracellular matrix interacting protein, enhances glycolysis and promotes pancreatic cancer cell migration.

Pancreatic ductal adenocarcinoma (PDAC) remains a deadly malignancy with no efficient therapy available up-to-date. Glycolysis is the main provider of energetic substrates to sustain cancer dissemination of PDAC. Accordingly, altering the glycolytic pathway is foreseen as a sound approach to trigger pancreatic cancer regression. Here, we show for the first time that high transforming growth factor beta-induced (TGFBI) expression in PDAC patients is associated with a poor outcome. We demonstrate that, although usually secreted by stromal cells, PDAC cells synthesize and secrete TGFBI in quantity correlated with their migratory capacity. Mechanistically, we show that TGFBI activates focal adhesion kinase signaling pathway through its binding to integrin αVß5, leading to a significant enhancement of glycolysis and to the acquisition of an invasive phenotype. Finally, we show that TGFBI silencing significantly inhibits PDAC tumor development in a chick chorioallantoic membrane assay model. Our study highlights TGFBI as an oncogenic extracellular matrix interacting protein that bears the potential to serve as a target for new anti-PDAC therapeutic strategies.

A dualistic model of primary anal canal adenocarcinoma with distinct cellular origins, etiologies, inflammatory microenvironments and mutational signatures: implications for personalised medicine.

BACKGROUND: Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. METHODS: In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. RESULTS: Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. CONCLUSIONS: Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits.

Methylglyoxal-Mediated Stress Correlates with High Metabolic Activity and Promotes Tumor Growth in Colorectal Cancer.

Cancer cells generally rely on aerobic glycolysis as a major source of energy. Methylglyoxal (MG), a dicarbonyl compound that is produced as a side product during glycolysis, is highly reactive and induces the formation of advanced glycation end-products that are implicated in several pathologies including cancer. All mammalian cells have an enzymatic defense against MG composed by glyoxalases GLO1 and GLO2 that converts MG to d-lactate. Colorectal cancer (CRC) is one of the most frequently occurring cancers with high morbidity and mortality. In this study, we used immunohistochemistry to examine the level of MG protein adducts, in a series of 102 CRC human tumors divided into four clinical stages. We consistently detected a high level of MG adducts and low GLO1 activity in high stage tumors compared to low stage ones suggesting a pro-tumor role for dicarbonyl stress. Accordingly, GLO1 depletion in CRC cells promoted tumor growth in vivo that was efficiently reversed using carnosine, a potent MG scavenger. Our study represents the first demonstration that MG adducts accumulation is a consistent feature of high stage CRC tumors. Our data point to MG production and detoxification levels as an important molecular link between exacerbated glycolytic activity and CRC progression.

Myoferlin plays a key role in VEGFA secretion and impacts tumor-associated angiogenesis in human pancreas cancer.

Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers with no satisfactory treatment to date. Recent studies have identified myoferlin, a ferlin family member, in human pancreas adenocarcinoma where its expression was associated to a bad prognosis. However, the function of myoferlin in pancreas adenocarcinoma has not been reported. In other cell types, myoferlin is involved in several key plasma membrane processes such as fusion, repair, endocytosis and tyrosine kinase receptor activity. In this study, we showed that myoferlin silencing in BxPC-3 human pancreatic cancer cells resulted in the inhibition of cell proliferation in vitro and in a significant reduction of the tumor volume in chick chorioallantoic membrane assay. In addition to be smaller, the tumors formed by the myoferlin-silenced cells showed a marked absence of functional blood vessels. We further demonstrated that this effect was due, at least in part, to an inhibition of VEGFA secretion by BxPC-3 myoferlin-silenced cells. Using immunofluorescence and electron microscopy, we linked the decreased VEGFA secretion to an impairment of VEGFA exocytosis. The clinical relevance of our results was further strengthened by a significant correlation between myoferlin expression in a series of human pancreatic malignant lesions and their angiogenic status evaluated by the determination of the blood vessel density.

Targeting osteopontin suppresses glioblastoma stem-like cell character and tumorigenicity in vivo.

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.

Organized proteomic heterogeneity in colorectal cancer liver metastases and implications for therapies.

UNLABELLED: Tumor heterogeneity is a major obstacle for developing effective anticancer treatments. Recent studies have pointed to large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Because to date no similar information is available at the protein (phenotype) level, we employed matrix assisted laser desorption ionization (MALDI) image-guided proteomics and explored the heterogeneity of extracellular and membrane subproteome in a unique collection of eight fresh human colorectal carcinoma (CRC) liver metastases. Monitoring the spatial distribution of over 1,000 proteins, we found unexpectedly that all liver metastasis lesions displayed a reproducible, zonally delineated pattern of functional and therapeutic biomarker heterogeneity. The peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis displayed increased cellular growth, movement, and drug metabolism, whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA-repair activity. From the aspect of therapeutic targeting, zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally, we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. We demonstrate their in vivo antibody-based targeting and highlight their potential usefulness for clinical applications. CONCLUSION: The proteome heterogeneity of human CRC liver metastases has a distinct, organized pattern. This particular hallmark can now be used as part of the strategy for developing rational therapies based on multiple sets of targetable antigens.

Impact of the structure of biocompatible aliphatic polycarbonates on siRNA transfection ability.

RNAi therapeutics are promising therapeutic tools that have sparked the interest of many researchers. In an effort to provide a safe alternative to PEI, we have designed a series of new guanidinium- and morpholino-functionalized biocompatible and biodegradable polycarbonate vectors. The impact of different functions (morpholino-, guanidinium-, hydrophobic groups) of the architecture (linear homopolymer to dumbbell-shape) and of the molecular weight of these copolymers on their capacity to form polyplexes and to decrease the expression of two epigenetic regulators of gene expression, HDAC7 and HDAC5, was evaluated. The use of one of these polymers combining morpholine and guanidine functions at the ratio >1 and hydrophobic trimethylene carbonate groups showed a significant decrease of mRNA and protein level in HeLa cells, similar to PEI. These results highlight the potential of polycarbonate vectors for future in vivo application as an anticancer therapy.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Acidosis-induced regulation of adipocyte G0S2 promotes crosstalk between adipocytes and breast cancer cells as well as tumor progression.

Bidirectional interactions between cancer cells and their microenvironment govern tumor progression. Among the stromal cells in this microenvironment, adipocytes have been reported to upregulate cancer cell migration and invasion by producing fatty acids. Conversely, cancer cells alter adipocyte phenotype notably via increased lipolysis. We aimed to identify the mechanisms through which cancer cells trigger adipocyte lipolysis and evaluate the functional consequences on cancer progression. Here, we show that cancer cell-induced acidification of the extracellular medium strongly promotes preadipocyte lipolysis through a mechanism that does not involve lipophagy but requires adipose triglyceride lipase (ATGL) activity. This increased lipolysis is triggered mainly by attenuation of the G0/G1 switch gene 2 (G0S2)-induced inhibition of ATGL. G0S2-mediated regulation in preadipocytes affects their communication with breast cancer cells, modifying the phenotype of the cancer cells and increasing their resistance to chemotherapeutic agents in vitro. Furthermore, we demonstrate that the adipocyte-specific overexpression of G0S2 impairs mammary tumor growth and lung metastasis formation in vivo. Our results highlight the importance of acidosis in cancer cell-adipocyte crosstalk and identify G0S2 as the main regulator of cancer-induced lipolysis, regulating tumor establishment and spreading.

Novel XBP1s-independent function of IRE1 RNase in HIF-1α-mediated glycolysis upregulation in human macrophages upon stimulation with LPS or saturated fatty acid.

In obesity, adipose tissue infiltrating macrophages acquire a unique pro-inflammatory polarization, thereby playing a key role in the development of chronic inflammation and Type 2 diabetes. Increased saturated fatty acids (SFAs) levels have been proposed to drive this specific polarization. Accordingly, we investigated the immunometabolic reprogramming in SFA-treated human macrophages. As expected, RNA sequencing highlighted a pro-inflammatory profile but also metabolic signatures including glycolysis and hypoxia as well as a strong unfolded protein response. Glycolysis upregulation was confirmed in SFA-treated macrophages by measuring glycolytic gene expression, glucose uptake, lactate production and extracellular acidification rate. Like in LPS-stimulated macrophages, glycolysis activation in SFA-treated macrophages was dependent on HIF-1α activation and fueled the production of pro-inflammatory cytokines. SFAs and LPS both induced IRE1α endoribonuclease activity, as demonstrated by XBP1 mRNA splicing, but with different kinetics matching HIF-1α activation and the glycolytic gene expression. Interestingly, the knockdown of IRE1α and/or the pharmacological inhibition of its RNase activity prevented HIF-1α activation and significantly decreased glycolysis upregulation. Surprisingly, XBP1s appeared to be dispensable, as demonstrated by the lack of inhibiting effect of XBP1s knockdown on glycolytic genes expression, glucose uptake, lactate production and HIF-1α activation. These experiments demonstrate for the first time a key role of IRE1α in HIF-1α-mediated glycolysis upregulation in macrophages stimulated with pro-inflammatory triggers like LPS or SFAs through XBP1s-independent mechanism. IRE1 could mediate this novel function by targeting other transcripts (mRNA or pre-miRNA) through a mechanism called regulated IRE1-dependent decay or RIDD. Deciphering the underlying mechanisms of this novel IRE1 function might lead to novel therapeutic targets to curtail sterile obesity- or infection-linked inflammation.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

Methylglyoxal: a novel upstream regulator of DNA methylation.

BACKGROUND: Aerobic glycolysis, also known as the Warburg effect, is predominantly upregulated in a variety of solid tumors, including breast cancer. We have previously reported that methylglyoxal (MG), a very reactive by-product of glycolysis, unexpectedly enhanced the metastatic potential in triple negative breast cancer (TNBC) cells. MG and MG-derived glycation products have been associated with various diseases, such as diabetes, neurodegenerative disorders, and cancer. Glyoxalase 1 (GLO1) exerts an anti-glycation defense by detoxifying MG to D-lactate. METHODS: Here, we used our validated model consisting of stable GLO1 depletion to induce MG stress in TNBC cells. Using genome-scale DNA methylation analysis, we report that this condition resulted in DNA hypermethylation in TNBC cells and xenografts. RESULTS: GLO1-depleted breast cancer cells showed elevated expression of DNMT3B methyltransferase and significant loss of metastasis-related tumor suppressor genes, as assessed using integrated analysis of methylome and transcriptome data. Interestingly, MG scavengers revealed to be as potent as typical DNA demethylating agents at triggering the re-expression of representative silenced genes. Importantly, we delineated an epigenomic MG signature that effectively stratified TNBC patients based on survival. CONCLUSION: This study emphasizes the importance of MG oncometabolite, occurring downstream of the Warburg effect, as a novel epigenetic regulator and proposes MG scavengers to reverse altered patterns of gene expression in TNBC.

Human papillomavirus E6/E7 oncoproteins promote radiotherapy-mediated tumor suppression by globally hijacking host DNA damage repair.

Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity (and, globally, on host DNA repair) remains mostly speculative. Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay (and validated by co-immunoprecipitation). The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed. Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets (CHEK2, CLK2, CLK2/3, ERCC3, MNAT1, PER1, RMI1, RPA1, UVSSA and XRCC6) for E6 and 11 (ALKBH2, CHEK2, DNA2, DUT, ENDOV, ERCC3, PARP3, PMS1, PNKP, POLDIP2 and RBBP8) for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy. Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities.

Differential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis.

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the setup of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment.

Cancer-Associated Fibroblast Diversity Shapes Tumor Metabolism in Pancreatic Cancer.

Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.