Cancer & Metabolism最新文献

Background: Glycosphingolipids (GSLs) are membrane lipids composed of a ceramide backbone linked to a glycan moiety. Ganglioside biosynthesis is a part of the GSL metabolism, which involves sequential reactions catalyzed by specific enzymes that in part have a poor substrate specificity. GSLs are deregulated in cancer, thus playing a role as potential biomarkers for personalized therapy or subtype classification. However, the analysis of GSL profiles is complex and requires dedicated technologies, that are currently not included in the commonly utilized high-throughput assays adopted in contexts such as molecular tumor boards.

Methods: In this study, we developed a method to discriminate the enzyme activity among the four series of the ganglioside metabolism pathway by incorporating transcriptome data and topological information of the metabolic network. We introduced three adjustment options for reaction activity scores (RAS) and demonstrated their application in both exploratory and comparative analyses by applying the method on neuroblastic tumors (NTs), encompassing neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). Furthermore, we interpreted the results in the context of earlier published GSL measurements in the same tumors.

Results: By adjusting RAS values using a weighting scheme based on network topology and transition probabilities (TPs), the individual series of ganglioside metabolism can be differentiated, enabling a refined analysis of the GSL profile in NT entities. Notably, the adjustment method we propose reveals the differential engagement of the ganglioside series between NB and GNB. Moreover, MYCN gene expression, a well-known prognostic marker in NTs, appears to correlate with the expression of therapeutically relevant gangliosides, such as GD2. Using unsupervised learning, we identified subclusters within NB based on altered GSL metabolism.

Conclusion: Our study demonstrates the utility of adjusting RAS values in discriminating ganglioside metabolism subtypes, highlighting the potential for identifying novel cancer subgroups based on sphingolipid profiles. These findings contribute to a better understanding of ganglioside dysregulation in NT and may have implications for stratification and targeted therapeutic strategies in these tumors and other tumor entities with a deregulated GSL metabolism.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease characterized by complex metabolic rewiring that enables growth in changing nutrient availability and oxygen conditions. Transcriptome-based prognostic PDAC tumor subtypes, known as 'basal-like' and 'classical' subtypes are associated with differences in metabolic gene expression including genes involved in glycolysis. Tumor subtype-specific metabolism phenotypes may provide new targets for treatment development in PDAC, but their functional relevance has not been fully elucidated. We aimed to investigate differences in metabolic profiles and transcriptomes in tumor models derived from patients with basal-like and classical tumors.

Methods: Patient-derived organoids (PDOs) were established from tumor biopsies collected from patients with metastatic PDAC, including three PDOs from basal-like and five PDOs from classical tumors. Metabolic analyses included assessment of differences in metabolic activity using Seahorse Glycolysis and Mito Stress tests and ¹³C-glucose metabolites tracing analysis. In order to investigate the influence of mitochondrial pyruvate transport on metabolic differences, PDOs were treated with the mitochondrial pyruvate carrier 1 (MPC1) inhibitor UK-5099. Prognostic relevance of MPC1 was determined using a tumor tissue microarray (TMA) in resectable, and proteomics profiling in metastatic PDAC datasets. Whole genome and transcriptome sequencing, differential gene expression and gene set enrichment analyses were performed in PDOs.

Results: Metastatic PDAC PDOs showed subtype-specific differences in glycolysis and oxidative phosphorylation (OXPHOS). Basal-like tumor-derived PDOs had a lower baseline extracellular acidification rate, but higher glycolytic reserves and oxygen consumption rate (OCR) than classical tumor-derived PDOs. OCR difference was eliminated following treatment with UK-5099. In the ¹³C-glucose metabolites tracing experiment, a basal-like tumor PDO showed lower fractions of some M + 2 metabolites but higher sensitivity to UK-5099 mediated reduction in M + 2 metabolites than a classical tumor PDO. Protein level analyses revealed lower MPC1 protein levels in basal-like PDAC cases and association of low MPC1 levels with clinicopathologic parameters of tumor aggressiveness in PDAC. PDO differential gene expression analyses identified additional subtype-specific cellular pathways and potential disease outcome biomarkers.

Conclusions: Our findings point to distinct metabolic profiles in PDAC subtypes with basal-like tumor PDOs showing higher OXPHOS and sensitivity to MPC1 inhibition. Subtypes-specific metabolic vulnerabilities may be exploited for selective therapeutic targeting.

Background: PAF1/PD2 deregulation contributes to tumorigenesis, drug resistance, and cancer stem cell maintenance in Pancreatic Cancer (PC). Recent studies demonstrate that metabolic reprogramming plays a role in PC progression, but the mechanism is poorly understood. Here, we focused on examining the role of PAF1/PD2 in the metabolic rewiring of PC.

Methods: Cell lines were transfected with shRNAs to knockdown PAF1/PD2. Metabolic genes regulated by PAF1/PD2 were identified by qPCR/western blot, and metabolic assays were performed. Immunoprecipitations/ChIP were performed to identify PAF1/PD2 protein partners and confirm PAF1/HIF1α sub-complex binding to LDHA.

Results: PAF1 and LDHA showed progressively increased expression in human pancreatic tumor sections. Aerobic glycolysis genes were downregulated in PAF1-depleted PC cells. Metabolic assays indicated a decreased lactate production and glucose uptake in knockdown cells. Furthermore, PAF1/PD2 depletion showed a reduced glycolytic rate and increased oxidative phosphorylation by ECAR and OCR analysis. Interestingly, we identified that HIF1α interacts and co-localizes with PAF1, specifically in PC cells. We also observed that the PAF1/PD2-HIF1α complex binds to the LDHA promoter to regulate its expression, reprogramming the metabolism to utilize the aerobic glycolysis pathway preferentially.

Conclusion: Overall, the results indicate that PAF1/PD2 rewires PC metabolism by interacting with HIF1α to regulate the expression of LDHA.

Background: Mitochondria are known to synthesize adenosine triphosphate (ATP) through oxidative phosphorylation. Understanding and accurately measuring mitochondrial ATP synthesis rate can provide insights into the functional status of mitochondria and how it contributes to overall cellular energy homeostasis. Traditional methods only estimate mitochondrial function by measuring ATP levels at a single point in time or through oxygen consumption rates. This study introduced the relative mitochondrial ATP synthesis response against inhibiting and stimulating substrates (MitoRAISE), designed to detect real-time changes in ATP levels as the cells respond to substrates.

Methods: The sensitivity and specificity of the MitoRAISE assay were verified under various conditions, including the isolation of mitochondria, variations in cell numbers, cells exhibiting mitochondrial damage, and heterogeneous mixtures. Using peripheral blood mononuclear cells (PBMCs), we analyzed MitoRAISE data from 19 patients with breast cancer and 23 healthy women.

Results: The parameters observed in the MitoRAISE data increased depending on the quantity of isolated mitochondria and cell count, whereas it remained unmeasured in mitochondrial-damaged cell lines. Basal ATP, rotenone response, malonate response, and mitochondrial DNA copy numbers were lower in PBMCs from patients with breast cancer than in those from healthy women.

Conclusions: The MitoRAISE assay has demonstrated its sensitivity and specificity by measuring relative ATP synthesis rates under various conditions. We propose MitoRAISE assay as a potential tool for monitoring changes in the mitochondrial metabolic status associated with various diseases.

Background: Prostate cancer (PCa) shows a rewired metabolism featuring increased fatty acid uptake and synthesis via de novo lipogenesis, both sharply related to mitochondrial physiology. The docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that exerts its antitumoral properties via different mechanisms, but its specific action on mitochondria in PCa is not clear. Therefore, we investigated whether the DHA modulates mitochondrial function in PCa cell lines.

Methods: Here, we evaluated mitochondrial function of non-malignant PNT1A and the castration-resistant (CRPC) prostate 22Rv1 and PC3 cell lines in response to DHA incubation. For this purpose, we used Seahorse extracellular flux assay to assess mitochondria function, [¹⁴C]-glucose to evaluate its oxidation as well as its contribution to fatty acid synthesis, ¹H-NMR for metabolite profile determination, MitoSOX for superoxide anion production, JC-1 for mitochondrial membrane polarization, mass spectrometry for determination of phosphatidylglycerol levels and composition, staining with MitoTracker dye to assess mitochondrial morphology under super-resolution in addition to Transmission Electron Microscopy, In-Cell ELISA for COX-I and SDH-A protein expression and flow cytometry (Annexin V and 7-AAD) for cell death estimation.

Results: In all cell lines DHA decreased basal respiratory activity, ATP production, and the spare capacity in mitochondria. Also, the omega-3 induced mitochondrial hyperpolarization, ROS overproduction and changes in membrane phosphatidylglycerol composition. In PNT1A, DHA led to mitochondrial fragmentation and it increased glycolysis while in cancer cells it stimulated glucose oxidation, but decreased de novo lipogenesis specifically in 22Rv1, indicating a metabolic shift. In all cell lines, DHA modulated several metabolites related to energy metabolism and it was incorporated in phosphatidylglycerol, a precursor of cardiolipin, increasing the unsaturation index in the mitochondrial membrane. Accordingly, DHA triggered cell death mainly in PNT1A and 22Rv1.

Conclusion: In conclusion, mitochondrial metabolism is significantly affected by the PUFA supplementation to the point that cells are not able to proliferate or survive under DHA-enriched condition. Moreover, combination of DHA supplementation with inhibition of metabolism-related pathways, such as de novo lipogenesis, may be synergistic in castration-resistant prostate cancer.

Background: The metabolic reprogramming of amino acids is critical for cancer cell growth and survival. Notably, intracellular accumulation of cysteine is often observed in various cancers, suggesting its potential role in alleviating the oxidative stress associated with rapid proliferation. The liver is the primary organ for cysteine biosynthesis, but much remains unknown about the metabolic alterations of cysteine and their mechanisms in hepatocellular carcinoma cells.

Methods: RNA-seq data from patients with hepatocarcinoma were analyzed using the TNMplot database. The underlying mechanism of the oncogenic alteration of cysteine metabolism was studied in mice implanted with BNL 1ME A.7 R.1 hepatocarcinoma.

Results: Database analysis of patients with hepatocellular carcinoma revealed that the expression of enzymes involved in de novo cysteine synthesis was down-regulated accompanying with increased expression of the cystine uptake transporter xCT. Similar alterations in gene expression have also been observed in a syngeneic mouse model of hepatocarcinoma. The enhanced expression of DNA methyltransferase in murine hepatocarcinoma cells caused methylation of the upstream regions of cysteine synthesis genes, thereby repressing their expression. Conversely, suppression of de novo cysteine synthesis in healthy liver cells induced xCT expression by up-regulating the oxidative-stress response factor NRF2, indicating that reduced de novo cysteine synthesis repulsively increases cystine uptake via enhanced xCT expression, leading to intracellular cysteine accumulation. Furthermore, the pharmacological inhibition of xCT activity decreased intracellular cysteine levels and suppressed hepatocarcinoma tumor growth in mice.

Conclusions: Our findings indicate an underlying mechanism of the oncogenic alteration of cysteine metabolism in hepatocarcinoma and highlight the efficacy of alteration of cysteine metabolism as a viable therapeutic target in cancer.

Background: N6-methyladenosine (m6A) regulates the progression of breast cancer (BC). We aimed to investigate the action and mechanism involved of methyltransferase-like protein 16 (METTL16) in BC growth and metastasis.

Methods: RT-qPCR, immunoblotting, and IHC were performed to test the levels of gene expression. CCK-8, clone formation, wound healing, and transwell assays were applied to measure the cell proliferation, migration, and invasion. m6A RNA methylation and MeRIP assay were utilized to confirm the m6A level of total RNA and FBXO5 mRNA. RIP was utilized to ascertain the interaction between METTL16 and FBXO5 mRNA. The in vivo murine subcutaneous tumor and metastasis model were constructed to further confirm the action of METTL16.

Results: METTL16 was overexpression in BC cells and tissues. Inhibition of METTL16 restrained the growth and metastasis of BC. Furthermore, the METTL16 level and FBXO5 level was positively correlated in BC tissues, and METTL16 aggrandized the stability of FBXO5 mRNA depending on the m6A modification. Overexpression of FBXO5 antagonized the restrained function of METTL16 knockdown on BC cells' proliferation, migration, invasion, and EMT.

Conclusion: METTL16 boosts the mRNA stability of FBXO5 via m6A modification to facilitate the malignant action of BC in vitro and in vivo, offering new latent targets for cure of BC.

Background: Stroma AReactive Invasion Front Areas (SARIFA) is a recently identified haematoxylin & eosin (H&E)based histopathologic biomarker in gastrointestinal cancers, including colorectal cancer (CRC), defined as direct contact between tumour cells and adipocytes at the tumour invasion front. The current study aimed at validating the prognostic relevance of SARIFA in a large population-based CRC series as well as at investigating the relationship between SARIFA-status and previously established Warburg-subtypes, both surrogates of the metabolic state of the tumour cells.

Methods: SARIFA-status (positive versus negative) was determined on H&E slides of 1,727 CRC specimens. Warburg-subtype (high versus moderate versus low) data was available from our previous study. The associations between SARIFA-status, Warburg-subtype, clinicopathological characteristics and CRC-specific as well as overall survival were investigated.

Results: 28.7% (n=496) CRC were SARIFA-positive. SARIFA-positivity was associated with more advanced disease stage, higher pT category, and more frequent lymph node involvement (all p<0.001). SARIFA-positivity was more common in Warburg-high CRC. 44.2% (n=219) of SARIFA-positive CRCs were Warburg-high compared to 22.8% (n=113) being Warburg-low and 33.1% (n=164) being Warburg-moderate (p<0.001). In multivariable-adjusted analysis, patients with SARIFA-positive CRCs had significantly poorer CRC-specific (HR_CRC-specific 1.65; 95% CI 1.41-1.93) and overall survival (HR_{overall survival} 1.46; 95% CI 1.28-1.67) independent of clinically known risk factors and independent of Warburg-subtype. Combining the SARIFA-status and the Warburg-subtype to a combination score (SARIFA-negative/Warburg-high versus SARIFA-positive/Warburg-low versus SARIFA-positive/Warburg-high, and so on) did not improve the survival prediction compared to the use of SARIFA-status alone (SARIFA-negative + Warburg-high: HR_CRC-specific 1.08; 95% CI 0.84-1.38; SARIFA-positive + Warburg-low: HR_CRC-specific 1.79; 95% CI 1.32-2.41; SARIFA-positive + Warburg-high: HR_CRC-specific 1.58; 95% CI 1.23-2.04).

Conclusions: Our current study is the by far largest external validation of SARIFA-positivity as a novel independent negative prognostic H&E-based biomarker in CRC. In addition, our study shows that SARIFA-positivity is associated with the Warburg-high subtype. Further research is warranted to provide a more mechanistic understanding of the underlying tumour biology. Based on our data, we conclude SARIFA-status should be implemented in pathologic routine practice to stratify CRC patients.

Background: Despite technological advances in radiotherapy, cancer cells at the tumor margin and in diffusive infiltrates can receive subcytotoxic doses of photons. Even if only a minority of cancer cells are concerned, phenotypic consequences could be important considering that mitochondrial DNA (mtDNA) is a primary target of radiation and that damage to mtDNA can persist. In turn, mitochondrial dysfunction associated with enhanced mitochondrial ROS (mtROS) production could promote cancer cell migration out of the irradiation field in a natural attempt to escape therapy. In this study, using MCF7 and MDA-MB-231 human breast cancer cells as models, we aimed to elucidate the molecular mechanisms supporting a mitochondrial contribution to cancer cell migration induced by subclinical doses of irradiation (< 2 Gy).

Methods: Mitochondrial dysfunction was tested using mtDNA multiplex PCR, oximetry, and ROS-sensitive fluorescent reporters. Migration was tested in transwells 48 h after irradiation in the presence or absence of inhibitors targeting specific ROS or downstream effectors. Among tested inhibitors, we designed a mitochondria-targeted version of human catalase (mtCAT) to selectively inactivate mitochondrial H₂O₂.

Results: Photon irradiation at subclinical doses (0.5 Gy for MCF7 and 0.125 Gy for MDA-MB-231 cells) sequentially affected mtDNA levels and/or integrity, increased mtROS production, increased MAP2K1/MEK1 gene expression, activated ROS-sensitive transcription factors NF-κB and AP1 and stimulated breast cancer cell migration. Targeting mtROS pharmacologically by MitoQ or genetically by mtCAT expression mitigated migration induced by a subclinical dose of irradiation.

Conclusion: Subclinical doses of photon irradiation promote human breast cancer migration, which can be countered by selectively targeting mtROS.