RNA demethylase ALKBH5 promotes colorectal cancer progression by posttranscriptional activation of RAB5A in an m6A-YTHDF2-dependent manner.

BACKGROUND: N6-methyladenosine (m6A) modification is an emerging epigenetic regulatory mechanism in tumourigenesis. Considering that AlkB homolog 5 (ALKBH5) is a well-described m6A demethylase in previous enzyme assays, we aimed to investigate the role of m6A methylation alteration conferred by disturbed ALKBH5 in colorectal cancer (CRC) development. METHODS: Expression of ALKBH5 and its correlation with clinicopathological characteristics of CRC were evaluated using the prospectively maintained institutional database. The molecular role and underlying mechanism of ALKBH5 in CRC were explored using in vitro and in vivo experiments with methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, RIP-qPCR and luciferase reporter assays. RESULTS: ALKBH5 expression was significantly upregulated in CRC tissues compared to the paired adjacent normal tissues, and higher expression of ALKBH5 was independently associated with worse overall survival in CRC patients. Functionally, ALKBH5 promoted the proliferative, migrative and invasive abilities of CRC cells in vitro and enhanced subcutaneous tumour growth in vivo. Mechanistically, RAB5A was identified as the downstream target of ALKBH5 in CRC development, and ALKBH5 posttranscriptionally activated RAB5A by m6A demethylation, which impeded the YTHDF2-mediated degradation of RAB5A mRNA. In addition, we demonstrated that dysregulation of the ALKBH5-RAB5A axis could affect the tumourigenicity of CRC. CONCLUSIONS: ALKBH5 facilitates the progression of CRC by augmenting the expression of RAB5A via an m6A-YTHDF2-dependent manner. Our findings suggested that ALKBH5-RAB5A axis might serve as valuable biomarkers and effective therapeutic targets for CRC.

Clinical atlas of rectal cancer highlights the barriers and insufficient interventions underlying the unfavorable outcomes in older patients.

Background: Aging confers an increased risk of developing cancer, and the global burden of cancer is cumulating as human longevity increases. Providing adequate care for old patients with rectal cancer is challenging and complex. Method: A total of 428 and 44,788 patients diagnosed with non-metastatic rectal cancer from a referral tertiary care center (SYSU cohort) and the Surveillance Epidemiology and End Results database (SEER cohort) were included. Patients were categorized into old (over 65 years) and young (aged 50-65 years) groups. An age-specific clinical atlas of rectal cancer was generated, including the demographic and clinicopathological features, molecular profiles, treatment strategies, and clinical outcomes. Results: Old and young patients were similar in clinicopathological risk factors and molecular features, including TNM stage, tumor location, tumor differentiation, tumor morphology, lymphovascular invasion, and perineural invasion. However, old patients had significantly worse nutritional status and more comorbidities than young patients. In addition, old age was independently associated with less systemic cancer treatment (adjusted odds ratio 0.294 [95% CI 0.184-0.463, P < 0.001]). We found that old patients had significantly worse overall survival (OS) outcomes in both SYSU (P < 0.001) and SEER (P < 0.001) cohorts. Moreover, the death and recurrence risk of old patients in the subgroup not receiving chemo/radiotherapy (P < 0.001 for OS, and P = 0.046 for time to recurrence [TTR]) reverted into no significant risk in the subgroup receiving chemo/radiotherapy. Conclusions: Although old patients had similar tumor features to young patients, they had unfavorable survival outcomes associated with insufficient cancer care from old age. Specific trials with comprehensive geriatric assessment for old patients are needed to identify the optimal treatment regimens and improve unmet cancer care. Study registration: The study was registered on the research registry with the identifier of researchregistry 7635.

Fasting-mimicking diet synergizes with ferroptosis against quiescent, chemotherapy-resistant cells.

BACKGROUND: More than ten randomized clinical trials are being tested to evaluate the efficacy, effectiveness and safety of a fasting-mimicking diet (FMD) combined with different antitumor agents. METHODS: UMI-mRNA sequencing, Cell-cycle analysis, Label retention, metabolomics, Multilabeling et al. were used to explore mechanisms. A tandem mRFP-GFP-tagged LC3B, Annexin-V-FITC Apoptosis, TUNEL, H&E, Ki-67 and animal model was used to search for synergistic drugs. FINDINGS: Here we showed that fasting or FMD retards tumor growth more effectively but does not increase 5-fluorouracil/oxaliplatin (5-FU/OXA) sensitivity to apoptosis in vitro and in vivo. Mechanistically, we demonstrated that CRC cells would switch from an active proliferative to a slow-cycling state during fasting. Furthermore, metabolomics shows cell proliferation was decreased to survive nutrient stress in vivo, as evidenced by a low level of adenosine and deoxyadenosine monophosphate. CRC cells would decrease proliferation to achieve increased survival and relapse after chemotherapy. In addition, these fasting-induced quiescent cells were more prone to develop drug-tolerant persister (DTP) tumor cells postulated to be responsible for cancer relapse and metastasis. Then, UMI-mRNA sequencing uncovered the ferroptosis pathway as the pathway most influenced by fasting. Combining fasting with ferroptosis inducer treatment leads to tumor inhibition and eradication of quiescent cells by boosting autophagy. INTERPRETATION: Our results suggest that ferroptosis could improve the antitumor activity of FMD + chemotherapy and highlight a potential therapeutic opportunity to avoid DTP cells-driven tumor relapse and therapy failure. FUNDING: A full list of funding bodies can be found in the Acknowledgements section.

Current Surveillance After Treatment is Not Sufficient for Patients With Rectal Cancer With Negative Baseline CEA.

BACKGROUND: Serum CEA has been widely used to screen for potential recurrent disease after resection in rectal cancer. However, the influence of baseline CEA on the performance of CEA in recurrence surveillance needs to be investigated. PATIENTS AND METHODS: This longitudinal cohort study included 484 patients with nonmetastatic rectal cancer from 18,013 patients in a prospectively enrolled institutional database program of colorectal disease. Baseline CEA levels were determined before treatment, and CEA-based follow-up tests and examinations were applied in the surveillance after treatment. RESULTS: A total of 62.6% (62/99) overall, 53.5% (23/43) local, and 64.9% (50/77) distant recurrences were seen in patients who had similar CEA levels with their baseline statuses. The sensitivity of elevated CEA levels during surveillance for overall recurrence was significantly lower in patients with negative baseline CEA than in those with elevated baseline CEA levels (41.3% vs 69.4%; P =.007). Moreover, similar results were observed in the surveillance for local (50% vs 61.5%; P =.048) and distant (39.6% vs 72.4%; P =.005) recurrences between these 2 patient groups. However, CEA had comparable and excellent specificity during surveillance for recurrent disease in these groups. The addition of CA19-9 to the CEA assay significantly improved the sensitivity in recurrence surveillance for patients with negative baseline CEA (49.2% vs 41.3%; P =.037). Finally, we identified a subgroup of CEA-turn recurrences characterized by negative CEA at baseline, elevated CEA at recurrence, and worse survival outcomes after recurrence (hazard ratio, 1.88; 95% CI, 1.07-3.30; P =.026). CONCLUSIONS: In patients with rectal cancer with negative baseline CEA, serum CEA had insufficient sensitivity in recurrence surveillance after treatment, and additional surveillance may improve oncologic outcomes. Baseline CEA should be considered before CEA-based surveillance can be applied in the follow-up trials.

Systemic Inflammation Status Relates to Anti-inflammatory Drug Benefit and Survival in Rectal Cancer.

BACKGROUND: Systemic inflammation status has been recognized as a sensitive marker associated with survival in cancers and anti-inflammatory treatment outcomes in inflammation-derived diseases. This study aimed to investigate the role of systemic inflammation status as a predictive marker for survival and anti-inflammatory treatment benefit in rectal cancer patients. METHODS: A total of 475 patients with stage I-III rectal cancer receiving curative resection were prospectively enrolled. The platelet-neutrophils to lymphocytes ratio (PNLR) that integrates neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios was applied to enable a comprehensive evaluation of systemic inflammation status and investigate its association with survival and nonsteroidal anti-inflammatory drugs (NSAIDs) benefit. Patients were grouped according to baseline PNLR and perioperative use of NSAIDs. RESULTS: The high-PNLR group had worse 5-y disease-free survival (DFS) compared with the low-PNLR group (61.2% versus 70.9%, P = 0.014). Multivariate analyses confirmed that PNLR was an independent predictor for DFS (hazard ratio [HR] 1.42, 95% CI: 1.03-1.97, P = 0.031). A nomogram including PNLR and other independent prognostic factors was developed and validated to predict DFS. In the high-PNLR subset, NSAIDs group had a 21.3% lower risk of recurrence than non-NSAIDs group (P = 0.009), and multivariate analysis confirmed the independently significant association of perioperative NSAIDs use with better DFS (hazard ratio 0.36, 95% CI 0.16-0.78, P = 0.010). However, this association was not significant in the low-PLR subset. CONCLUSIONS: Baseline PNLR could be used to predict DFS and NSAIDs benefit in rectal cancer patients. This study highlights the potential survival benefit from the anti-inflammatory treatment in the patients with elevated systemic inflammation status in cancer patients.

Nomograms for Prediction of Molecular Phenotypes in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) patients with different molecular phenotypes, including microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and somatic mutations in BRAF and KRAS gene, vary in treatment response and prognosis. However, molecular phenotyping under adequate quality control in a community-based setting may be difficult. We aimed to build the nomograms based on easily accessible clinicopathological characteristics to predict molecular phenotypes. METHODS: Three hundred and six patients with pathologically confirmed stage I-IV CRC were included in the cohort. The assays for MSI, CIMP, and mutations in BRAF and KRAS gene were performed using resected tumor samples. The candidate predictors were identified from clinicopathological variables using multivariate Logistic regression analyses to construct the nomograms that could predict each molecular phenotype. RESULTS: The incidences of MSI, CIMP, BRAF mutation and KRAS mutation were 25.3% (72/285), 2.5% (7/270), 3.4% (10/293), and 34.8% (96/276) respectively. In the multivariate Logistic analysis, poor differentiation and high neutrophil/lymphocyte ratio (NLR) were independently associated with MSI; poor differentiation, high NLR and high carcinoembryonic antigen/tumor size ratio (CSR) were independently associated with CIMP; poor differentiation, lymphovascular invasion and high CSR were independently associated with BRAF mutation; poor differentiation, proximal tumor, mucinous tumor and high NLR were independently associated with KRAS mutation. Four nomograms for MSI, CIMP, BRAF mutation and KRAS mutation were developed based on these independent predictors, the C-indexes of which were 61.22% (95% CI: 60.28-62.16%), 95.57% (95% CI: 95.20-95.94%), 83.56% (95% CI: 81.54-85.58%), and 69.12% (95% CI: 68.30-69.94%) respectively. CONCLUSION: We established four nomograms using easily accessible variables that could well predict the presence of MSI, CIMP, BRAF mutation and KRAS mutation in CRC patients.

Potential Genes Related to Levofloxacin Resistance in Mycobacterium tuberculosis Based on Transcriptome and Methylome Overlap Analysis.

Drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become an increasingly serious public health problem and has complicated tuberculosis (TB) treatment. Levofloxacin (LOF) is an ideal anti-tuberculosis drug in clinical applications. However, the detailed molecular mechanisms of LOF-resistant M. tuberculosis in TB treatment have not been revealed. Our study performed transcriptome and methylome sequencing to investigate the potential biological characteristics of LOF resistance in M. tuberculosis H37Rv. In the transcriptome analysis, 953 differentially expressed genes (DEGs) were identified; 514 and 439 DEGs were significantly downregulated and upregulated in the LOF-resistant group and control group, respectively. The KEGG pathway analysis revealed that 97 pathways were enriched in this study. In the methylome analysis, 239 differentially methylated genes (DMGs) were identified; 150 and 89 DMGs were hypomethylated and hypermethylated in the LOF-resistant group and control group, respectively. The KEGG pathway analysis revealed that 74 pathways were enriched in this study. The overlap study suggested that 25 genes were obtained. It was notable that nine genes expressed downregulated mRNA and upregulated methylated levels, including pgi, fadE4, php, cyp132, pckA, rpmB1, pfkB, acg, and ctpF, especially cyp132, pckA, and pfkB, which were vital in LOF-resistant M. tuberculosis H37Rv. The overlapping genes between transcriptome and methylome could be essential for studying the molecular mechanisms of LOF-resistant M. tuberculosis H37Rv. These results may provide informative evidence for TB treatment with LOF.

The Stress Response Regulator AflSkn7 Influences Morphological Development, Stress Response, and Pathogenicity in the Fungus Aspergillus flavus.

This study focused on AflSkn7, which is a stress response regulator in the aflatoxin-producing Aspergillus flavus. The ΔAflSkn7 mutants exhibited partially defective conidial formation and a complete inability to generate sclerotia, indicating AflSkn7 affects A. flavus asexual and sexual development. The mutants tolerated osmotic stress but were partially susceptible to the effects of cell wall stress. Additionally, the ΔAflSkn7 mutants were especially sensitive to oxidative stress. These observations confirmed that AflSkn7 influences oxidative stress responses rather than osmotic stress responses. Additionally, AflSkn7 was observed to increase aflatoxin biosynthesis and seed infection rates. These results indicate AflSkn7 affects A. flavus morphological development, stress response, aflatoxin production, and pathogenicity. The results of this study may facilitate the development of new methods to manage A. flavus infections.

Inhibition of aflatoxin metabolism and growth of Aspergillus flavus in liquid culture by a DNA methylation inhibitor.

Aflatoxins (AFs) are a group of highly oxygenated polyketidese-derived toxins mainly produced by Aspergillus flavus and A. parasiticus, whose biosynthesis mechanisms are extremely sophisticated. Methylation is known as the major form of epigenetic regulation, which is correlated with gene expression. As the DNA methylation inhibitor 5-azacytidine (5-AC) blocks AF production, we studied AFB1 metabolism and morphological changes of A. flavus by treatment with 5-AC in liquid culture. The results show that 5-AC caused a decrease in AF production and concurrent changes in morphology. In addition, we isolated a non-aflatoxigenic mutant of A. flavus, showing a significant reduction in pigment production, after 5-AC treatment. This mutant showed significant reduction in the expression of genes in the AF biosynthesis pathway, and conidia formation. Furthermore, as AF biosynthesis and oxidative stress are intimately related events, we assessed the viability of A. flavus to oxidative stress after treatment with 5-AC, which showed that the mutant was more sensitive to the strong oxidant hydrogen peroxide. We found that the non-aflatoxigenic mutant showed a decrease in reactive oxygen species (ROS) and metabolites indicative of oxidative stress, which may be caused by the disruption of the defence system against excessive ROS formation after 5-AC treatment. These data indicate that 5-AC, as an inactivator of DNA methyltransferase, plays a very important role in AFB1 metabolism and the development of A. flavus, which might provide an effective strategy to pre- or post-harvest control of AFs.