METTL3 Inhibition Suppresses Cell Growth and Survival in Colorectal Cancer via ASNS Downregulation.

Background: Colorectal cancer (CRC) presents a significant global health burden, with high rates of incidence and mortality, and an urgent need to improve prognosis. STM2457, a novel small molecule inhibitor specific for N6-methyladenosine (m6A) catalytic enzyme Methyltransferase-like 3 (METTL3) has implicated significant treatment potentials in a few of types of cancer. However, its impact and underlying mechanism are still unclear in CRC cells. Methods: We used CCK-8 and colony formation assay to observe cell growth, flow cytometry and TUNEL approaches to detect cell apoptosis under the treatment of STM2457 on CRC cells in vitro or in vivo. RNA-sequencing, qRT-PCR and western blotting were performed to explore downstream effectors of STM2457. Messenger RNA stability was evaluated by qRT-PCR after treatment with actinomycin D. The methylated RNA immunoprecipitation (MeRIP) qPCR, dual-luciferase reporter analyses and m6A dot blotting were carried out to measure the m6A modification. Associated gene expression pattern and clinical relevance in CRC clinical tissue samples were analyzed using online database. Results: STM2457 exhibited a strong influence on cell growth suppression and apoptosis of CRC cells in vitro and subcutaneous xenograft growth in vivo. Asparagine synthetase (ASNS) was markedly downregulated upon STM2457 treatment or METTL3 knockdown and exogenous overexpression of ASNS could rescue the biological defects induced by STM2457. Mechanistically, the downregulation of ASNS by STM2457 may be due to the decrease of m6A modification level in ASNS mRNA mediated by METTL3. Conclusions: Our findings suggest that STM2457 may serve as a potential therapeutic agent and ASNS may be a new promising therapeutic target for CRC.

High USP32 expression contributes to cancer progression and is correlated with immune infiltrates in hepatocellular carcinoma.

BACKGROUND: Ubiquitin-specific protease 32 (USP32) is a highly conserved gene that promotes cancer progression. However, its role in hepatocellular carcinoma (HCC) is not well understood. The aim of this project is to explore the clinical significance and functions of USP32 in HCC. METHODS: The expression of USP32 in HCC was evaluated using data from TCGA, GEO, TISCH, tissue microarray, and human HCC samples from our hospital. Survival analysis, PPI analysis and GSEA analysis were performed to evaluate USP32-related clinical significance, key molecules and enrichment pathways. Using the ssGSEA algorithm and TIMER, we investigated the relationships between USP32 and immune infiltrates in the TME. Univariate and multivariate Cox regression analyses were then used to identify key USP32-related immunomodulators and constructed a USP32-related immune prognostic model. Finally, CCK8, transwell and colony formation assays of HCC cells were performed and an HCC nude mouse model was established to verify the oncogenic role of USP32. RESULTS: USP32 is overexpressed in HCC and its expression is an independent predictive factor for outcomes of HCC patients. USP32 is associated with pathways related to cell behaviors and cancer signaling, and its expression is significantly correlated with the infiltration of immune cells in the TME. We also successfully constructed a USP32-related immune prognostic model using 5 genes. Wet experiments confirmed that knockdown of USP32 could repress the proliferation, colony formation and migration of HCC cells in vitro and inhibit tumor growth in vivo. CONCLUSION: USP32 is highly expressed in HCC and closely correlates with the TME of HCC. It is a potential target for improving the efficacy of chemotherapy and developing new strategies for targeted therapy and immunotherapy in HCC.

PIAS3 promotes ferroptosis by regulating TXNIP via TGF-ß signaling pathway in hepatocellular carcinoma.

Ferroptosis has been suggested to play a potential role in cancer therapy as an iron-dependent programmed cell death mechanism distinct from other forms. Hepatocellular carcinoma (HCC) remains a great threat, with high mortality and limited therapeutic options. The induction of ferroptosis has emerged as a novel and promising therapeutic strategy for HCC. In the present study, we identified protein inhibitor of activated STAT3 (PIAS3) as a driver of ferroptosis in HCC using TMT-based quantitative proteomics and ferroptosis-related functional assays. Mechanistically, thioredoxin-interacting protein (TXNIP) was confirmed to be PIAS3 in promoting ferroptotic cell death, based on RNA-seq analysis. Knockdown of TXNIP degrades ferroptotic susceptibility caused by PIAS3-overexpression, whereas transfection-forced reexpression of TXNIP restores sensitivity to ferroptosis in PIAS3-downregulated cells. PIAS3 interacts with SMAD2/3 to activate transforming growth factor (TGF)-ß signaling, leading to increased TXNIP expression. Our study revealed the critical role of PIAS3 in ferroptosis and a novel actionable axis-PIAS3/TGF-ß/TXNIP that could govern ferroptotic sensitivity, paving the path for using ferroptosis as an efficient approach in HCC therapies.

UHMK1 aids colorectal cancer cell proliferation and chemoresistance through augmenting IL-6/STAT3 signaling.

UHMK1, a serine/threonine kinase with a U2AF homology motif, is implicated in RNA processing and protein phosphorylation. Increasing evidence has indicated its involvement in tumorigenesis. However, it remains to be elucidated whether UHMK1 plays a role in the development of colorectal cancer (CRC). Here, we demonstrated that UHMK1 was frequently upregulated in CRC samples compared with adjacent normal tissue and high expression of UHMK1 predicted poor outcomes. Knockdown of UHMK1 by siRNAs restrained CRC cell proliferation and increased oxaliplatin sensitivity, whereas overexpression of UHMK1 promoted CRC cell growth and oxaliplatin resistance, suggesting that UHMK1 plays important oncogenic roles in CRC. Mechanistically, we showed that UHMK1 had a significant effect on IL6/STAT3 signaling by interacting with STAT3. The interaction of UHMK1 with STAT3 enhanced STAT3 activity in regulating gene transcription. Furthermore, we found that STAT3 could in turn transcriptionally activate UHMK1 expression in CRC cells. The complementary experiments for cell growth and oxaliplatin resistance indicated the interdependent relationship between UHMK1 and STAT3. Thus, these collective findings uncovered a new UHMK1/STAT3 positive feedback regulatory loop contributing to CRC development and chemoresistance.

Transcriptome Profiling Analysis Identifies LCP1 as a Contributor for Chidamide Resistance in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) remains a significant health problem and carries with it substantial morbidity and mortality. Chidamide is a novel and orally administered histone deacetylase (HDAC) inhibitor and has been demonstrated its anti-tumor efficacy on different kinds of hematological and solid tumors. However, the underlying mechanism of chidamide resistance is still poorly characterized. METHODS: We established chidamide resistant GC cell lines, AGS ChiR and MGC803 ChiR and investigated the toxicologic effects through cell survival, colony formation and flow cytometry assays in vitro, and a subcutaneous xenograft model in vivo. RNA-sequence was then performed to screen chidamide resistance-associated genes between AGS and AGS ChiR cells. The role of Lymphocyte cytosolic protein 1 (LCP1) in chidamide resistance was explored by gain- and loss-of-function analyses. RESULTS: We found that chidamide significantly inhibited cell proliferation and induced the apoptosis in a concentration-dependent manner in wild-type GC cell lines as compared to chidamide resistant cell lines. The transcriptomic profiling, quantitative RT-PCR, and western blot data revealed that LCP1 was upregulated in AGS ChiR cells compared with parental cells. Overexpression of LCP1 conferred and knockdown of LCP1 attenuated the chidamide resistance of GC cells. Epigenetic derepression of LCP1 by chidamide may be a possible reason for the contribution of LCP1 to chidamide resistance. CONCLUSIONS: These findings illustrated that LCP1 may play a chidamide resistance role in GC, suggesting that LCP1 could be a potential target for the therapy of GC combined with chidamide.

PIK3CA mutations-mediated downregulation of circLHFPL2 inhibits colorectal cancer progression via upregulating PTEN.

BACKGROUND: PIK3CA mutation and PTEN suppression lead to tumorigenesis and drug resistance in colorectal cancer (CRC). There is no research on the role of circular RNAs (circRNAs) in regulating PIK3CA mutation and MEK inhibitor resistance in CRC. METHODS: The expression of circLHFPL2 in PIK3CA-mutant and wild-type cells and tissues was quantified by RNA-sequencing and qRT-PCR. CCK-8 assay and colony formation assay were used to evaluate cell viability. Annexin V/PI staining was implemented to assess cell apoptosis. Luciferase assay, biotin-coupled microRNA capture, and RIP assay were used to validate the interaction among potential targets. Western blotting and qRT-PCR assays were used to evaluate the expression of involved targets. Xenograft tumor in a nude mouse model was used to explore the role of circRNAs in vivo. RESULTS: RNA sequencing defined downregulated expression of circLHFPL2 in both PIK3CAH1047R (HCT116) and PIK3CAE545K (DLD1) cells. CircLHFPL2 was also downregulated in PIK3CA-mutant CRC primary cells and tissues, which was correlated with poor prognosis. CircLHFPL2 was mainly localized in the cytoplasm and its downregulation was attributed to the PI3K/AKT signaling pathway activated by phosphorylating Foxo3a. CircLHFPL2 inhibited PI3KCA-Mut CRC progression both in vitro and in vivo. Furthermore, our work indicated that circLHFPL2 acts as a ceRNA to sponge miR-556-5p and miR-1322 in CRC cells and in turn modulate the expression of PTEN. Importantly, circLHFPL2 was able to overcome PIK3CA-mediated MEK inhibitor resistance in CRC cells. CONCLUSIONS: Downregulation of circLHFPL2 sustains the activation of the PI3K/AKT signaling pathway via a positive feedback loop in PIK3CA-mutant CRC. In addition, downregulation of circLHFPL2 leads to MEK inhibitor resistance in CRC. Therefore, targeting circLHFPL2 could be an effective approach for the treatment of CRC patients harboring oncogenic PIK3CA mutations.

Honokiol improves cognitive impairment in APP/PS1 mice through activating mitophagy and mitochondrial unfolded protein response.

Activated mitophagy and mitochondrial unfolded protein response (UPRmt) has been reported to protect against mitochondrial dysfunction, which is closely related to the onset of Alzheimer's disease (AD). Honokiol (HKL, C18H18O2) is a kind of natural extraction from bark of Magnolia officinalis with anti-AD effect, and our study aims to explore the effect of HKL on mitophagy and UPRmt in AD. Briefly, male APP/PS1 mice and Aß oligmer (AßO)-treated primary hippocampal neurons were respectively used to mimic AD in vivo and in vitro. It was determined that HKL significantly ameliorated cognitive impairment and synaptic damages in APP/PS1 mice. Besides, the activated mitophagy and UPRmt together with inhibited oxidative stress and improved mitochondrial dynamic disorder were further validated in hippocampus of HKL-treated APP/PS1 mice. Meanwhile, HKL-treated mice displayed much higher hippocampal expression and activity of mitochondrial sirtuin 3 (SIRT3). Therefore, SIRT3 knockdown was further achieved in primary hippocampal neurons by effective shRNA, and we determined that HKL improved synaptic damage, mitochondrial dysfunction, mitophagy and UPRmt in AßO-treated primary hippocampal neurons in a SIRT3-dependent manner. In summary, our study validates the protective effect of HKL on AD, and highlights that HKL exerts anti-AD effect by activating mitophagy and UPRmt.

Blockade of enhancer of zeste homolog 2 alleviates renal injury associated with hyperuricemia.

Hyperuricemia has been identified as an independent risk factor for chronic kidney disease (CKD) and is associated with the progression of kidney diseases. It remains unknown whether enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 methyltransferase, can regulate metabolism of serum uric acid and progression of renal injury induced by hyperuricemia. In this study, we demonstrated that blockade of EZH2 with 3-DZNeP, a selective EZH2 inhibitor, or silencing of EZH2 with siRNA inhibited uric acid-induced renal fibroblast activation and phosphorylation of Smad3, epidermal growth factor receptor (EGFR), and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured renal fibroblasts. Inhibition of EZH2 also suppressed proliferation of renal fibroblasts and epithelial-mesenchymal transition of tubular cells. In a mouse model of renal injury induced by hyperuricemia, EZH2 and trimethylation of histone H3 at lysine27 expression levels were enhanced, which was coincident with renal damage and increased expression of lipocalin-2 and cleaved caspase-3. Inhibition of EZH2 with 3-DZNeP blocked all these responses. Furthermore, 3-DZNeP treatment decreased the level of serum uric acid and xanthine oxidase activity, alleviated renal interstitial fibrosis, inhibited activation of transforming growth factor-ß/Smad3, EGFR/ERK1/2, and nuclear factor-κB signaling pathways, as well as reduced expression of multiple chemokines/cytokines. Collectively, EZH2 inhibition can reduce the level of serum uric acid and alleviate renal injury and fibrosis through a mechanism associated with inhibition of multiple signaling pathways. Targeting EZH2 may be a novel strategy for the treatment of hyperuricemia-induced CKD.

EGF Receptor Inhibition Alleviates Hyperuricemic Nephropathy.

Hyperuricemia is an independent risk factor for CKD and contributes to kidney fibrosis. In this study, we investigated the effect of EGF receptor (EGFR) inhibition on the development of hyperuricemic nephropathy (HN) and the mechanisms involved. In a rat model of HN induced by feeding a mixture of adenine and potassium oxonate, increased EGFR phosphorylation and severe glomerular sclerosis and renal interstitial fibrosis were evident, accompanied by renal dysfunction and increased urine microalbumin excretion. Administration of gefitinib, a highly selective EGFR inhibitor, prevented renal dysfunction, reduced urine microalbumin, and inhibited activation of renal interstitial fibroblasts and expression of extracellular proteins. Gefitinib treatment also inhibited hyperuricemia-induced activation of the TGF-ß1 and NF-κB signaling pathways and expression of multiple profibrogenic cytokines/chemokines in the kidney. Furthermore, gefitinib treatment suppressed xanthine oxidase activity, which mediates uric acid production, and preserved expression of organic anion transporters 1 and 3, which promotes uric acid excretion in the kidney of hyperuricemic rats. Thus, blocking EGFR can attenuate development of HN via suppression of TGF-ß1 signaling and inflammation and promotion of the molecular processes that reduce uric acid accumulation in the body.