Lactate enhances NMNAT1 lactylation to sustain nuclear NAD+ salvage pathway and promote survival of pancreatic adenocarcinoma cells under glucose-deprived conditions.

The aim of this study was to investigate the underlying molecular mechanism behind the promotion of cell survival under conditions of glucose deprivation by l-lactate. To accomplish this, we performed tissue microarray and immunohistochemistry staining to analyze the correlation between the abundance of pan-Lysine lactylation and prognosis. In vivo evaluations of tumor growth were conducted using the KPC and nude mice xenograft tumor model. For mechanistic studies, multi-omics analysis, RNA interference, and site-directed mutagenesis techniques were utilized. Our findings robustly confirmed that l-lactate promotes cell survival under glucose deprivation conditions, primarily by relying on GLS1-mediated glutaminolysis to support mitochondrial respiration. Mechanistically, we discovered that l-lactate enhances the NMNAT1-mediated NAD+ salvage pathway while concurrently inactivating p-38 MAPK signaling and suppressing DDIT3 transcription. Notably, Pan-Kla abundance was significantly upregulated in patients with Pancreatic adenocarcinoma (PAAD) and associated with poor prognosis. We identified the 128th Lysine residue of NMNAT1 as a critical site for lactylation and revealed EP300 as a key lactyltransferase responsible for catalyzing lactylation. Importantly, we elucidated that lactylation of NMNAT1 enhances its nuclear localization and maintains enzymatic activity, thereby supporting the nuclear NAD+ salvage pathway and facilitating cancer growth. Finally, we demonstrated that the NMNAT1-dependent NAD+ salvage pathway promotes cell survival under glucose deprivation conditions and is reliant on the activity of Sirt1. Collectively, our study has unraveled a novel molecular mechanism by which l-lactate promotes cell survival under glucose deprivation conditions, presenting a promising strategy for targeting lactate and NAD+ metabolism in the treatment of PAAD.

LonP1 Links Mitochondria-ER Interaction to Regulate Heart Function.

Interorganelle contacts and communications are increasingly recognized to play a vital role in cellular function and homeostasis. In particular, the mitochondria-endoplasmic reticulum (ER) membrane contact site (MAM) is known to regulate ion and lipid transfer, as well as signaling and organelle dynamics. However, the regulatory mechanisms of MAM formation and their function are still elusive. Here, we identify mitochondrial Lon protease (LonP1), a highly conserved mitochondrial matrix protease, as a new MAM tethering protein. The removal of LonP1 substantially reduces MAM formation and causes mitochondrial fragmentation. Furthermore, deletion of LonP1 in the cardiomyocytes of mouse heart impairs MAM integrity and mitochondrial fusion and activates the unfolded protein response within the ER (UPRER). Consequently, cardiac-specific LonP1 deficiency causes aberrant metabolic reprogramming and pathological heart remodeling. These findings demonstrate that LonP1 is a novel MAM-localized protein orchestrating MAM integrity, mitochondrial dynamics, and UPRER, offering exciting new insights into the potential therapeutic strategy for heart failure.

Identification of three metabolic subtypes in gastric cancer and the construction of a metabolic pathway-based risk model that predicts the overall survival of GC patients.

Gastric cancer (GC) is highly heterogeneous and GC patients have low overall survival rates. It is also challenging to predict the prognosis of GC patients. This is partly because little is known about the prognosis-related metabolic pathways in this disease. Hence, our objective was to identify GC subtypes and genes related to prognosis, based on changes in the activity of core metabolic pathways in GC tumor samples. Differences in the activity of metabolic pathways in GC patients were analyzed using Gene Set Variation Analysis (GSVA), leading to the identification of three clinical subtypes by non-negative matrix factorization (NMF). Based on our analysis, subtype 1 showed the best prognosis while subtype 3 exhibited the worst prognosis. Interestingly, we observed marked differences in gene expression between the three subtypes, through which we identified a new evolutionary driver gene, CNBD1. Furthermore, we used 11 metabolism-associated genes identified by LASSO and random forest algorithms to construct a prognostic model and verified our results using qRT-PCR (five matched clinical tissues of GC patients). This model was found to be both effective and robust in the GSE84437 and GSE26253 cohorts, and the results from multivariate Cox regression analyses confirmed that the 11-gene signature was an independent prognostic predictor (p < 0.0001, HR = 2.8, 95% CI 2.1-3.7). The signature was found to be relevant to the infiltration of tumor-associated immune cells. In conclusion, our work identified significant GC prognosis-related metabolic pathways in different GC subtypes and provided new insights into GC-subtype prognostic assessment.

WZ35 inhibits gastric cancer cell metastasis by depleting glutathione to promote cellular metabolic remodeling.

This study aimed at elucidating the crosstalk between redox reaction and metabolic remodeling through uncovering the mechanism underlying WZ35-mediated reactive oxygen species (ROS) production and regulation of amino acid metabolism to inhibit gastric cancer (GC) cell metastasis. The activity and biosafety of curcumin analog, WZ35, were verified in vitro and in vivo. The potential molecular mechanism underlying WZ35-mediated enhanced radiotherapeutic sensitivity by reduced Glutathione (GSH) depletion was elucidated by RNA sequencing, single-cell sequencing (scRNA-seq), metabolic mass spectrometry, and other molecular experiments. Compared to curcumin, WZ35 proved more potent anti-proliferative and anti-metastasis properties. Importantly, we demonstrated that WZ35 could consume GSH in multiple ways, including by reduction of raw materials and consumption reserves, inhibition of reformation, and enhanced decomposition. Mechanistically, we identify that WZ35 maintains the GSH depletion phenotype through the ROS-YAP-AXL-ALKBH5-GLS2 loop, further backing the relevance of metabolic remodeling in the tumor microenvironment with tumor metastasis and the role of m6A in tumor metastasis. Collectively, our study identified WZ35 as a novel GSH depletion agent and a previously undiscovered GSH depletion loop mechanism in GC cell metastasis.

SRSF3 and HNRNPH1 Regulate Radiation-Induced Alternative Splicing of Protein Arginine Methyltransferase 5 in Hepatocellular Carcinoma.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3'- splicing site on intron 2 and the alternative 3'- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.

A novel small molecule glycolysis inhibitor WZ35 exerts anti-cancer effect via metabolic reprogramming.

BACKGROUND: Liver cancer is the fifth leading cause of cancer death worldwide, but early diagnosis and treatment of liver cancer remains a clinical challenge. How to screen and diagnose liver cancer early and prolong the survival rate is still the focus of researchers. METHODS: Cell experiments were used to detect the effect of WZ35 on the colony formation ability and proliferation activity of hepatoma cells, nude mouse experiment to observe the in vivo anticancer activity and toxic side effects of WZ35; metabolomics analysis, glucose metabolism experiment and Seahorse analysis of liver cancer cells treated with WZ35; cell experiments combined with bioinformatics analysis to explore the mechanism of WZ35-mediated metabolic reprogramming to exert anticancer activity; tissue microarray and case analysis to evaluate the clinical significance of biomarkers for early diagnosis, treatment and prognosis evaluation of liver cancer. RESULTS: WZ35 inhibited the proliferation activity of various cell lines of liver cancer, and showed good therapeutic effect in nude mice model of hepatocellular carcinoma without obvious toxic and side effects; WZ35 inhibited the absorption of glucose in hepatoma cells, and the drug effect glycolysis, phosphorylation and purine metabolism are relatively seriously damaged; WZ35 mainly inhibits YAP from entering the nucleus as a transcription factor activator by activating oxidative stress in liver cancer cells, reducing the transcription of GLUT1, and finally reducing its GLUT1. Tissue microarray and case analysis showed that GLUT1 and YAP were highly expressed and correlated in liver cancer patients, and were associated with poor patient prognosis. The GLUT1-YAP risk model had a high score in predicting prognosis. CONCLUSION: The study confirms that WZ35 is a small molecule glycolysis inhibitor, and through its properties, it mediates metabolic reprogramming dominated by impaired glycolysis, oxidative phosphorylation and purine metabolism to inhibit the proliferation activity of liver cancer cells. Our findings present novel insights into the pathology of liver cancer and potential targets for new therapeutic strategies. GLUT1-YAP has important reference significance for predicting the stages of disease progression in liver cancer patients and have the potential to serve as novel biomarkers for the diagnosis and treatment of liver cancer.

Cold protection allows local cryotherapy in a clinical-relevant model of traumatic optic neuropathy.

Therapeutic hypothermia (TH) is potentially an important therapy for central nervous system (CNS) trauma. However, its clinical application remains controversial, hampered by two major factors: (1) Many of the CNS injury sites, such as the optic nerve (ON), are deeply buried, preventing access for local TH. The alternative is to apply TH systemically, which significantly limits the applicable temperature range. (2) Even with possible access for 'local refrigeration', cold-induced cellular damage offsets the benefit of TH. Here we present a clinically translatable model of traumatic optic neuropathy (TON) by applying clinical trans-nasal endoscopic surgery to goats and non-human primates. This model faithfully recapitulates clinical features of TON such as the injury site (pre-chiasmatic ON), the spatiotemporal pattern of neural degeneration, and the accessibility of local treatments with large operating space. We also developed a computer program to simplify the endoscopic procedure and expand this model to other large animal species. Moreover, applying a cold-protective treatment, inspired by our previous hibernation research, enables us to deliver deep hypothermia (4 °C) locally to mitigate inflammation and metabolic stress (indicated by the transcriptomic changes after injury) without cold-induced cellular damage, and confers prominent neuroprotection both structurally and functionally. Intriguingly, neither treatment alone was effective, demonstrating that in situ deep hypothermia combined with cold protection constitutes a breakthrough for TH as a therapy for TON and other CNS traumas.

Hypothermic therapy is a radical type of treatment that involves cooling a person's core body temperature several degrees below normal to protect against brain damage. Lowering body temperature slows blood flow, which reduces inflammation, and eases metabolic demands, similar to hibernation. It can also reduce lasting damage to the brain and aid recovery when used to treat people who have gone into cardiac arrest, where their heart suddenly stops beating. Recently, there has been renewed interest in using hypothermic therapy to treat people who have sustained traumatic brain injuries, which can cause brain swelling, and other nerve injuries. However, its use remains controversial because clinical trials have failed to show that inducing mild hypothermia provides any benefit for people with severe nerve injuries. This might be because cooling cells to near-freezing temperatures can damage their internal structural supports, called microtubules, thwarting any therapeutic benefit. Traumatic optical neuropathy is a type of injury in which the optic nerve ­ the nerve that connects the eyes to the brain ­ is damaged or severed, causing vision loss. There is currently no clinically proven treatment for this condition, nor is there a system that can test local treatments in large animals as a prior test to using the treatment in the clinic. Therefore, Zhang et al. wanted to establish such a animal model and test whether local hypothermic therapy could help protect the optic nerve. Zhang et al. used a surgical tool guided by an endoscope (a thin plastic tube with a light and camera attached to it) to injure the optic nerves of goats, and then deliver hypothermic therapy. To cool the surgically-injured nerves to a chilly 4C, Zhang et al. applied a deep-cooling agent, using a second reagent (a cocktail of protease inhibitors) to protect the cells' microtubules from cold-induced damage, an insight gained from a previous study of hibernating animals. This was critical, as the hypothermic therapy was only effective when the secondary protective agent was applied. The combination therapy developed by Zhang et al. relieved some aspects of nerve degeneration at the injury site and activated an anti-inflammatory response in cells, but did not restore vision. To simplify surgical techniques, Zhang et al. also developed a computer program which generates virtual surgical paths for up-the-nose endoscopic procedures based on brain scans of an animal's skull. This program was successfully applied in a range of large animals, including goats and macaque monkeys. Zhang et al.'s work establishes a method to study treatments for traumatic optical neuropathy using large animals, including hypothermic therapy. The methods developed could also be useful to study other optic nerve disorders, such as optic neuritis or ischemic optic neuropathy.

Curcumin micelles suppress gastric tumor cell growth by upregulating ROS generation, disrupting redox equilibrium and affecting mitochondrial bioenergetics.

Gastric cancer is the fourth most common cancer and the second most frequent cause of cancer death worldwide. Chemotherapy is an important treatment. However, traditional chemotherapy drugs have low bioavailability and targeting ability. Therefore, we developed curcumin-encapsulated micelles for the treatment of gastric cancer and investigated their antitumor efficacy and active mechanism. Gastric cancer cells were treated with different concentrations of curcumin micelles. MTS cell proliferation assays, flow cytometry (FCM), real time cellular analysis (RTCA) and nude mice xenografts were used to evaluate the effects of curcumin micelles on gastric cancer cell growth in vitro and in vivo. Western blotting was performed to analyze the protein levels of the indicated molecules. A Seahorse bioenergetics analyzer was used to investigate alterations in oxygen consumption and the aerobic glycolysis rate. Curcumin micelles significantly inhibited proliferation and colony formation and induced apoptosis in gastric tumor cells compared to the control groups. We further investigated the mechanism of curcumin micelles on gastric tumor cells and demonstrated that curcumin micelles acted on mitochondrial proteins, causing changes in mitochondrial function and affecting mitochondrial bioenergetics. Furthermore, curcumin micelles decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) generation and disrupted redox equilibrium. The nude mouse model verified that curcumin micelle treatment significantly attenuated tumor growth in vivo. Curcumin micelles suppress gastric tumor cell growth in vitro and in vivo. The mechanism may be related to increasing ROS generation, disrupting redox equilibrium and affecting mitochondrial bioenergetics.

The curcumin analogue WZ35 affects glycolysis inhibition of gastric cancer cells through ROS-YAP-JNK pathway.

To investigate the anti-tumor activities of WZ35 and its possible molecular mechanism, bioinformatics analysis and the hematoxylin-eosin (HE) staining were applied to evaluate the Yes-associated-protein (YAP) level in gastric cancer. Cell counting kit-8 (CCK-8) was used to examine cell viability. Apoptosis was determined by flow cytometry analysis. Seahorse bioenergetics analyzer was used to investigate the alteration of oxygen consumption and aerobic glycolysis rate. SiRNA transfection was applied to silence endogenous YAP. Western blot was performed to detect indicated proteins. We found that treatment of gastric cancer cells with WZ35 exerted stronger anti-tumor activities than curcumin. Mechanistically, our research showed that WZ35 inhibited glycolysis, and induced reactive oxygen species (ROS) generation, resulting in Jun N-terminal Kinase (JNK) activation through downregulation of YAP in gastric cancer cells. ROS mediated YAP downregulation and JNK activation was regulated by glycolysis. Abrogation of ROS production markedly attenuated WZ35 induced anti-tumor activities as well as YAP downregulation and JNK activation. Similarly, the JNK inhibitor significantly reversed WZ35 induced anti-tumor activities in gastric cancer cells. Our study reveals a novel anti-gastric cancer mechanism of WZ35 by inhibiting glycolysis through the ROS-YAP-JNK pathway. WZ35 might be a potential therapeutics for the treatment of gastric cancer.

Curcumin derivative WZ35 inhibits tumor cell growth via ROS-YAP-JNK signaling pathway in breast cancer.

BACKGROUND: Breast cancer is the most prevalent cancer among women worldwide. WZ35, an analog of curcumin, has been demonstrated to remarkably improve the pharmacokinetic profiles in vivo compared with curcumin. WZ35 exhibits promising antitumor activity in gastric cancer, HCC, colon cancer. However, antitumor effects of WZ35 in breast cancer and its underlying molecular mechanisms remain unclear. METHODS: CCK8, Flow cytometry and transwell assays were used to measure cell proliferation, cell cycle arrest, apoptosis, cell migration and invasion. We constructed xenograft mouse model and lung metastasis model to assess the antitumor activities of WZ35 in vivo. To explore the underlying molecular mechanisms of WZ35, we performed a series of overexpression and knockdown experiments. The cellular oxygen consumption rates (OCRs) was measured to assess mitochondrial dysfunction. RESULTS: We found that treatment of breast cancer cells with WZ35 exerts stronger anti-tumor activities than curcumin both in vitro and in vivo. Mechanistically, our research showed that WZ35 induced reactive oxygen species (ROS) generation and subsequent YAP mediated JNK activation in breast cancer cells. Abrogation of ROS production markedly attenuated WZ35 induced anti-tumor activities as well as YAP and JNK activation. In addition, ROS mediated YAP and JNK activation induced mitochondrial dysfunction in breast cancer cells. CONCLUSION: Our study showed that novel anti-cancer mechanisms of WZ35 in breast cancer cells and ROS-YAP-JNK pathway might be a potential therapeutic target for the treatment of breast cancer patients.