Lactylated Apolipoprotein C-II Induces Immunotherapy Resistance by Promoting Extracellular Lipolysis.

Mortality rates due to lung cancer are high worldwide. Although PD-1 and PD-L1 immune checkpoint inhibitors boost the survival of patients with non-small-cell lung cancer (NSCLC), resistance often arises. The Warburg Effect, which causes lactate build-up and potential lysine-lactylation (Kla), links immune dysfunction to tumor metabolism. The role of non-histone Kla in tumor immune microenvironment and immunotherapy remains to be clarified. Here, global lactylome profiling and metabolomic analyses of samples from patients with NSCLC is conducted. By combining multi-omics analysis with in vitro and in vivo validation, that intracellular lactate promotes extracellular lipolysis through lactyl-APOC2 is revealed. Mechanistically, lactate enhances APOC2 lactylation at K70, stabilizing it and resulting in FFA release, regulatory T cell accumulation, immunotherapy resistance, and metastasis. Moreover, the anti-APOC2K70-lac antibody that sensitized anti-PD-1 therapy in vivo is developed. This findings highlight the potential of anti lactyl-APOC2-K70 approach as a new combination therapy for sensitizing immunotherapeutic responses.

CPT1A mediates chemoresistance in human hypopharyngeal squamous cell carcinoma via ATG16L1-dependent cellular autophagy.

Hypopharyngeal squamous cell carcinoma (HSCC) is a highly aggressive malignancy that constitutes approximately 95% of all hypopharyngeal carcinomas, and it carries a poor prognosis. The primary factor influencing the efficacy of anti-cancer drugs for this type of carcinoma is chemoresistance. Carnitine palmitoyltransferase 1A (CPT1A) has been associated with tumor progression in various cancers, including breast, gastric, lung, and prostate cancer. The inhibition or depletion of CPT1A can lead to apoptosis, curbing cancer cell proliferation and chemoresistance. However, the role of CPT1A in HSCC is not yet fully understood. In this study, we discovered that CPT1A is highly expressed in HSCC and is associated with an advanced T-stage and a poor 5-year survival rate among patients. Furthermore, the overexpression of CPT1A contributes to HSCC chemoresistance. Mechanistically, CPT1A can interact with the autophagy-related protein ATG16L1 and stimulate the succinylation of ATG16L1, which in turn drives autophagosome formation and autophagy. We also found that treatment with 3-methyladenine (3-MA) can reduce cisplatin resistance in HSCC cells that overexpress CPT1A. Our findings also showed that a CPT1A inhibitor significantly enhances cisplatin sensitivity both in vitro and in vivo. This study is the first to suggest that CPT1A has a regulatory role in autophagy and is linked to poor prognosis in HSCC patients. It presents novel insights into the roles of CPT1A in tumorigenesis and proposes that CPT1A could be a potential therapeutic target for HSCC treatment.

Lactylation of METTL16 promotes cuproptosis via m6A-modification on FDX1 mRNA in gastric cancer.

Cuproptosis, caused by excessively high copper concentrations, is urgently exploited as a potential cancer therapeutic. However, the mechanisms underlying the initiation, propagation, and ultimate execution of cuproptosis in tumors remain unknown. Here, we show that copper content is significantly elevated in gastric cancer (GC), especially in malignant tumors. Screening reveals that METTL16, an atypical methyltransferase, is a critical mediator of cuproptosis through the m6A modification on FDX1 mRNA. Furthermore, copper stress promotes METTL16 lactylation at site K229 followed by cuproptosis. The process of METTL16 lactylation is inhibited by SIRT2. Elevated METTL16 lactylation significantly improves the therapeutic efficacy of the copper ionophore- elesclomol. Combining elesclomol with AGK2, a SIRT2-specific inhibitor, induce cuproptosis in gastric tumors in vitro and in vivo. These results reveal the significance of non-histone protein METTL16 lactylation on cuproptosis in tumors. Given the high copper and lactate concentrations in GC, cuproptosis induction becomes a promising therapeutic strategy for GC.

Upregulation of KLF14 expression attenuates kidney fibrosis by inducing PPARα-mediated fatty acid oxidation.

Tubulointerstitial fibrosis (TIF) is essential during the development of end-stage kidney disease (ESKD) and is associated with the impairment of fatty acid oxidation (FAO). Kruppel-like factor 14 (KLF14) is an important gene in lipid metabolism, but its role in TIF remains unknown. TGF-ß-stimulated HK-2 cells and mouse unilateral ureteral obstruction (UUO) were used as renal fibrosis models. The role of KLF14 in the process of renal fibrosis was verified by gene knockout mice, genetic or pharmacological interference in animal model and cell model respectively. In the current study, we found that KLF14 expression increased after activation of the TGF-ß signaling pathway during TIF. In KLF14-/- mice, more severe fibrosis was observed after unilateral ureteral obstruction (UUO) was induced. In human HK2 cells, knockdown of KLF14 led to more severe fibrosis induced by TGF-ß1, while overexpression of KLF14 partially attenuated this process. Specifically, KLF14 deficiency decreased mitochondrial FAO activity, resulting in lipid accumulation. Thus, the energy supply to the cells was insufficient, finally resulting in TIF. We further proved that KLF14 could target peroxisome proliferator activated receptor alpha (PPARα) as a transcriptional activator. This study identified the upregulation of KLF14 expression in response to kidney stress during the process of fibrosis. Upon TIF, the activated TGF-ß signaling pathway can enhance KLF14 expression, while the upregulation of KLF14 expression can decrease the degree of TIF by improving FAO activity in tubular epithelial cells and recovering the energy supply mediated by PPARα.

The ATM and ATR kinases regulate centrosome clustering and tumor recurrence by targeting KIFC1 phosphorylation.

Drug resistance and tumor recurrence are major challenges in cancer treatment. Cancer cells often display centrosome amplification. To maintain survival, cancer cells achieve bipolar division by clustering supernumerary centrosomes. Targeting centrosome clustering is therefore considered a promising therapeutic strategy. However, the regulatory mechanisms of centrosome clustering remain unclear. Here we report that KIFC1, a centrosome clustering regulator, is positively associated with tumor recurrence. Under DNA damaging treatments, the ATM and ATR kinases phosphorylate KIFC1 at Ser26 to selectively maintain the survival of cancer cells with amplified centrosomes via centrosome clustering, leading to drug resistance and tumor recurrence. Inhibition of KIFC1 phosphorylation represses centrosome clustering and tumor recurrence. This study identified KIFC1 as a prognostic tumor recurrence marker, and revealed that tumors can acquire therapeutic resistance and recurrence via triggering centrosome clustering under DNA damage stresses, suggesting that blocking KIFC1 phosphorylation may open a new vista for cancer therapy.

The Caspase-3/PKCδ/Akt/VEGF-A Signaling Pathway Mediates Tumor Repopulation during Radiotherapy.

PURPOSE: Tumor repopulation is known as a major cause of treatment failure and/or tumor recurrence after radiotherapy. The underlying mechanism remains unclear. Our previous study demonstrated that irradiated apoptotic cells mediated tumor repopulation, in which caspase-3 played an important role. Herein, we investigated downstream effectors of caspase-3 involved in this process. EXPERIMENTAL DESIGN: A dominant-negative protein kinase Cδ (DN_PKCδ) mutant that could not be cleaved by caspase-3 and therefore could not be activated by irradiation-induced apoptosis was constructed. DN_PKCδ stably transduced tumor cells were compared with wild-type tumor cells for their growth stimulation effects in in vitro and in vivo tumor repopulation models. Downstream effectors of caspase-3 and PKCδ were investigated. The role of PKCδ was further verified in human colorectal tumor specimens. RESULTS: Inactivation of caspase-3 or caspase-7 attenuated tumor repopulation and weakened PKCδ cleavage. Both DN_PKCδ and PKCδ inhibitors restrained tumor repopulation both in vitro and in vivo. Phosphorylated Akt was attenuated in caspase-3-, caspase-7-, or PKCδ-inactivated tumor cells. Furthermore, expression of vascular endothelial growth factor (VEGF)-A but not hypoxia-inducible factor 1α (HIF1α) was decreased in PKCδ- or Akt-inactivated tumor cells. In addition, inhibition of p-Akt, HIF1α, VEGF-A, or VEGF-A receptor reduced tumor repopulation significantly. Finally, increased nuclear translocation of PKCδ in colorectal tumor specimens was associated with worse patient prognosis. CONCLUSIONS: The caspase-3/PKCδ/Akt/VEGF-A axis is involved in tumor repopulation and could be exploited as a potential target to enhance the efficacy of radiotherapy.

HMGB1 released by irradiated tumor cells promotes living tumor cell proliferation via paracrine effect.

Tumor repopulation during therapy is an important cause of treatment failure. Strategies to overcome repopulation are arising in parallel with advances in the comprehension of underlying biological mechanisms. Here, we reveal a new mechanism by which high mobility group box 1 (HMGB1) released by dying cells during radiotherapy or chemotherapy could stimulate living tumor cell proliferationInhibition or genetic ablation of HMGB1 suppressed tumor cell proliferation. This effect was due to binding of HMGB1with the member receptor for advanced glycation end-products (RAGE), which activated downstream ERK and p38 signaling pathway and promoted cell proliferation. Furthermore, higher HMGB1 expression in tumor tissue correlated with poor overall survival and higher HMGB1 concentration was detected in serum of patients who accepted radiotherapy. Collectively, the results from this study suggested that interaction between dead cells and surviving cells might influence the fate of tumor. HMGB1 could be a novel tumor promoter with therapeutic and prognostic relevance in cancers.

SIRT1 Functions as a Negative Regulator of Eukaryotic Poly(A)RNA Transport.

Most eukaryotic mRNAs are polyadenylated in the nucleus, and the poly(A)-tail is required for efficient mRNA export and translation. However, mechanisms governing mRNA transport remain unclear. Here, we report that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 acts as an energy sensor and negatively regulates poly(A)RNA transport via deacetylating a poly(A)-binding protein, PABP1. Upon energy starvation, SIRT1 interacts with and deacetylates PABP1 and deactivates its poly(A)RNA binding, leading to nuclear accumulation of PABP1 and poly(A)RNA and thus facilitating eukaryotic cells to attenuate protein synthesis and energy consumption to adapt to energy stress. Moreover, AMPK-directed SIRT1 phosphorylation is required for energy starvation-induced PABP1-SIRT1 association, PABP1 deacetylation, and poly(A)RNA nuclear retention. In addition, the SIRT1-PABP1 association is not specific to energy starvation but represents a common stress response. These observations provide insights into dynamic modulation of eukaryotic mRNA transport and translation, suggesting that the poly(A)-tail also provides a basis for eukaryotes to effectively shut down mature mRNA transport and thereby tailor protein synthesis to maintain energy homeostasis under stress conditions.

Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome.

Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits.

Loss of KLF14 triggers centrosome amplification and tumorigenesis.

Centrosome amplification is frequent in cancer, but the underlying mechanisms remain unclear. Here we report that disruption of the Kruppel-like factor 14 (KLF14) gene in mice causes centrosome amplification, aneuploidy and spontaneous tumorigenesis. Molecularly, KLF14 functions as a transcriptional repressor of Plk4, a polo-like kinase whose overexpression induces centrosome overduplication. Transient knockdown of KLF14 is sufficient to induce Plk4-directed centrosome amplification. Clinically, KLF14 transcription is significantly downregulated, whereas Plk4 transcription is upregulated in multiple types of cancers, and there exists an inverse correlation between KLF14 and Plk4 protein expression in human breast and colon cancers. Moreover, KLF14 depletion promotes AOM/DSS-induced colon tumorigenesis. Our findings reveal that KLF14 reduction serves as a mechanism leading to centrosome amplification and tumorigenesis. On the other hand, forced expression of KLF14 leads to mitotic catastrophe. Collectively, our findings identify KLF14 as a tumour suppressor and highlight its potential as biomarker and therapeutic target for cancer.

The REGγ proteasome regulates hepatic lipid metabolism through inhibition of autophagy.

The ubiquitin-proteasome and autophagy-lysosome systems are major proteolytic pathways, whereas function of the Ub-independent proteasome pathway is yet to be clarified. Here, we investigated roles of the Ub-independent REGγ-proteasome proteolytic system in regulating metabolism. We demonstrate that mice deficient for the proteasome activator REGγ exhibit dramatic autophagy induction and are protected against high-fat diet (HFD)-induced liver steatosis through autophagy. Molecularly, prevention of steatosis in the absence of REGγ entails elevated SirT1, a deacetylase regulating autophagy and metabolism. REGγ physically binds to SirT1, promotes its Ub-independent degradation, and inhibits its activity to deacetylate autophagy-related proteins, thereby inhibiting autophagy under normal conditions. Moreover, REGγ and SirT1 dissociate from each other through a phosphorylation-dependent mechanism under energy-deprived conditions, unleashing SirT1 to stimulate autophagy. These observations provide a function of the REGγ proteasome in autophagy and hepatosteatosis, underscoring mechanistically a crosstalk between the proteasome and autophagy degradation system in the regulation of lipid homeostasis.

Triterpenoids and steroids from the fruits of Melia toosendan and their cytotoxic effects on two human cancer cell lines.

Ten new triterpenoids, named meliasenins I-R (1-10), one new steroid (11), and 11 related known compounds (12-22) were isolated from fruits of Melia toosendan. The structures of the new compounds were established on the basis of spectroscopic methods, including 2D NMR techniques and mass spectrometry. The relative configuration of 1, (20R*,23E)-25-hydroperoxyeupha-7,23-diene-3ß,16ß-diol (meliasenin I), was confirmed by single-crystal X-ray diffraction analysis. All isolated triterpenoids (1-10, 12-15) and two steroids (11, 20) were tested for their cytotoxicity against U20S human osteosarcoma and MCF-7 human breast cancer cells using the MTT assay, and some of them were significantly cytotoxic (IC(50) <10 µg/mL). The insecticidal properties of compounds 1-15 and 20 were also briefly evaluated.

[Construction and analysis of subtractive cDNA library of Phellodendron amurense under drought stress].

With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300-800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.