MPC1 and MPC2 are two well-known components of the mitochondrial pyruvate carrier (MPC) complex maintaining MPC activity to transport pyruvate into mitochondria for tricarboxylic acid (TCA) cycle entry in mammalian cells. It is currently unknown whether there is an additional MPC component crucially maintaining MPC complex activity for pyruvate mitochondrial import. Here we show that ALDH4A1, a proline-metabolizing enzyme localized in mitochondria, serves as a previously unrecognized MPC component maintaining pyruvate mitochondrial import and the TCA cycle independently of its enzymatic activity. Loss of ALDH4A1 in mammalian cells impairs pyruvate entry to mitochondria, resulting in defective TCA cycle entry. ALDH4A1 forms an active trimeric complex with MPC1-MPC2 to maintain the integrity and oligomerization of MPC1-MPC2 and facilitates pyruvate transport in an in vitro system. ALDH4A1 displays tumour suppression by maintaining MPC complex activity. Our study identifies ALDH4A1 as an essential component of MPC for pyruvate mitochondrial import, TCA cycle entry and tumour suppression.

Passive diffusion does not explain why many drugs are too large and/or too polar for rule-breaking membrane penetration, such as proteolysis-targeting chimeras (PROTACs, generally of a molecular weight > 800 Da). Here, using biotinylated chemical-probe-based target fishing and genetic knockdown/knockin approaches, we discovered that the membrane cluster of differentiation 36 (CD36) binds to and facilitates the uptake and efficacy of diverse PROTACs (e.g., SIM1-Me, MZ1, and clinical ARV-110) and large and/or polar small-molecule drugs (e.g., rapalink-1, rapamycin, navitoclax, birinapant, tubacin, and doxorubicin) via the CD36-mediated early endosome antigen 1 (EEA1)/Ras-related protein 5A (Rab5) endosomal cascade in vitro and/or in vivo. We then devised a novel chemical endocytic medicinal chemistry strategy to improve binding of PROTACs to CD36 using structural modifications via the prodrug approach, markedly enhancing PROTAC anti-tumor efficacy through spontaneously augmenting permeability and solubility.

AMP-activated protein kinase (AMPK) is a protein kinase that plays versatile roles in response to a variety of physiological stresses, including glucose deprivation, hypoxia, and ischemia. As a kinase with pleiotropic functions, it plays a complex role in tumor progression, exhibiting both tumor-promoting and tumor-suppressing activities. On one hand, AMPK enhances cancer cell proliferation and survival, promotes cancer metastasis, and impairs anti-tumor immunity. On the other hand, AMPK inhibits cancer cell growth and survival and stimulates immune responses in a context-dependent manner. Apart from these functions, AMPK plays a key role in orchestrating aging and aging-related disorders, including cardiovascular diseases (CVD), Osteoarthritis (OA), and Diabetes. In this review article, we summarized the functions of AMPK pathway based on its oncogenic and tumor-suppressive roles and highlighted the importance of AMPK pathway in regulating cellular aging. We also spotlighted the significant role of various signaling pathways, activators, and inhibitors of AMPK in serving as therapeutic strategies for anti-cancer and anti-aging therapy.

Acquisition of prostate cancer stem cells (PCSCs) manifested during androgen ablation therapy (ABT) contributes to castration-resistant prostate cancer (CRPC). However, little is known about the specific metabolites critically orchestrating this process. Here, we show that IMPA1-derived inositol enriched in PCSCs is a key metabolite crucially maintaining PCSCs for CRPC progression and ABT resistance. Notably, conditional Impa1 knockout in the prostate abrogates the pool and properties of PCSCs to orchestrate CRPC progression and prolong the survival of TRAMP mice. IMPA1-derived inositol serves as a cofactor that directly binds to and activates IMPDH2, which synthesizes guanylate nucleotides for maintaining PCSCs with ARlow/- features leading to CRPC progression and ABT resistance. IMPA1/inositol/IMPDH2 axis is upregulated in human prostate cancer, and its overexpression predicts poor survival outcomes. Genetically and pharmacologically targeting the IMPA1/inositol/IMPDH2 axis abrogates CRPC and overcomes ABT resistance in various CRPC xenografts, patient-derived xenograft (PDX) tumor models, and TRAMP mouse models. Our study identifies IMPDH2 as an inositol sensor whose activation by inositol represents a key mechanism for maintaining PCSCs for CRPC and ABT resistance.

The PI3K/Akt pathway,one of the most frequently dysregu-lated signaling pathways in various cancers,plays a key role in tumorigenesis and resistance to chemotherapy.Recent discovery highlights an important regulatory step where Smurf1-mediated PDK1 neddylation through poly-Nedd8 chains is an early event for oncogenic Akt activation in KRAS-mutant colorectal cancer.

Oncogene Moesin plays critical role in initiation, progression, and metastasis of multiple cancers. It exerts oncogenic activity due to its high-level expression as well as posttranslational modification in cancer. However, factors responsible for its high-level expression remain elusive. In this study, we identified positive as well as negative regulators of Moesin. Our study reveals that Moesin is a cellular target of F-box protein FBXW2. We showed that FBXW2 suppresses breast cancer progression through directing proteasomal degradation of Moesin. In contrast, AKT kinase plays an important role in oncogenic function of Moesin by protecting it from FBXW2-mediated proteasomal degradation. Mechanistically, AKT phosphorylates Moesin at Thr-558 and thereby prevents its degradation by FBXW2 via weakening the association between FBXW2 and Moesin. Further, accumulated Moesin prevents FBXW2-mediated degradation of oncogene SKP2, showing that Moesin functions as an upstream regulator of oncogene SKP2. In turn, SKP2 stabilizes Moesin by directing its non-degradable form of polyubiquitination and therefore AKT-Moesin-SKP2 oncogenic axis plays crucial role in breast cancer progression. Collectively, our study reveals that FBXW2 functions as a tumor suppressor in breast cancer by restricting AKT-Moesin-SKP2 axis. Thus, AKT-Moesin-SKP2 axis may be explored for the development of therapeutics for cancer treatment.

Glucose metabolism is known to orchestrate oncogenesis. Whether glucose serves as a signaling molecule directly regulating oncoprotein activity for tumorigenesis remains elusive. Here, we report that glucose is a cofactor binding to methyltransferase NSUN2 at amino acid 1-28 to promote NSUN2 oligomerization and activation. NSUN2 activation maintains global m5C RNA methylation, including TREX2, and stabilizes TREX2 to restrict cytosolic dsDNA accumulation and cGAS/STING activation for promoting tumorigenesis and anti-PD-L1 immunotherapy resistance. An NSUN2 mutant defective in glucose binding or disrupting glucose/NSUN2 interaction abolishes NSUN2 activity and TREX2 induction leading to cGAS/STING activation for oncogenic suppression. Strikingly, genetic deletion of the glucose/NSUN2/TREX2 axis suppresses tumorigenesis and overcomes anti-PD-L1 immunotherapy resistance in those cold tumors through cGAS/STING activation to facilitate apoptosis and CD8+ T cell infiltration. Our study identifies NSUN2 as a direct glucose sensor whose activation by glucose drives tumorigenesis and immunotherapy resistance by maintaining TREX2 expression for cGAS/STING inactivation.

Here, we present optimized approaches to identify the efficiency of cancer cell phagocytosis by macrophages in vitro and in vivo. We describe the preparation and co-culture of macrophages and cancer cells, followed by in vitro phagocytosis assay using flow cytometry and confocal microscopy, respectively. We then detail the establishment of xenograft tumor mouse model and the in vivo detecting of phagocytosis efficiency by flow cytometry and qRT-PCR. This protocol provides a convenient way to assess macrophage-mediated phagocytosis of cancer cells. For complete details on the use and execution of this protocol, please refer to Xu et al.1.

Cancer stem-like cells (CSLCs) acquire enhanced immune checkpoint responses to evade immune cell killing and promote tumor progression. Here we showed that signal regulatory protein γ (SIRPγ) determined CSLC properties and immune evasiveness in a small population of lung adenocarcinoma (LUAD) cancer cells. A SIRPγhi population displayed CSLC properties and transmitted the immune escape signal through sustaining CD47 expression in both SIRPγhi and SIRPγlo/– tumor cells. SIRPγ bridged MST1 and PP2A to facilitate MST1 dephosphorylation, resulting in Hippo/YAP activation and leading to cytokine release by CSLCs, which stimulated CD47 expression in LUAD cells and consequently inhibited tumor cell phagocytosis. SIRPγ promoted tumor growth and metastasis in vivo through YAP signaling. Notably, SIRPγ targeting with genetic SIRPγ knockdown or a SIRPγ-neutralizing antibody inhibited CSLC phenotypes and elicited phagocytosis that suppressed tumor growth in vivo. SIRPG was upregulated in human LUAD and its overexpression predicted poor survival outcome. Thus, SIRPγhi cells serve as CSLCs and tumor immune checkpoint–initiating cells, propagating the immune escape signal to the entire cancer cell population. Our study identifies Hippo/YAP signaling as the first mechanism by which SIRPγ is engaged and reveals that targeting SIRPγ represents an immune- and CSLC-targeting strategy for lung cancer therapy.

Metabolic reprogramming is an important cancer hallmark that plays a key role in cancer malignancies and therapy resistance. Cancer cells reprogram the metabolic pathways to generate not only energy and building blocks but also produce numerous key signaling metabolites to impact signaling and epigenetic/transcriptional regulation for cancer cell proliferation and survival. A deeper understanding of the mechanisms by which metabolic reprogramming is regulated in cancer may provide potential new strategies for cancer targeting. Recent studies suggest that deregulated transcription factors have been observed in various human cancers and significantly impact metabolism and signaling in cancer. In this review, we highlight the key transcription factors that are involved in metabolic control, dissect the crosstalk between signaling and transcription factors in metabolic reprogramming, and offer therapeutic strategies targeting deregulated transcription factors for cancer treatment.

Arsenic trioxide (ATO), a potent anti-neoplastic drug, is known to prevent cancer cell growth through induction of autophagic cell death. However, importance of cellular factors in ATO-mediated autophagic cell death is poorly understood. In this study, using biochemical and immunofluorescence techniques, we show that F-box protein FBXO41 plays a critical role in anti-proliferative activity of ATO. Our study reveals the importance of FBXO41 in induction of autophagic death of cancer cells by ATO. Further, we show that the autophagic cell death induced by FBXO41 is distinct and independent of apoptosis and necrosis, showing that FBXO41 may play vital role in inducing autophagic death of apoptosis resistant cancer cells. Overall, our study elucidates the importance of FBXO41 in ATO induced autophagic cell death to prevent cancer progression, which could be explored to develop promising cancer therapeutic strategy.

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3 alpha/beta, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3 alpha/beta modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

Aberrant activation of β‐catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/β‐catenin‐signaling pathways is still poorly understood. In this study, we show that F‐box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1‐Cullin1‐F‐box protein) complex, which targets the nuclear β‐catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C‐terminal domain of β‐catenin and promotes its lysine 48‐linked polyubiquitination. In addition, it inhibits epithelial‐to‐mesenchymal transition (EMT) by attenuating the level of β‐catenin. Therefore, depletion of FBXO16 leads to increased levels of β‐catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and β‐catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCFFBXO16 complex that targets nuclear β‐catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant β‐catenin activation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

BRCA1 is a nuclear phosphoprotein involved in genome integrity by regulating cell cycle checkpoints, DNA repair and apoptosis. BRCA1 down regulation occurs in sporadic breast cancer (BC). Posttranscriptional regulation of gene expression has evolved as a means of fine-tuning of protein levels. There are several posttranscriptional regulatory motifs, including CU-rich, U-rich and AU-rich elements, which are usually located in the 5' and 3' UTR's. Post-transcriptional regulation of BRCA1 is poorly characterized and underappreciated. To elucidate the molecular mechanism of AnxA2 mediated posttranscriptional regulation of BRCA1 mRNA in sporadic breast cancer. In order to investigate BRCA1 mRNA levels in MDAMB-231 & MCF-7 cells, we conducted DRB chase experiment. The BRCA1 mRNA destabilization is significantly increased upon AnxA2 induction in MCF-7 cells. Further we knocked down AnxA2 in MDAMB-231 cells we found, BRCA1 mRNA stabilization which proves AnxA2 destabilizes BRCA1 mRNA. In RIP-CHIP experiment we found BRCA1 immunoprecipitated with AnxA2. This supports our finding that AnxA2 has role in the regulation of BRCA1 at mRNA level. Luciferase reporter assay showed decrease in luciferase activity with BRCA1 3'UTR. On treating with AnxA2 binding oligo from BRCA1 3'UTR showed decrease in cell viability.

Two Schiff bases HL and HL′, having potential tridentate O,N,N′ donor sets, have been used for the synthesis of two copper(II) complexes, namely [Cu(HL)(pdc)]2 (1) and [Cu(L′)2]2 (2′), where HL = 2-([2-(piperazin-yl)ethylimino]methyl)phenol, pdc = py-2,5-dicarboxylate and HL′ = 2-(((2-(di-isopropylamino)ethyl)imino)methyl)phenol. X-ray single crystal analysis of complex 1 shows a centro-symmetric dimer. It crystallizes with a number of lattice water molecules that form a network of H-bonds, also involving the protonated piperazinium fragment, giving rise to a 3D supramolecular architecture. Complex 2′ also crystallizes as dinuclear, formed through mutual bridging phenol oxygen atoms as [Cu(L′)2]2, whilst an ESI mass spectrometry study evidences that in solution the complex exists as mononuclear [Cu(L′)2] (2). The interaction of complexes 1 and 2 with calf thymus DNA (CT-DNA) and with bovine serum albumin (BSA) was investigated using electronic absorption and fluorescence spectroscopic techniques. In both studies the results show a higher binding affinity of complex 1 in comparison to 2. The anticancer activity of the complexes against human breast (MCF7) cancer cell lines reveals that complex 1 has moderate growth suppression activity against these cells with an IC50 value of 24 ± 6.24 μM.

The Schiff base 2-[(3-Methylamino-propylimino)-methyl]-phenol (HL) has been used for the synthesis of three dinuclear Cu(II) complexes, namely [Cu2L2(dca)(2)] (1), [Cu2L2(2,5-pdc)(2)]center dot 6H(2)O (2), [Cu2L2(tp)] (3), where dca = dicyanamide, 2,5-pdc = pyridine-2,5-dicarboxylate and tp = terephthalate. In 2, HL acts as bidentate (O, N) chelating ligand, while tridentate (O, N, N) in 1 and 3. In all the complexes copper(II) is in its five coordination with distorted trigonal bipyramidal geometry in 1, whereas distorted square pyramidal in 2 and 3. Non covalent interactions give rise supramolecular architectures in all complexes. Cytotoxicity of the complexes was examined using breast cell lines MCF7 and MDA-MB-231. Complexes 1 and 2 show dose dependent suppression of MCF7 and MDA-MB-231 cells and the related IC50 values are 26 +/- 2.9 mu M and 51 +/- 4.7 mu M for MCF7 cell, 16.4 +/- 1.6 mu M and 43.2 +/- 3.1 mu M for MDA-MB-231 cell. Interaction of complexes 1-3 with bovine serum albumin (BSA), human serum albumin (HSA) and calf thymus DNA have been studied by electronic absorption and fluorescence spectroscopic technique. Molecular docking study has been used to explain the interaction of complexes with serum albumins.