P38 kinase in gastrointestinal cancers.

Gastrointestinal cancers are a leading cause of cancer morbidity and mortality worldwide with 4.2 million new cases and 3.2 million deaths estimated in 2020. Despite the advances in primary and adjuvant therapies, patients still develop distant metastases and require novel therapies. Mitogen­activated protein kinase (MAPK) cascades are crucial signaling pathways that regulate many cellular processes, including proliferation, differentiation, apoptosis, stress responses and cancer development. p38 Mitogen Activated Protein Kinases (p38 MAPKs) includes four isoforms: p38α (MAPK14), p38ß (MAPK11), p38γ (MAPK12), and p38δ (MAPK13). p38 MAPK was first identified as a stress response protein kinase that phosphorylates different transcriptional factors. Dysregulation of p38 pathways, in particular p38γ, are associated with cancer development, metastasis, autophagy and tumor microenvironment. In this article, we provide an overview of p38 and p38γ with respect to gastrointestinal cancers. Furthermore, targeting p38γ is also discussed as a potential therapy for gastrointestinal cancers.

Epigenetic Control of Translation Checkpoint and Tumor Progression via RUVBL1-EEF1A1 Axis.

Epigenetic dysregulation is reported in multiple cancers including Ewing sarcoma (EwS). However, the epigenetic networks underlying the maintenance of oncogenic signaling and therapeutic response remain unclear. Using a series of epigenetics- and complex-focused CRISPR screens, RUVBL1, the ATPase component of NuA4 histone acetyltransferase complex, is identified to be essential for EwS tumor progression. Suppression of RUVBL1 leads to attenuated tumor growth, loss of histone H4 acetylation, and ablated MYC signaling. Mechanistically, RUVBL1 controls MYC chromatin binding and modulates the MYC-driven EEF1A1 expression and thus protein synthesis. High-density CRISPR gene body scan pinpoints the critical MYC interacting residue in RUVBL1. Finally, this study reveals the synergism between RUVBL1 suppression and pharmacological inhibition of MYC in EwS xenografts and patient-derived samples. These results indicate that the dynamic interplay between chromatin remodelers, oncogenic transcription factors, and protein translation machinery can provide novel opportunities for combination cancer therapy.

Flavopiridol (Alvocidib), a Cyclin-dependent Kinases (CDKs) Inhibitor, Found Synergy Effects with Niclosamide in Cutaneous T-cell Lymphoma.

Flavopiridol (FVP; Alvocidib), a CDKs inhibitor, is currently undergoing clinical trials for treatment of leukemia and other blood cancers. Our studies demonstrated that FVP also inhibited p38 kinases activities with IC50 (µM) for p38α: 1.34; p38 ß: 1.82; p38γ: 0.65, and p38δ: 0.45. FVP showed potent cytotoxicity in cutaneous T-cell lymphoma (CTCL) Hut78 cells, with IC50 <100 nM. NMR analysis revealed that FVP bound to p38γ in the ATP binding pocket, causing allosteric perturbation from sites surrounding the ATP binding pocket. Kinomic profiling with the PamGene platform in both cell-based and cell-free analysis further revealed dosage of FVP significantly affects downstream pathways in treated CTCL cells, which suggested a need for development of synergistic drugs with FVP to prevent its clinically adverse effects. It led us discover niclosamide as a synergistic drug of FVP for our future in vivo study.

Targeting the non-ATP-binding pocket of the MAP kinase p38γ mediates a novel mechanism of cytotoxicity in cutaneous T-cell lymphoma (CTCL).

We describe here for the first time a lipid-binding-domain (LBD) in p38γ mitogen-activated protein kinase (MAPK) involved in the response of T cells to a newly identified inhibitor, CSH71. We describe how CSH71, which binds to both the LBD and the ATP-binding pocket of p38γ, is selectively cytotoxic to CTCL Hut78 cells but spares normal healthy peripheral blood mononuclear (PBMC) cells, and propose possible molecular mechanisms for its action. p38γ is a key player in CTCL development, and we expect that the ability to regulate its expression by specifically targeting the lipid-binding domain will have important clinical relevance. Our findings characterize novel mechanisms of gene regulation in T lymphoma cells and validate the use of computational screening techniques to identify inhibitors for therapeutic development.

DNMT1 and p38γ are inversely expressed in reactive non-metastatic lymph nodes burdened with colorectal adenocarcinoma.

Lymph nodes are important front-line defense immune tissues, which also act against inflammatory diseases and cancer. Lymph nodes undergo extensive upheavals within newly formed germinal centers (GCs) when exposed to antigens, the molecular mechanisms of which remain elusive. Recently, p38γ was identified as an important target for multiple cancers, including cutaneous T-cell lymphoma (CTCL). We previously observed that p38γ is overexpressed in CTCL versus normal cells, but it is not clear if p38γ is expressed in B or T lymphocytes of GCs of patients in response to a stress such as cancer. Therefore, in this study, we obtained non-metastatic reactive lymph nodes adjacent to cancer lesions (colorectal adenocarcinoma), then performed multicolor immunohistochemical staining for p38γ and other relevant markers. We observed for the first time that p38γ was expressed in the light zone of activated B cells and T helper cells in GCs, whereas DNA-methyltransferase 1 (DNMT1), a marker for GC B cells, was highly expressed in centrocytes and in the dark zone of GCs. This inverse relationship suggests a novel function for p38γ in T cells that cross-talk to B cells in response to stress.

Multi-Kinase Inhibitor with Anti-p38γ Activity in Cutaneous T-Cell Lymphoma.

Current cutaneous T-cell lymphoma (CTCL) therapies are marked by an abbreviated response, subsequent drug resistance, and poor prognosis for patients with advanced disease. An understanding of molecular regulators involved in CTCL is needed to develop effective targeted therapies. One candidate regulator is p38γ, a mitogen-activated protein kinase crucial for malignant T-cell activity and growth. p38γ gene expression is selectively increased in CTCL patient samples and cell lines but not in healthy T cells. In addition, gene silencing of p38γ reduced CTCL cell viability, showing a key role in CTCL pathogenesis. Screening p38γ inhibitors is critical for understanding the mechanism of CTCL tumorigenesis and developing therapeutic applications. We prioritized a potent p38γ inhibitor (F7, also known as PIK75) through a high-throughput kinase inhibitor screen. At nanomolar concentrations, PIK75, a multiple kinase inhibitor, selectively killed CD4+ malignant CTCL cells but spared healthy CD4+ cells; induced significant reduction of tumor size in mouse xenografts; and effectively inhibited p38γ enzymatic activity and phosphorylation of its substrate, DLGH1, in CTCL cells and mouse xenografts. Here, we report that PIK75 has a potential clinical application to serve as a scaffold molecule for the development of a more selective p38γ inhibitor.

Ethanol induction of CYP2A5: role of CYP2E1-ROS-Nrf2 pathway.

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 (-/-)) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 (-/-) mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 (-/-) KI) mice but not in CYP2E1 knockout (Cyp2e1 (-/-)) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 (-/-) KI mice but not in Cyp2e1 (-/-) mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 (-/-)) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 (-/-) mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 (-/-) KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis.

Advanced glycation end products inhibit glucose-stimulated insulin secretion through nitric oxide-dependent inhibition of cytochrome c oxidase and adenosine triphosphate synthesis.

Advanced glycation end products (AGEs) are implicated in diabetic complications. However, their role in beta-cell dysfunction is less clear. In this study we examined the effects of AGEs on islet function in mice and in isolated islets. AGE-BSA or BSA was administered ip to normal mice twice a day for 2 wk. We showed that AGE-BSA-treated mice exhibited significantly higher glucose levels and lower insulin levels in response to glucose challenge than did BSA-treated mice, although there were no significant differences in insulin sensitivity and islet morphology between two groups. Glucose-stimulated insulin secretion by islets of the AGE-BSA-treated mice or AGE-BSA-treated normal islets was significantly lower than that by islets isolated from the BSA-treated mice or BSA-treated normal islets. Furthermore, AGE treatment of islet beta-cells inhibited ATP production, and glimepiride, a sulfonylurea derivative, restored glucose-stimulated insulin secretion. Further investigation indicated that AGEs inhibited cytochrome c oxidase activity by inducing the expression of inducible nitric oxide synthase (iNOS). Blocking the formation of nitric oxide with an iNOS selective inhibitor aminoguanidine reversed the inhibitory effects of AGEs on ATP production and insulin secretion. We conclude that AGEs inhibit cytochrome c oxidase and ATP production, leading to the impairment of glucose-stimulated insulin secretion through iNOS-dependent nitric oxide production.

The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR.

The G1 phase of the cell cycle is marked by the rapid turnover of phospholipids. This turnover is regulated by CTP:phosphocholine-cytidylyltransferase (CCT) and group VIA Ca(2+)-independent-phospholipase A(2) (iPLA(2)). We previously reported that inhibition of iPLA(2) arrests cells in G1 phase of the cell cycle by activating the p53-p21 checkpoint. Here we further characterize the mechanism of p53 activation. We show that specific inhibition of iPLA(2) induces a time dependent phosphorylation of Ser15 in p53 in the absence of DNA damage. This phosphorylation requires the kinase ataxia-telangiectasia and Rad-3-related (ATR) but not the ataxia-telangiectasia-mutated (ATM) kinase. Moreover, we identify in cell membranes a significant increase of phosphatidylcholines (PCs) containing chains of polyunsaturated fatty acids and a decrease of PCs containing saturated fatty acids in response to inhibition of iPLA(2). The time course of phosphorylation of Ser15 in p53 correlates with increasing levels of PCs containing polyunsaturated fatty acids. We further demonstrate that the PCs with linoleic acid in their sn-2 position (18:2n6) induce phosphorylation of Ser15 in p53 in an ATR-dependent manner. Our findings establish that cells can regulate the levels of polyunsaturated fatty acids in phospholipids through iPLA(2)-mediated deacylation of PCs. Disruption of this regulation increases the proportions of PCs containing polyunsaturated fatty acids and activates the ATR-p53 signalling pathway.

Disruption of G1-phase phospholipid turnover by inhibition of Ca2+-independent phospholipase A2 induces a p53-dependent cell-cycle arrest in G1 phase.

The G1 phase of the cell cycle is characterized by a high rate of membrane phospholipid turnover. Cells regulate this turnover by coordinating the opposing actions of CTP:phosphocholine cytidylyltransferase and the group VI Ca2+-independent phospholipase A2 (iPLA2). However, little is known about how such turnover affects cell-cycle progression. Here, we show that G1-phase phospholipid turnover is essential for cell proliferation. Specific inhibition of iPLA2 arrested cells in the G1 phase of the cell cycle. This G1-phase arrest was associated with marked upregulation of the tumour suppressor p53 and the expression of cyclin-dependent kinase inhibitor p21cip1. Inactivation of iPLA2 failed to arrest p53-deficient HCT cells in the G1 phase and caused massive apoptosis of p21-deficient HCT cells, suggesting that this G1-phase arrest requires activation of p53 and expression of p21cip1. Furthermore, downregulation of p53 by siRNA in p21-deficient HCT cells reduced the cell death, indicating that inhibition of iPLA2 induced p53-dependent apoptosis in the absence of p21cip1. Thus, our study reveals hitherto unrecognized cooperation between p53 and iPLA2 to monitor membrane-phospholipid turnover in G1 phase. Disrupting the G1-phase phospholipid turnover by inhibition of iPLA2 activates the p53-p21cip1 checkpoint mechanism, thereby blocking the entry of G1-phase cells into S phase.

Calcium-independent phospholipase A2 localizes in and protects mitochondria during apoptotic induction by staurosporine.

Mitochondria-mediated production of reactive oxygen species (ROS) plays a key role in apoptosis. Mitochondrial phospholipid cardiolipin molecules are likely the main target of ROS because they are particularly rich in polyunsaturated fatty acids. They are also located in the inner mitochondrial membrane near the ROS-producing sites. Under physiological conditions mitochondria can repair peroxidative damage in part through a remodeling mechanism via the deacylation-reacylation cycle mediated by phospholipase A2 (PLA2) and acyl-coenzyme A-dependent monolysocardiolipin acyltransferase. Here we investigate whether group VIA Ca2+-independent PLA2 (iPLA2) plays a role in the protection of mitochondrial function from damage caused by mitochondrially generated ROS during apoptotic induction by staurosporine (STS). We show that iPLA2-expressing cells were relatively resistant to STS-induced apoptosis. iPLA2 localized to mitochondria even before apoptotic induction, and most iPLA2-associated mitochondria were intact in apoptotic resistant cells. Expression of iPLA2 in INS-1 cells prevented the loss of mitochondrial membrane potential, attenuated the release of cytochrome c, Smac/DIABLO, and apoptosis inducing factor from mitochondria, and reduced mitochondrial reactive oxygen species production. Inhibition of caspase 8 has little effect on STS-induced apoptosis in INS-1 cells. Finally, we found that STS down-regulated endogenous iPLA2 transcription in both INS-1 and iPLA2-expressing INS-1 cells without affecting the expression of group IV Ca2+-dependent PLA2. Together, our data indicate that iPLA2 is important for the protection of mitochondrial function from oxidative damage during apoptotic induction. Down-regulation of endogenous iPLA2 by STS may result in the loss of mitochondrial membrane repair functions and lead to mitochondrial failure and apoptosis.