RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

mTOR inhibition acts as an unexpected checkpoint in p53-mediated tumor suppression.

Here, we showed that the acetylation-defective p53-4KR mice, lacking the ability of cell cycle arrest, senescence, apoptosis, and ferroptosis, were tumor prone but failed to develop early-onset tumors. By identifying a novel p53 acetylation site at lysine K136, we found that simultaneous mutations at all five acetylation sites (p53-5KR) diminished its remaining tumor suppression function. Moreover, the embryonic lethality caused by the deficiency of mdm2 was fully rescued in the background of p535KR/5KR , but not p534KR/4KR background. p53-4KR retained the ability to suppress mTOR function but this activity was abolished in p53-5KR cells. Notably, the early-onset tumor formation observed in p535KR/5KR and p53-null mice was suppressed upon the treatment of the mTOR inhibitor. These results suggest that p53-mediated mTOR regulation plays an important role in both embryonic development and tumor suppression, independent of cell cycle arrest, senescence, apoptosis, and ferroptosis.

REDD1-dependent GSK3ß dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice.

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3ß and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3ß variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3ß knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3ß to promote canonical NF-κB signaling and the development of retinal inflammation.

REDD1 Deletion Suppresses NF-κB Signaling in Cardiomyocytes and Prevents Deficits in Cardiac Function in Diabetic Mice.

Activation of the transcription factor NF-κB in cardiomyocytes has been implicated in the development of cardiac function deficits caused by diabetes. NF-κB controls the expression of an array of pro-inflammatory cytokines and chemokines. We recently discovered that the stress response protein regulated in development and DNA damage response 1 (REDD1) was required for increased pro-inflammatory cytokine expression in the hearts of diabetic mice. The studies herein were designed to extend the prior report by investigating the role of REDD1 in NF-κB signaling in cardiomyocytes. REDD1 genetic deletion suppressed NF-κB signaling and nuclear localization of the transcription factor in human AC16 cardiomyocyte cultures exposed to TNFα or hyperglycemic conditions. A similar suppressive effect on NF-κB activation and pro-inflammatory cytokine expression was also seen in cardiomyocytes by knocking down the expression of GSK3ß. NF-κB activity was restored in REDD1-deficient cardiomyocytes exposed to hyperglycemic conditions by expression of a constitutively active GSK3ß variant. In the hearts of diabetic mice, REDD1 was required for reduced inhibitory phosphorylation of GSK3ß at S9 and upregulation of IL-1ß and CCL2. Diabetic REDD1+/+ mice developed systolic functional deficits evidenced by reduced ejection fraction. By contrast, REDD1-/- mice did not exhibit a diabetes-induced deficit in ejection fraction and left ventricular chamber dilatation was reduced in diabetic REDD1-/- mice, as compared to diabetic REDD1+/+ mice. Overall, the results support a role for REDD1 in promoting GSK3ß-dependent NF-κB signaling in cardiomyocytes and in the development of cardiac function deficits in diabetic mice.

Stress response protein REDD1 promotes diabetes-induced retinal inflammation by sustaining canonical NF-κB signaling.

Inflammation contributes to the progression of retinal pathology caused by diabetes. Here, we investigated a role for the stress response protein regulated in development and DNA damage response 1 (REDD1) in the development of retinal inflammation. Increased REDD1 expression was observed in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes, and REDD1 was essential for diabetes-induced pro-inflammatory cytokine expression. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented increased pro-inflammatory cytokine expression in response to hyperglycemic conditions. REDD1 deletion promoted nuclear factor erythroid-2-related factor 2 (Nrf2) hyperactivation; however, Nrf2 was not required for reduced inflammatory cytokine expression in REDD1-deficient cells. Rather, REDD1 enhanced inflammatory cytokine expression by promoting activation of nuclear transcription factor κB (NF-κB). In WT cells exposed to tumor necrosis factor α (TNFα), inflammatory cytokine expression was increased in coordination with activating transcription factor 4 (ATF4)-dependent REDD1 expression and sustained activation of NF-κB. In both Müller cell cultures exposed to TNFα and in the retina of STZ-diabetic mice, REDD1 deletion promoted inhibitor of κB (IκB) expression and reduced NF-κB DNA-binding activity. We found that REDD1 acted upstream of IκB by enhancing both K63-ubiquitination and auto-phosphorylation of IκB kinase complex. In contrast with STZ-diabetic REDD1+/+ mice, IκB kinase complex autophosphorylation and macrophage infiltration were not observed in the retina of STZ-diabetic REDD1-/- mice. The findings provide new insight into how diabetes promotes retinal inflammation and support a model wherein REDD1 sustains activation of canonical NF-κB signaling.

PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here, we investigated the role of the stress response protein regulated in development and DNA damage 1 (REDD1) as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 wk, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the proinflammatory cytokines IL-1ß, IL-6, and TNFα. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase protein kinase RNA-like endoplasmic reticulum kinase (PERK) or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced proinflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.NEW & NOTEWORTHY Interplay between endoplasmic reticulum stress and inflammation contributes to cardiovascular disease progression. The studies here identify the stress response protein known as REDD1 as a missing molecular link that connects the development of endoplasmic reticulum stress with increased production of proinflammatory cytokines in the hearts of obese mice.

DDIT4 Downregulation by siRNA Approach Increases the Activity of Proteins Regulating Fatty Acid Metabolism upon Aspirin Treatment in Human Breast Cancer Cells.

Repositioning of aspirin for a more effective breast cancer (BC) treatment requires identification of predictive biomarkers. However, the molecular mechanism underlying the anticancer activity of aspirin remains fully undefined. Cancer cells enhance de novo fatty acid (FA) synthesis and FA oxidation to maintain a malignant phenotype, and the mechanistic target of rapamycin (mTORC1) is required for lipogenesis. We, therefore, aimed to test if the expression of mTORC1 suppressor DNA damage-inducible transcript (DDIT4) affects the activity of main enzymes in FA metabolism after aspirin treatment. MCF-7 and MDA-MB-468 human BC cell lines were transfected with siRNA to downregulate DDIT4. The expression of carnitine palmitoyltransferase 1 A (CPT1A) and serine 79-phosphorylated acetyl-CoA carboxylase 1 (ACC1) were analyzed by Western Blotting. Aspirin enhanced ACC1 phosphorylation by two-fold in MCF-7 cells and had no effect in MDA-MB-468 cells. Aspirin did not change the expression of CPT1A in either cell line. We have recently reported DDIT4 itself to be upregulated by aspirin. DDIT4 knockdown resulted in 1.5-fold decreased ACC1 phosphorylation (dephosphorylation activates the enzyme), 2-fold increased CPT1A expression in MCF-7 cells, and 2.8-fold reduced phosphorylation of ACC1 following aspirin exposure in MDA-MB-468 cells. Thus, DDIT4 downregulation raised the activity of main lipid metabolism enzymes upon aspirin exposure which is an undesired effect as FA synthesis and oxidation are linked to malignant phenotype. This finding may be clinically relevant as DDIT4 expression has been shown to vary in breast tumors. Our findings justify further, more extensive investigation of the role of DDIT4 in aspirin's effect on fatty acid metabolism in BC cells.

The RNA polymerase II subunit B (RPB2) functions as a growth regulator in human glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor with a poor prognosis. The growth of GBM cells depends on the core transcriptional apparatus, thus rendering RNA polymerase (RNA pol) complex as a candidate therapeutic target. The RNA pol II subunit B (POLR2B) gene encodes the second largest subunit of the RNA pol II (RPB2); however, its genomic status and function in GBM remain unclear. Certain GBM data sets in cBioPortal were used for investigating the genomic status and expression of POLR2B in GBM. The function of RPB2 was analyzed following knockdown of POLR2B expression by shRNA in GBM cells. The cell counting kit-8 assay and PI staining were used for cell proliferation and cell cycle analysis. A xenograft mouse model was established to analyze the function of RPB2 in vivo. RNA sequencing was performed to analyze the RPB2-regulated genes. GO and GSEA analyses were applied to investigate the RPB2-regulated gene function and associated pathways. In the present study, the genomic alteration and overexpression of the POLR2B gene was described in glioblastoma. The data indicated that knockdown of POLR2B expression suppressed tumor cell growth of glioblastoma in vitro and in vivo. The analysis further demonstrated the identification of the RPB2-regulated gene sets and highlighted the DNA damage-inducible transcript 4 gene as the downstream target of the POLR2B gene. The present study provides evidence indicating that RPB2 functions as a growth regulator in glioblastoma and could be used as a potential therapeutic target for the treatment of this disease.

Downregulation of DDIT4 ameliorates abnormal behaviors in autism by inhibiting ferroptosis via the PI3K/Akt pathway.

Autism spectrum disorder (ASD) is a complex disease with unclear etiology. Studies have shown that ferroptosis is also related to ASD progression, but the specific mechanism is still unclear. Valproic acid (VPA) induced neuronal ferroptosis in vitro. Mechanistic studies showed that both VPA and ferroptosis inducers promoted the expression of DDIT4 in neurons, thereby inhibiting the activation of the PI3K/Akt pathway. DDIT4 increased the accumulation of ROS, MDA and Fe2+, inhibited neuronal viability and downregulated GPX4 expression by inactivating the PI3K/Akt pathway. Ferroptosis inhibitors reversed the anti-survival effect of DDIT4, indicating that DDIT4 enhances ferroptosis through the PI3K/Akt pathway, thereby inhibiting neuronal viability. Further in vivo experiments found that autistic mice had high levels of ROS, MDA and Fe2+, increased DDIT4 expression, and downregulated expression levels of GPX4, p-PI3K and p-Akt; after downregulation of DDIT4 expression, the accumulation of ROS, MDA and Fe2+ was significantly reduced, while the expression levels of GPX4, p-PI3K and p-Akt were upregulated, indicating that DDIT4 knockdown reduces ferroptosis in autistic mice. In addition, DDIT4 downregulation, PI3K/Akt pathway activation, and ferroptosis inhibitors all improved social behavior deficits, repetitive stereotyped and compulsive behaviors, anxiety and exploratory behaviors in autistic mice, but PI3K/Akt pathway inhibitors significantly blocked the rescue of abnormal behaviors by DDIT4 downregulation in autistic mice. Therefore, downregulation of DDIT4 expression ameliorates abnormal behaviors in autism by inhibiting ferroptosis via the PI3K/Akt pathway, indicating that DDIT4, the PI3K/Akt pathway and ferroptosis have key roles in autism.

DDIT4 promotes malignancy of head and neck squamous cell carcinoma.

This study investigated the cancer-promoting effect of ferroptosis regulator DNA damage-inducible transcript 4 (DDIT4) and its relevant mechanisms. Vital ferroptosis-related genes were identified using bioinformatic methods on the basis of data collected from TCGA and seven other online databases. Cell Counting Kit-8 (CCK8), colony formation, wound-healing and transwell assays, and western blot analysis were conducted for verifying the biological role of DDIT4 in vitro. The immune score and tumor purity were calculated using R package "estimate." The relationship was identified between DDIT4 expression and immune cell infiltration using ssGSEA and CIBERSORT algorithms. R package "Seurat" was used to perform unsupervised clustering of the single cells, and "SingleR" was utilized for annotation. R package "STUtility" was employed to plot the spatial expression of DDIT4. For trajectory analysis, monocle was used to predict cell differentiation and demonstrate the expression of DDIT4 at each state. Here, DDIT4 overexpression was observed in Head and Neck Squamous Cell Carcinoma (HNSCC) cohort, and DDIT4 upregulation showed a positive correlation with larger tumor size, lymph node metastasis, more advanced TNM stage and higher tumor mutational burden (TMB). Moreover, DDIT4 knockdown could markedly inhibit the proliferation, colony formation, invasion and migration of HNSCC cells, as well as suppress the expression of HIF-1a, VEGF and vimentin. In comparison, DDIT4 overexpression showed a negative correlation with immune score and infiltrations of several immune cells. DDIT4 played crucial roles in the differentiation of CAFs and T cells. Collectively, this study demonstrates that DDIT4 contributes a critical role in HNSCC progression. The positive feedback regulation between DDIT4 and HIF-1a may be a potential target for HNSCC treatment.

Small extracellular vesicles-transported lncRNA TDRKH-AS1 derived from AOPPs-treated trophoblasts initiates endothelial cells pyroptosis through PDIA4/DDIT4 axis in preeclampsia.

BACKGROUND: Substantial studies have demonstrated that oxidative stress placenta and endothelial injury are considered to inextricably critical events in the pathogenesis of preeclampsia (PE). Systemic inflammatory response and endothelial dysfunction are induced by the circulating factors released from oxidative stress placentae. As a novel biomarker of oxidative stress, advanced oxidation protein products (AOPPs) levels are strongly correlated with PE characteristics. Nevertheless, the molecular mechanism underlying the effect of factors is still largely unknown. METHODS: With the exponential knowledge on the importance of placenta-derived extracellular vesicles (pEVs), we carried out lncRNA transcriptome profiling on small EVs (sEVs) secreted from AOPPs-treated trophoblast cells and identified upregulated lncRNA TDRKH-AS1 as a potentially causative factor for PE. We isolated and characterized sEVs from plasma and trophoblast cells by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. The expression and correlation of lncRNA TDRKH-AS1 were evaluated using qRT-PCR in plasmatic sEVs and placentae from patients. Pregnant mice injected with TDRKH-AS1-riched trophoblast sEVs was performed to detect the TDRKH-AS1 function in vivo. To investigate the potential effect of sEVs-derived TDRKH-AS1 on endothelial function in vitro, transcriptome sequencing, scanning electron Microscopy (SEM), immunofluorescence, ELISA and western blotting were conducted in HUVECs. RNA pulldown, mass spectrometry, RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP) and coimmunoprecipitation (Co-IP) were used to reveal the latent mechanism of TDRKH-AS1 on endothelial injury. RESULTS: The expression level of TDRKH-AS1 was significantly increased in plasmatic sEVs and placentae from patients, and elevated TDRKH-AS1 in plasmatic sEVs was positively correlated with clinical severity of the patients. Moreover, pregnant mice injected with TDRKH-AS1-riched trophoblast sEVs exhibited a hallmark feature of PE with increased blood pressure and systemic inflammatory responses. Pyroptosis, an inflammatory form of programmed cell death, is involved in the development of PE. Indeed, our in vitro study indicated that sEVs-derived TDRKH-AS1 secreted from AOPPs-induced trophoblast elevated DDIT4 expression levels to trigger inflammatory response of pyroptosis in endothelial cells through interacting with PDIA4. CONCLUSIONS: Herein, results in the present study supported that TDRKH-AS1 in sEVs isolated from oxidative stress trophoblast may be implicated in the pathogenesis of PE via inducing pyroptosis and aggravating endothelial dysfunction.

DDIT4 is essential for DINP-induced autophagy of ovarian granulosa cells.

As one of the most important phthalates, di-isononyl phthalate (DINP) has been widely used as a common plasticizer in the food and personal care products sectors. In our previous study, we found that DINP can induce autophagy of ovarian granulosa cells; while the underlying mechanism is unclear. In the study, we showed that DINP exposure could induce autophagy of ovarian granulosa cells and KGN cells, accompanied with the increase in the mRNA and protein level of DDIT4. Furthermore, overexpression of DDIT4 were shown to induce autophagy of KGN cells; while knockdown of DDIT4 inhibited DINP-induced autophagy, implying that DDIT4 played an important role in DINP-induced autophagy of ovarian granulosa cells. There were three putative binding sites of transcription factor ATF4 in the promoter region of DDIT4 gene, suggesting that DDIT4 might be regulated by ATF4. Herein, we found that overexpression of ATF4 could upregulate the expression of DDIT4 in KGN cells, while knockdown of ATF4 inhibited its expression. Subsequently, ATF4 was identified to bind to the promoter region of DDIT4 gene and promote its transcription. The expression of ATF4 was also increased in the DINP-exposed granulosa cells, and ATF4 overexpression promoted autophagy of KGN cells; whereas knockdown of ATF4 alleviated DINP-induced upregulation of DDIT4 and autophagy of the cells. Taken together, DINP triggered autophagy of ovarian granulosa cells through activating ATF4/DDIT4 signals.

The regulatory roles of DDIT4 in TDCIPP-induced autophagy and apoptosis in PC12 cells.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is a commonly used organophosphate-based flame retardant and can bio-accumulate in human tissues and organs. As its structure is similar to that of neurotoxic organophosphate pesticides, the neurotoxicity of TDCIPP has raised widespread concerns. TDCIPP can increase neuronal apoptosis and induce autophagy. However, its regulatory mechanism remains unclear. In this study, we found that the expression upregulation of the DNA Damage-Inducible Transcript 4 (DDIT4) protein, which might play essential roles in TDCIPP-induced neuronal autophagy and apoptosis, was observed in TDCIPP-treated differentiated rat PC12 cells. Furthermore, we determined the protective effect of the DDIT4 suppression on the autophagy and apoptosis induced by TDCIPP using Western blot (WB) and Flow cytometry (FACS) analysis. We observed that TDCIPP treatment increased the DDIT4, the autophagy marker Beclin-1, and the microtubule-associated protein light chain 3-II (LC3II) expressions and decreased the mTOR phosphorylation levels. Conversely, the suppression of DDIT4 expression increased the p-mTOR expression and decreased cell autophagy and apoptosis. Collectively, our results revealed the function of DDIT4 in cell death mechanisms triggered by TDCIPP through the mTOR signaling axis in differentiated PC12 cells. Thus, this study provided vital evidence necessary to explain the mechanism of TDCIPP-induced neurotoxicity in differentiated PC12 cells.

Smurf2 exerts neuroprotective effects on cerebral ischemic injury.

The present study aimed to explore specific mechanisms involved in mediating the neuroprotective effects of Smad ubiquitination regulatory factor 2 (Smurf2) in cerebral ischemic injury. A middle cerebral artery occlusion (MCAO) mouse model and an oxygen-glucose deprivation (OGD)-treated neuron model were developed. The expression of Smurf2, Yin Yang 1 (YY1), hypoxia-inducible factor-1 alpha (HIF1α), and DNA damage-inducible transcript 4 gene (DDIT4) was analyzed. Thereafter, the expression of Smurf2, YY1, HIF1α, and DDIT4 was altered in the MCAO mice and OGD-treated neurons. Apoptosis in tissues and cerebral infarction were assessed. In neurons, the expression of apoptosis-related proteins, viability, and apoptosis were assessed, followed by evaluation of lactate dehydrogenase leakage rate. The interaction between Smurf2 and YY1 was analyzed by coimmunoprecipitation assay and that between YY1 ubiquitination by in vivo ubiquitination experiment. The results showed downregulation of Smurf2 and upregulation of YY1, HIF1α, and DDIT4 in both MCAO mice and OGD-treated neurons. Smurf2 elevated YY1 ubiquitination and degradation, and YY1 increased HIF1α expression to promote DDIT4 in neurons. Overexpressed Smurf2 or downregulated YY1, HIF1α, or DDIT4 reduced the volume of cerebral infarction and apoptosis in MCAO mice, while enhancing cell viability and reducing apoptosis and lactate dehydrogenase leakage in OGD-treated neurons. In summary, our findings elucidated a neuroprotective role of Smurf2 in cerebral ischemic injury via inactivation of the YY1/HIF1α/DDIT4 axis.

The m6A demethylase ALKBH5-mediated upregulation of DDIT4-AS1 maintains pancreatic cancer stemness and suppresses chemosensitivity by activating the mTOR pathway.

BACKGROUND: Chemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors associated with chemoresistance and a very promising direction for cancer treatment. However, the exact molecular mechanisms of cancer stemness have not been completely elucidated. METHODS: m6A-RNA immunoprecipitation and sequencing were used to screen m6A-related mRNAs and lncRNAs. qRT-PCR and FISH were utilized to analyse DDIT4-AS1 expression. Spheroid formation, colony formation, Western blot and flow cytometry assays were performed to analyse the cancer stemness and chemosensitivity of PDAC cells. Xenograft experiments were conducted to analyse the tumour formation ratio and growth in vivo. RNA sequencing, Western blot and bioinformatics analyses were used to identify the downstream pathway of DDIT4-AS1. IP, RIP and RNA pulldown assays were performed to test the interaction between DDIT4-AS1, DDIT4 and UPF1. Patient-derived xenograft (PDX) mouse models were generated to evaluate chemosensitivities to GEM. RESULTS: DDIT4-AS1 was identified as one of the downstream targets of ALKBH5, and recruitment of HuR onto m6A-modified sites is essential for DDIT4-AS1 stabilization. DDIT4-AS1 was upregulated in PDAC and positively correlated with a poor prognosis. DDIT4-AS1 silencing inhibited stemness and enhanced chemosensitivity to GEM (Gemcitabine). Mechanistically, DDIT4-AS1 promoted the phosphorylation of UPF1 by preventing the binding of SMG5 and PP2A to UPF1, which decreased the stability of the DDIT4 mRNA and activated the mTOR pathway. Furthermore, suppression of DDIT4-AS1 in a PDX-derived model enhanced the antitumour effects of GEM on PDAC. CONCLUSIONS: The ALKBH5-mediated m6A modification led to DDIT4-AS1 overexpression in PDAC, and DDIT-AS1 increased cancer stemness and suppressed chemosensitivity to GEM by destabilizing DDIT4 and activating the mTOR pathway. Approaches targeting DDIT4-AS1 and its pathway may be an effective strategy for the treatment of chemoresistance in PDAC.

APEX2-Mediated Proximity Labeling Resolves the DDIT4-Interacting Proteome.

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins' molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings.

Overexpression of DDIT4 and TPTEP1 are associated with metastasis and advanced stages in colorectal cancer patients: a study utilizing bioinformatics prediction and experimental validation.

BACKGROUND: Various diagnostic and prognostic tools exist in colorectal cancer (CRC) due to multiple genetic and epigenetic alterations causing the disease. Today, the expression of RNAs is being used as prognostic markers for cancer. METHODS: In the current study, various dysregulated RNAs in CRC were identified via bioinformatics prediction. Expression of several of these RNAs were measured by RT-qPCR in 48 tissues from CRC patients as well as in colorectal cancer stem cell-enriched spheroids derived from the HT-29 cell line. The relationships between the expression levels of these RNAs and clinicopathological features were analyzed. RESULTS: Our bioinformatics analysis determined 11 key mRNAs, 9 hub miRNAs, and 18 lncRNAs which among them 2 coding RNA genes including DDIT4 and SULF1 as well as 3 non-coding RNA genes including TPTEP1, miR-181d-5p, and miR-148b-3p were selected for the further investigations. Expression of DDIT4, TPTEP1, and miR-181d-5p showed significantly increased levels while SULF1 and miR-148b-3p showed decreased levels in CRC tissues compared to the adjacent normal tissues. Positive relationships between DDIT4, SULF1, and TPTEP1 expression and metastasis and advanced stages of CRC were observed. Additionally, our results showed significant correlations between expression of TPTEP1 with DDIT4 and SULF1. CONCLUSIONS: Our findings demonstrated increased expression levels of DDIT4 and TPTEP1 in CRC were associated with more aggressive tumor behavior and more advanced stages of the disease. The positive correlations between TPTEP1 as non-coding RNA and both DDIT4 and SULF1 suggest a regulatory effect of TPTEP1 on these genes.

DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

Ataxia telangiectasia (AT) is a rare, severe, and ineluctably progressive multisystemic neurodegenerative disease. Histone deacetylase 4 (HDAC4) nuclear accumulation has been related to neurodegeneration in AT. Since treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome, the effects of dexamethasone on HDAC4 were investigated. In this paper, we describe a novel nonepigenetic function of HDAC4 induced by dexamethasone, through which it can directly modulate HIF-1a activity and promote the upregulation of the DDIT4 gene and protein expression. This new HDAC4 transcription regulation mechanism leads to a positive effect on autophagic flux, an AT-compromised biological pathway. This signaling was specifically induced by dexamethasone only in AT cell lines and can contribute in explaining the positive effects of dexamethasone observed in AT-treated patients.

Exploring vitamin D signalling within skin cancer.

Sunlight exposure of the skin is associated with both risks and benefits. On one hand, sunlight ultraviolet (UV) radiation can cause skin cancer through signature DNA mutations. On the other hand, it can be absorbed in the skin by 7-dehydrocholesterol to instigate endogenous synthesis of vitamin D to regulate anticancer effects. Thus, protecting one's skin from sunlight to avoid skin cancer may lead to impaired vitamin D levels arguing for sensible sun exposure practices. To limit cancer, vitamin D metabolites can promote uncharacterized and diverse sets of events such as repair responses to DNA damage, apoptosis of malignant cells, and suppression of immune surveillance, proliferation and angiogenesis. Recent findings also suggest that part of the anticancer effects of vitamin D within squamous cell carcinoma-a type of skin cancer most directly linked to sun exposure-involves the DDIT4-mTOR catabolic signalling pathway to enhance cell autophagy. As mTOR activity and cellular metabolism are modulated as part of the DNA damage response, insights into the means by which mTOR can be controlled by vitamin D to suppress cancer is of molecular and clinical importance. Overall, the research so far suggests that presence of vitamin D through sunlight exposure and supplementation are beneficial for human health in the face of cancer.

NNT-AS1 modulates prostate cancer cell proliferation, apoptosis and migration through miR-496/DDIT4 axis.

BACKGROUND: Emerging studies have disclosed long non-coding RNAs (lncRNAs) as pivotal modulators in the progression of prostate cancer (PCa). Current research planned to figure out the involvement of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in PCa. METHODS: RNA expression was examined using RT-qPCR in PCa cells. Functional assays assessed the viability, proliferation, apoptosis and migration of PCa cells. RNA pull down and luciferase reporter experiments detected the interplay between miRNA and lncRNA or mRNA. RESULTS: NNT-AS1 was apparently upregulated in PCa cells. NNT-AS1 deficiency abrogated PCa cell viability, proliferation and migration but promoted apoptosis. Besides, miR-496 could be sequestered by NNT-AS1 to elevate the expression of DNA damage inducible transcript 4 (DDIT4) in PCa. Rescue assays indicated that overexpressed DDIT4 or restrained miR-496 could reverse the influence of NNT-AS1 depletion on malignant processes in PCa cells. CONCLUSION: NNT-AS1 contributes to the malignant phenotypes of PCa cells through targeting miR-496 to boost DDIT4 expression.