Expression Of Death Receptor Research Articles

CXCR4 intracellular protein promotes drug resistance and tumorigenic potential by inversely regulating the expression of Death Receptor 5

Chemokine receptor CXCR4 overexpression in solid tumors has been strongly associated with poor prognosis and adverse clinical outcome. However, blockade of CXCL12-CXCR4 signaling axis by inhibitors like Nox-A12, FDA approved CXCR4 inhibitor drug AMD3100 have shown limited clinical success in cancer treatment. Therefore, exclusive contribution of CXCR4-CXCL12 signaling in pro-tumorigenic function is questionable. In our pursuit to understand the impact of chemokine signaling in carcinogenesis, we reveal that instead of CXCR4-CXCL12 signaling, presence of CXCR4 intracellular protein augments paclitaxel resistance and pro-tumorigenic functions. In search of pro-apoptotic mechanisms for CXCR4 mediated drug resistance; we discover that DR5 is a new selective target of CXCR4 in breast and colon cancer. Further, we detect that CXCR4 directs the differential recruitment of transcription factors p53 and YY1 to the promoter of DR5 in course of its transcriptional repression. Remarkably, inhibiting CXCR4-ligand-mediated signals completely fails to block the above phenotype. Overexpression of different mutant versions of CXCR4 lacking signal transduction capabilities also result in marked downregulation of DR5 expression in colon cancer indeed confirms the reverse relationship between DR5 and intracellular CXCR4 protein expression. Irrespective of CXCR4 surface expression, by utilizing stable gain and loss of function approaches, we observe that intracellular CXCR4 protein selectively resists and sensitizes colon cancer cells against paclitaxel therapy in vitro and in vivo. Finally, performing TCGA data mining and using human breast cancer patient samples, we demonstrate that expression of CXCR4 and DR5 are inversely regulated. Together, our data suggest that targeting CXCR4 intracellular protein may be critical to dampen the pro-tumorigenic functions of CXCR4.

Sodium fluoride activates the extrinsic apoptosis via regulating NOX4/ROS-mediated p53/DR5 signaling pathway in lung cells both in vitro and in vivo

An extensive body of research has demonstrated that pulmonary toxicity induced by fluoride is related to cell apoptosis. Although induction of death receptor-initiated extrinsic apoptosis by sodium fluoride (NaF) has been reported, its mechanism of action is still not clearly defined. Herein, we found that NaF treatment induced activation of caspase-8 in BEAS-2B cells, resulting in apoptosis, which was markedly reduced by blocking caspase-8 using small interfering RNA (siRNA). In this study, we report that death receptor 5 (DR5), a major component of the extrinsic apoptotic pathway, is markedly induced upon NaF stimulation. Enhanced DR5 induction was necessary for the apoptotic effects of NaF, inasmuch as transfected BEAS-2B cells with DR5 siRNA attenuated NaF-induced caspase-8 activation in lung cells. Mechanism investigation indicated that the induction of DR5, following NaF exposure, was mediated by tumor protein 53 (p53)-dependent transcriptional activation. Notably, we demonstrated that NaF could induce a significant increase in intracellular reactive oxygen species (ROS) level derived from nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). Specifically, NOX4 knockdown inhibited NaF-induced the activation of p53/DR5 axis by reducing NOX4-derived ROS production. Further in vivo investigation demonstrated that NOX4 deficiency markedly attenuates NaF-induced lung injury, apoptosis, and ROS levels in the lung. Moreover, the expressions of p53 and DR5 were significantly reduced after NaF treatment in NOX4 knockout mice compared with the wild type mice. Taken together, our findings provide a novel insight into for the pulmonary apoptosis in response to NaF exposure.

LCT-3d Induces Oxidative Stress-Mediated Apoptosis by Upregulating Death Receptor 5 in Gastric Cancer Cells.

Gastric cancer is a global health problem. In this study, we investigate the role of a novel Indole derivative, named LCT-3d, in inhibiting the growth of gastric cancer cells by MTT assay. The Western blotting results showed that LCT-3d modulated the mitochondrial-related proteins and Cleaved-Caspases 3/9, to induce cell apoptosis. The up-regulation of Death receptor 5 (DR5) in MGC803 cells was observed with LCT-3d treatment. Knockdown of DR5 on MGC803 cells partially reversed the LCT-3d-induced mitochondrial apoptosis. The level of Reactive Oxygen Species (ROS) in MGC803 cells was increased with LCT-3d treatment and could be blocked with the pretreatment of the ROS inhibitor N-Acetylcysteine (NAC). The results demonstrate that the elevating ROS can up-regulate the expression of DR5, resulting in apoptosis via mitochondrial pathway. Although the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway served an important role in protecting gastric cancer cells against the injury of ROS, it can’t reverse LCT-3d-induced cell apoptosis. Taken together, our study showed that LCT-3d induced apoptosis via DR5-mediated mitochondrial apoptotic pathway in gastric cancer cells. LCT-3d could be a novel lead compound for development of anti-cancer activity in gastric cancer.

Recombinant protein TRAIL-Mu3 enhances the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway.

Pancreatic cancer is a highly malignant type of cancer and its treatment remains a major challenge. The novel recombinant protein TNF-related apoptosis-inducing ligand (TRAIL)-Mu3 has been shown to exert stronger tumor inhibitory effects in colon cancer in vitro and in vivo compared with TRAIL. The present study investigated the antitumor effects of TRAIL-Mu3 on pancreatic cancer cells, and the possible mechanisms were further examined. Compared with TRAIL, TRAIL-Mu3 exhibited significantly higher cytotoxic effects on pancreatic cancer cell lines. The inhibitory effect of TRAIL-Mu3 on the viability of PANC-1 cells was shown to be a caspase-dependent process. The affinity of TRAIL-Mu3 to PANC-1 cell membranes was significantly enhanced compared with TRAIL. In addition, TRAIL-Mu3 upregulated death receptor (DR) expression in PANC-1 cells and promoted the redistribution of DR5 in lipid rafts. Western blotting results demonstrated that TRAIL-Mu3 activated the caspase cascade in a faster and more efficient manner compared with TRAIL in PANC-1 cells. Therefore, TRAIL-Mu3 enhanced the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway. The present study indicated the potential of TRAIL-Mu3 for the treatment of pancreatic cancer.

Matrine inhibits ovarian cancer cell viability and promotes apoptosis by regulating the ERK/JNK signaling pathway via p38MAPK.

Ovarian cancer displays the highest mortality rate among all types of gynecological cancer worldwide. The survival of patients with ovarian cancer remains poor due to poor responses to anticancer treatments. The present study aimed to investigate the therapeutic effects and potential mechanism underlying matrine in ovarian cancer tissues, ovarian cancer cells and a CAOV-3-derived tumor-bearing mouse model. MTT, migration, invasion, flow cytometry, immunofluorescence and immunohistochemistry assays were performed to assess the inhibitory effects of matrine on ovarian cancer. A xenograft ovarian cancer mouse model was established and treated with matrine or PBS. The results demonstrated that compared with the control group, matrine significantly induced ovarian cancer cell apoptosis by upregulating caspase-8 and Fas cell surface death receptor (Fas) expression levels, and downregulating Bcl-2 and Bcl-xl expression levels. Moreover, compared with the control group, matrine significantly inhibited ovarian cancer cell viability, migration and invasion by downregulating metastasis associated protein-1, fibronectin, angiotensin II type 2 receptor–interacting protein 3a and H high mobility group AT-hook 2 expression levels. Compared with the control group, matrine significantly increased p38MAPK, phosphorylated (p)ERK/ERK and pJNK/JNK expression levels in ovarian cancer cells. p38MAPK knockdown significantly downregulated p38MAPK, pERK/ERK and pJNK/JNK expression levels compared with the control group, which significantly promoted ovarian cancer cell viability, migration and invasion. In vivo experiments demonstrated that compared with the control group, matrine significantly suppressed tumor growth by markedly upregulating p38MAPK, ERK and JNK expression levels. The immunohistochemistry results demonstrated that caspase-8 and Fas expression levels were notably increased, whereas Bcl-2 and Bcl-xl expression levels were obviously decreased in matrine-treated tumors compared with PBS-treated tumors. In conclusion, the present study demonstrated that matrine inhibited ovarian cancer cell viability, migration and invasion, but induced cell apoptosis, suggesting that matrine may serve as a promising anticancer agent for the treatment of ovarian cancer.

Alternol Sensitizes Renal Carcinoma Cells to TRAIL-Induced Apoptosis.

Purpose: Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a member of the TNF family, can selectively induce cancer cell death while sparing normal cells. However, the application of TRAIL-based antitumor therapies has been hindered due to drug resistance. Alternol is a new compound isolated from microbial fermentation that possesses antitumor activity in different tumors. In our research, we discovered that alternol can sensitize TRAIL-induced apoptosis in renal carcinoma cells (RCCs). Materials and Methods: Cytotoxic activity was measured by MTT assay. Apoptosis was probed using the PI/annexin V method. Real-time PCR and western blot were used to test the levels of mRNA and protein, respectively. Luciferase assay was used to investigate whether CHOP regulated the expression of death receptor (DR) 5 through transcription. A xenogeneic tumor transplantation model was used to evaluate the anticancer effects of alternol/TRAIL in vivo. Results: When the mechanisms were investigated, we discovered that alternol increased DR5 expression. DR5 knockdown by siRNA eliminated the enhanced effect of alternol on TRAIL-mediated apoptosis. Alternol reduced the expression of antiapoptotic proteins and increased the levels of proapoptotic proteins. Moreover, alternol increased the level of CHOP, which is necessary for the enhancing effect of alternol on TRAIL-induced apoptosis, given that downregulation of CHOP abrogated the synergistic effect. DR5 upregulation induced by alternol required the production of reactive oxygen species (ROS). Removing ROS inhibited the induction of DR5 and blocked the antiapoptotic proteins induced by alternol. Conclusion: Taken together, our research suggested that alternol increased TRAIL-mediated apoptosis via inhibiting antiapoptotic proteins and upregulating DR5 levels via ROS generation and the CHOP pathway.

Resistance to Cell Death in Mucinous Colorectal Cancer-A Review.

Simple SummaryMucinous colorectal cancer is characterised by abundant mucin glycoprotein production and is associated with resistance to chemotherapy-based treatments. This review outlines mechanisms for cell death resistance present in mucinous colorectal cancer including glycoprotein interactions with cell survival and cell apoptotic pathways. Further mechanisms are explored, including alterations in the expression of chemotherapy metabolism and resistance genes. This review identifies directions for future investigation of mucinous colorectal cancer and novel treatment strategies.Mucinous colorectal cancer (CRC) is estimated to occur in approximately 10–15% of CRC cases and is characterized by abundant extracellular mucin. Mucinous CRC is frequently associated with resistance to apoptosis. Inferior prognosis is observed in mucinous CRC, particularly in rectal cancer and metastatic cases. Mucins are heavily glycosylated secretory or transmembrane proteins that participate in protection of the colonic epithelium. MUC2 overexpression is a hallmark of mucinous CRCs. Mucinous CRC is associated with KRAS and BRAF mutation, microsatellite instability and the CpG island methylator phenotype. Mutations of the APC gene and p53 mutations which are characteristic non-mucinous colorectal adenocarcinoma are less common in mucinous CRC. Both physical and anti-apoptotic properties of mucin provide mechanisms for resistance to cell death. Mucin glycoproteins are associated with decreased expression of pro-apoptotic proteins, increased expression of anti-apoptotic proteins and increased cell survival signaling. The role for BCL-2 proteins, including BCL-XL, in preventing apoptosis in mucinous CRC has been explored to a limited extent. Additional mechanisms opposing cell death include altered death receptor expression and altered mutation rates in genes responsible for chemotherapy resistance. The roles of alternate cell death programs including necroptosis and pyroptosis are not well understood in mucinous CRC. While the presence of MUC2 is associated with an immunosuppressive environment, the tumor immune environment of mucinous CRC and the role of immune-mediated tumor cell death likewise require further investigation. Improved understanding of cell death mechanisms in mucinous CRC may allow modification of currently used regimens and facilitate targeted treatment.

Taxanes Sensitize Prostate Cancer Cells to TRAIL-Induced Apoptotic Synergy via Endoplasmic Reticulum Stress.

Docetaxel and cabazitaxel are guideline-chemotherapy treatments for metastatic castration-resistant prostate cancer (mCRPC), which comprises the majority of prostate cancer deaths. TNF-related apoptosis inducing ligand (TRAIL) is an anticancer agent that is selectively cytotoxic to cancer cells; however, many human cancers are resistant to TRAIL. In this study, we sensitized androgen-independent and TRAIL-resistant prostate cancer cells to TRAIL-mediated apoptosis via taxane therapy and examined the mechanism of sensitization. DU145 and PC3 cells displayed no significant reduction in cell viability when treated with soluble TRAIL, docetaxel, or cabazitaxel alone indicating that both cell lines are resistant to TRAIL and taxanes individually. Taxane and TRAIL combination synergistically amplified apoptosis strongly suggesting that taxanes sensitize prostate cancer cells to TRAIL. A Jun N-terminal kinases (JNK) inhibitor inhibited apoptosis in treated cells and significantly reduced death receptor expression indicating JNK activation by ER stress sensitizes PCa cells to TRAIL-induced apoptosis by upregulating DR4/DR5 expression. In addition, suppression of C/EBP homologous protein (CHOP) reduced TRAIL sensitization in both cell lines indicating that ER stress-related apoptosis is mediated, in part, by CHOP. Cytochrome c knockdown showed a significant decrease in sensitivity in PC3 cells, but not in Bax-deficient DU145 cells. A computational model was used to simulate apoptosis for cells treated with taxane and TRAIL therapy as demonstrated in in vitro experiments. Pretreatment with taxanes sensitized cells to apoptosis induced by TRAIL-mediated apoptosis, demonstrating that combining TRAIL with ER stress inducers is a promising therapy to reverse TRAIL resistance to treat mCRPC.

Abstract PS19-18: Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis

Abstract Introduction: Triple-negative breast cancer (TNBC) does not respond to conventional targeted therapy, necessitating novel treatment options. Metformin possesses unique anti-proliferative and pro-apoptotic properties in TNBC cells. However, the molecular mechanism through which metformin may induce apoptosis is incompletely understood. In the current study, we aim to determine whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptotic signaling is involved in metformin-induced cytotoxicity on TNBC cells. Methods: TNBC cell lines HCC70, MDA-MB-468, BT549 and MDA-MB-231 were used in the study. A cell death detection ELISA kit was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes). Western blot analyses were carried out to assess the expression of TRAIL, TRAIL receptors DR5 and DR4, and apoptosis-related proteins (PARP, Caspase-8 and Caspase-3). Flow cytometric analyses were performed to define the expression of cell surface TRAIL receptors DR5 and DR4. Secreted TRAIL protein level in the conditioned medium (CM) of cell culture was measured by using a highly specific ELISA and western blot. MDA-MB-231 cells were treated with concentrated CM from metformin-treated cells to test the activity of secreted TRAIL in inducing cell apoptosis. A soluble recombinant TRAIL decoy receptor, which contains a normal extracellular domain of death receptor 5 (DR5) but a truncated intracellular domain and thus is unable to transduce death signals, was used to block TRAIL function. Infection with lentivirus containing specific shRNAs was performed to knockdown TRAIL expression. Results: Metformin induced apoptosis in a panel of TNBC cell lines in a dose-dependent manner, as evidenced by the increase of cytoplasmic histone-associated DNA fragments and the cleavage of PARP, caspase-8 and caspase-3. Interestingly, during the process of TNBC cells undergoing apoptosis, metformin elicited a dose-dependent upregulation of TRAIL protein levels (both cellular TRAIL and secreted TRAIL) without altering the expression of TRAIL receptors DR5 and DR4. The secreted TRAIL from concentrated CM of metformin-treated cells showed activity in inducing apoptosis of MDA-MB-231 cells. To determine if the induction of TRAIL was essential for metformin-induced cytotoxicity effects on TNBC cells, we examined whether inhibition of TRAIL function by a decoy receptor or specific knockdown of TRAIL expression with shRNAs would affect metformin-induced apoptosis. In all three cell lines (HCC70, MDA-MB-468, BT549) we tested, both the TRAIL decoy receptor and TRAIL-specific shRNAs significantly attenuated metformin-induced DNA fragmentation and cleavage of PARP, caspase-8, and caspase-3. Conclusion: We demonstrate that metformin promotes TNBC cells undergoing apoptosis via induction of TRAIL expression. Our data suggest that TRAIL-mediated apoptosis critically contributes to metformin's antitumor activity against TNBC. Keywords: Metformin, TRAIL, Apoptosis, triple-negative breast cancer Citation Format: Shuang Liu, Erik V Polsdofer, Lukun Zhou, Susan Edgerton, Ann Thor, Bolin Liu. Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-18.

Expression of Death Receptor 3 (DR3) on Circulating CD4 Positive Lymphocytes on Patients with Vitiligo

Vitiligo is the most common depigmenting skin disorder with an estimated prevalence of 0.5–2% of the population in both adults and children worldwide. Death receptor 3 is a typical member of TNFRSF which play a role in apoptotic signalling and differentiation. DR3 is considered the primary activating receptor of TL1A and is predominantly expressed on leucocytes. DR3 expression is significantly upregulated on various activated cells in the immune system, including T lymphocytes, B lymphocytes, natural killer T (NKT) cells, dendritic cells and macrophages. The aim of this study was to study the expression of death receptor 3 (DR3) on CD4 positive lymphocytes on patients with vitiligo . This study was a case control study that included 50 patients with vitiligo in addition to 25 apparently healthy individuals of matched age and sex as a control group. All patients were recruited from the outpatient clinic of Dermatology, Venereology and Andrology Department of Benha University Hospitals. DR3 showed fair AUC (AUC=0.794). At cut off value of 23.8, sensitivity was72%, specificity was 80%, PPV was 87.8%, NPV was 58.8%, and accuracy was 74.7%. DR3 expression level was significantly higher in vitiligo cases when compared to control group. DR3 expression level was significantly correlated with BSA %. DR3 expression level was suggested to be independent predictor of vitiligo susceptibility and Extent.

Sesamol Upregulates Death Receptors and Acts as a Chemosensitizer in Solid Ehrlich Carcinoma Model in Mice

Aims The aim of the present study was to investigate the anti-tumor effect of sesamol (SML), a nutritional phenolic compound of sesame, in solid Ehrlich carcinoma (SEC) model in mice and its ability to enhance doxorubicin (DOX) anti-tumor activity. Moreover, we analyzed the ability of SML to protect against DOX-induced cardiotoxicity. Main methods SML (70 mg/kg), DOX (2 mg/kg) and their combination were given to mice bearing SEC for 21 day. The mRNA level of Fas, FasL, TRAILR2, TRAIL, caspase-3 and Bcl-2 were assessed by qPCR. Tumor and cardiac tissues were examined for histopathological changes by hematoxylin and eosin. Active caspase-3 was scored by immunohistochemical analysis. Key findings SML treatment significantly decreased solid tumor size and weight. In addition, SML enhanced DOX anti-tumor activity. SML treatment either alone or in combination with DOX induced upregulation of Fas/FasL and TRAILR2/TRAIL gene expression. Moreover, SML increased caspase-3 protein and gene expressions and decreased Bcl-2 gene expression. Significance SML upregulates death receptors expression and enhances apoptosis induction in tumor cells that may explain its anti-tumor activity. Not only that, but SML also enhances DOX anti-tumor activity and attenuates its cardiotoxicity.

Therapy Induced Senescence Promote Expression of Death Receptors in Breast Cancer Cells

Chemotherapeutic agents that cause DNA damage also induce cellular senescence known as therapy-induced senescence (TIS). Cells undergoing senescence may exert detrimental effects by promoting tumor progression in healthy cells or supporting metastases in cancer cells due to “senesence-associated secretory phenotype” (SASP), involving secretion of chemokines, cytokines, metalloproteinases, and growth factors. Death receptors belong to the tumor necrosis factor receptor superfamily and implicated in induction of apoptosis via activation of extrinsic pathway. The most recognized death receptors are FAS (CD95), TNFR1 and TRAIL-R1 / 2 (DR4-DR5) etc. and capable of directly inducing apoptosis in the cell. In this study we aim to investigate the expression of cell death receptors in response to TIS of breast cancer cells for their potential use in elimination of senescent cells. Doxorubicin and etoposide were used to induce senescence selectively in MCF7 breast cancer cell line. Senescence induction was confirmed by β-galactosidase staining and cell cycle analysis. Activations of p53, p21, and γ-H2AX and expression levels of cell death receptors (FAS (CD95), TNFR1-2 and DR5 were tested by western blot analysis. Apoptosis was measured by Annexin V/7AAD analysis. Here, we show that chemotherapy agents etoposide and doxorubicin induced senescence by arresting MCF-12A and MCF-7 cells in G1 and G2/M phases of cell cycle., respectively. In addition, Induction of senescence is confirmed by SA-β-gal staining and by activation of g-H2AX, p53 and p21 proteins. Neither etoposide nor doxorubicin induced significant apoptosis in MCF12A or MCF-7 cells. Importantly, TIS increased the protein levels of TNFR1, TNFR2 and DR5 receptors selectively in MCF-7 cells but not in MCF-12A cells. These data suggest that chemotherapy agents induce senescence increased the expression of death receptors in breast cancer cell line MCF-7 thus provide a basis for further investigation of death receptor mediated targeting of senescent cells as potential therapeutic strategy.

Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation

Heat-modified citrus pectin, a water-soluble indigestible polysaccharide fiber derived from citrus fruits and modified by temperature treatment, has been reported to exhibit anticancer effects. However, the bioactive fractions and their mechanisms remain unclear. In this current study, we isolated an active compound, trans-4,5-dihydroxy-2-cyclopentene-l-one (DHCP), from heat-treated citrus pectin, and found that is induces cell death in colon cancer cells via induction of mitochondrial ROS. On the molecular level, DHCP triggers ROS production by inhibiting the activity of succinate ubiquinone reductase (SQR) in mitochondrial complex II. Furthermore, cytotoxicity, apoptotic activity, and activation of caspase cascades were determined in HCT116 and HT-29 cell-based systems, the results indicated that DHCP enhances the sensitivity of cancer cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), with DHCP-induced ROS accounting for the synergistic effect between DHCP and TRAIL. Furthermore, the combination of DHCP and TRAIL inhibits the growth of HCT116 and HT-29 xenografts synergistically. ROS significantly increases the expression of TRAIL death receptor 5 (DR5) via the p53 and C/EBP homologous protein pathways. Collectively, our findings indicate that DHCP has a favorable toxicity profile and is a new TRAIL sensitizer that shows promise in the development of pectin-based pharmaceuticals, nutraceuticals, and dietary agents aimed at combating human colon cancer.

Downregulation of death receptor 4 is tightly associated with positive response of EGFR mutant lung cancer to EGFR-targeted therapy and improved prognosis.

Death receptor 4 (DR4), a cell surface receptor, mediates apoptosis or induces inflammatory cytokine secretion upon binding to its ligand depending on cell contexts. Its prognostic impact in lung cancer and connection between EGFR-targeted therapy and DR4 modulation has not been reported and thus was the focus of this study.Methods: Intracellular protein alterations were measured by Western blotting. Cell surface protein was detected with antibody staining and flow cytometry. mRNA expression was monitored with qRT-PCR. Gene transactivation was analyzed with promoter reporter assay. Drug dynamic effects in vivo were evaluated using xenografts. Gene modulations were achieved with gene overexpression and knockdown. Proteins in human archived tissues were stained with immunohistochemistry.Results: EGFR inhibitors (e.g., osimertinib) decreased DR4 levels only in EGFR mutant NSCLC cells and tumors, being tightly associated with induction of apoptosis. This modulation was lost once cells became resistant to these inhibitors. Increased levels of DR4 were detected in cell lines with acquired osimertinib resistance and in NSCLC tissues relapsed from EGFR-targeted therapy. DR4 knockdown induced apoptosis and augmented apoptosis when combined with osimertinib in both sensitive and resistant cell lines, whereas enforced DR4 expression significantly attenuated osimertinib-induced apoptosis. Mechanistically, osimertinib induced MARCH8-mediated DR4 proteasomal degradation and suppressed MEK/ERK/AP-1-dependent DR4 transcription, resulting in DR4 downregulation. Moreover, we found that DR4 positive expression in human lung adenocarcinoma was significantly associated with poor patient survival.Conclusions: Collectively, we suggest that DR4 downregulation is coupled to therapeutic efficacy of EGFR-targeted therapy and predicts improved prognosis, revealing a previously undiscovered connection between EGFR-targeted therapy and DR4 modulation.

Membrane expression and significance of TRAIL death receptors DR4 and DR5 in Pancreatic cancer

Introduction: Tumor necrosis factor related-apoptosis-inducing ligand (TRAIL) is a powerful and selective activator of apoptosis in many cancer cells. We aim to investigate the expression and significance of TRAIL death receptor DR4 and DR5 in pancreatic cancer (PC) tissues.
 Method: Twenty-eight histologically verified samples of PC tissue were collected between 2018 and 2019. TRAIL death receptor expression profiles were determined by immunohistochemistry.
 Result: Death receptor DR4 and DR5 were expressed in the PC tissue and the adjacent non-cancerous pancreatic tissues, the expression of DR4 and DR5 in the PC tissue was significantly higher than that of the adjacent non-cancerous pancreatic tissues (p<0.05). Additionally, in both the tissue group, the expression of DR4 was significantly stronger than the DR5 (p<0.05). To assess the relationship between DR4 and DR5 expression, differentiation, and tumor staging of PC, the result reveals that the expression of DR4 and DR5 was significantly higher in stage I tumors than the stage II, III, IV tumors (p<0.05). In contrast, the expression of DR4 and DR5 was decreased with a decrease in the degree of differentiation of tumors. However, the difference was not statistically significant.
 Conclusion: The membrane expression of TRAIL death receptor DR4 and DR5 is greater in PC than in the adjacent non-cancerous pancreatic tissues. Furthermore, increased membrane expression of TRAIL death receptor DR4 and DR5 in stage I PC and well-differentiated PC may predict the prognosis and feasibility of using TRAIL gene therapy as a treatment option for early PC.

TRNA-derived fragments tRFGlnCTG induced by arterial injury promote vascular smooth muscle cell proliferation

tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) are originated from the specific cleavage of endogenous tRNAs or their precursors and regulate gene expression when the cells are in stressful circumstances. Here, we replicated the rat common carotid artery (CCA) intimal hyperplasia model and investigated the expression of tRFs/tiRNAs in the artery. The normal and the balloon-injured rat CCAs were subjected to small RNA sequencing, and then the differentially expressed tRFs/tiRNAs were identified and analyzed. The expression profiles of tRFs/tiRNAs in the healthy and injured CCAs were remarkably different. tRNAGlnCTG-derived fragments (tRFGlnCTG) were found to be overexpressed with a high abundance in the injured CCA. In in vitro experiments, the synthetic tRFGlnCTG mimetics elevated the proliferation and migration of rat vascular smooth muscle cells (VSMCs). Through bioinformatics analysis and an overexpression experiment, tRFGlnCTG was found to negatively regulate the expression of FAS cell surface death receptor (FAS). This study revealed that tRFGlnCTG is a crucial regulator in promoting VSMC proliferation. The investigation of the roles of tRFs/tiRNAs is of significance for understanding the mechanism, diagnosis, and treatment of intimal hyperplasia.

JDP2 is directly regulated by ATF4 and modulates TRAIL sensitivity by suppressing the ATF4-DR5 axis.

Jun dimerization protein 2 (JDP2) is a bZip-type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4), enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.

RALB GTPase: a critical regulator of DR5 expression and TRAIL sensitivity in KRAS mutant colorectal cancer

RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.

DR3 stimulation of adipose resident ILC2s ameliorates type 2 diabetes mellitus

Disturbances in glucose homeostasis and low-grade chronic inflammation culminate into metabolic syndrome that increase the risk for the development of type 2 diabetes mellitus (T2DM). The recently discovered group 2 innate lymphoid cells (ILC2s) are capable of secreting copious amounts of type 2 cytokines to modulate metabolic homeostasis in adipose tissue. In this study, we have established that expression of Death Receptor 3 (DR3), a member of the TNF superfamily, on visceral adipose tissue (VAT)-derived murine and peripheral blood human ILC2s is inducible by IL-33. We demonstrate that DR3 engages the canonical and/or non-canonical NF-κB pathways, and thus stimulates naïve and co-stimulates IL-33-activated ILC2s. Importantly, DR3 engagement on ILC2s significantly ameliorates glucose tolerance, protects against insulin-resistance onset and remarkably reverses already established insulin-resistance. Taken together, these results convey the potent role of DR3 as an ILC2 regulator and introduce DR3 agonistic treatment as a novel therapeutic avenue for treating T2DM.

Artemisinin Derivatives Stimulate DR5-Specific TRAIL-Induced Apoptosis by Regulating Wildtype P53.

Simple SummaryThe risk of developing colorectal cancer at a younger age has increased, but current therapies are either risky or limited. We aim to demonstrate that the combination treatment of artemisinin derivatives and TRAIL could be a potential therapy to kill colon cancer cells. We found that artemisinin derivatives increase death receptor production and further sensitize colon cancer cells to TRAIL-induced apoptosis. Furthermore, we explored the role of P53 in response to artemisinin derivatives, which transactivates Death Receptor 5 (DR5) and the cyclin-dependent kinase inhibitor P21. Finally, a 3D tumor spheroid model also confirmed the efficacy of the combination treatment.Artemisinin derivatives, widely known as commercial anti-malaria drugs, may also have huge potential in treating cancer cells. It has been reported that artemisinin derivatives can overcome resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in liver and cervical cancer cells. In our study, we demonstrated that artesunate (ATS) and dihydroartemisinin (DHA) are more efficient in killing colon cancer cells compared to artemisinin (ART). ATS/DHA induces the expression of DR5 in a P53 dependent manner in HCT116 and DLD-1 cells. Both ATS and DHA overcome the resistance to DHER-induced apoptosis in HCT116, mainly through upregulating death receptor 5 (DR5). We also demonstrate that DHA sensitizes HCT116 cells to DHER-induced apoptosis via P53 regulated DR5 expression in P53 knockdown assays. Nevertheless, a lower effect was observed in DLD-1 cells, which has a single Ser241Phe mutation in the P53 DNA binding domain. Thus, the status of P53 could be one of the determinants of TRAIL resistance in some cancer cells. Finally, the combination treatment of DHA and the TRAIL variant DHER increases cell death in 3D colon cancer spheroid models, which shows its potential as a novel therapy.