Decreased Expression Of c-Myc Research Articles

WDR20 prevents hepatocellular carcinoma senescence by orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc

The dysfunction of the ubiquitin-proteasome system (UPS) facilitates the malignant progression of hepatocellular carcinoma (HCC). While targeting the UPS for HCC therapy has been proposed, identifying effective targets has been challenging. In this study, we conducted a focused screen of siRNA libraries targeting UPS-related WD40 repeat (WDR) proteins and found that silencing WDR20, a deubiquitinating enzyme activating factor, selectively inhibited the proliferation of HCC cells without affecting normal hepatocytes. Moreover, the downregulation of WDR20 expression induced HCC cellular senescence and suppressed tumor progression in xenograft, sleeping beauty transposon/transposase, and hydrodynamic tail vein injection-induced HCC models, and Alb-Cre+/MYC+ HCC transgenic mouse models. Mechanistically, we found that WDR20 silencing disturbed the protein stability of c-Myc, orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc, thereby promoting the transcriptional activation of CDKN1A. Further investigation revealed a positive coexpression of WDR20 and c-Myc in a tissue microarray with 88 HCC clinical samples. By employing three patient-derived organoids from individuals with HCC, we have validated the decrease in c-Myc expression and the significant induction of senescence and growth inhibition following silencing of WDR20. This study not only uncovers the biological function of WDR20 and elucidates the molecular mechanism underlying its negative regulation of HCC cellular senescence but also highlight the potential of WDR20 as a promising target for HCC therapy.

Xanthine negatively regulates c-MYC through the PI3K/AKT signaling pathway and inhibits the proliferation, invasion, and migration of breast cancer cells.

Breast cancer is a prevalent and aggressive malignancy associated with elevated mortality rates worldwide. Dysregulation of the c-MYC oncogene and aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway are common features in breast cancer progression, rendering them attractive therapeutic targets. Here, we assessed the effects of the plant derivative, xanthine, on breast cancer cells and explored the molecular mechanisms underlying its activity. Breast cancer cell lines were treated with xanthine, followed by assessment of c-MYC expression levels. Cell proliferation, invasion, and migration were analyzed to assess the effects of xanthine treatment on breast cancer cell behavior. Xanthine treatment induced a decrease in c-MYC expression, resulting in significant inhibition of breast cancer cell proliferation, invasion, and migration. Mechanistic investigations revealed that these effects were mediated by suppression of the PI3K/AKT signaling pathway. Xanthine shows great potential for breast cancer treatment by targeting c-MYC via the PI3K/AKT signaling pathway. Our findings indicate that development of xanthine as a novel treatment option for breast cancer, which acts by influencing key oncogenic pathways involved in tumor progression, may be warranted.

Abstract 6799: The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with rapidly acquired chemotherapy resistance. While the inflamed SCLC subtype is associated with a significantly improved response to immunotherapy, the non-inflamed SCLC subtypes, encompassing most SCLC patients, experience only modest survival gains with chemo-immunotherapy. Wnt signaling pathway activation is associated with resistance to chemotherapy and an immune checkpoint inhibitor in several tumors. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers, but the efficacy of TNIK inhibition in SCLC has not previously been researched. We previously demonstrated that single agent TNIK inhibitors are effective in a subset of SCLC. Here, we hypothesize that targeting TNIK will show an anti-tumor effect based on specific biomarkers and also modify the tumor immune environment in SCLC. Methods: We evaluated susceptibility to a small molecule TNIK inhibitor, NCB-0846, in 29 human-derived SCLC cell lines using 96-hour proliferation assays. To investigate efficacy biomarkers, we correlated NCB-0846 IC50 values with proteomic expression (profiled using reverse phase protein array). cMYC and FOXK1 expression was reduced by siRNA. Chemokine concentrations were determined by ELISA. Results: SCLC cell lines with high cMYC levels, were more sensitive to the TNIK inhibitor, NCB-0846 (F.C.=-2.07, p=0.01), while SCLC cell lines with high expression of TTF-1 were more resistant (F.C.=2.48, p<0.01). Knockdown of cMYC conferred resistance to NCB-0846 in cMYC-high cells, while NCB-0846 treatment resulted in decreased cMYC expression in a concentration-dependent manner. Additionally, NCB-0846 treatment decreased the immunosuppressive chemokine CCL2 levels in the supernatant of human-derived SCLC cell lines and GEMM-derived cell lines. The expression of FOXK1, a transcription factor of CCL2, was also reduced in cell lines treated with NCB-0846. Conclusions: These findings show that TNIK inhibition is more effective in cMYC-high SCLC, acting through downregulating cMYC levels. TNIK inhibition also decreases the production of CCL2, an immunosuppressive chemokine implicated in resistance to anti-PD-L1 immunotherapy. Together, these findings highlight the promising potential of TNIK inhibitors in cMYC-high SCLC. Additionally, suppressing CCL2 by TNIK inhibition supports the rationale for combining the TNIK inhibitor and an anti-PD-L1 antibody in the non-inflamed SCLC subtypes. Citation Format: Azusa Tanimoto, Kavya Ramkumar, Allison Stewart, Bingnan Zhang, Robert Cardnell, Shen Li, Qi Wang, Jing Wang, Carl Gay, Lauren Averett Byers. The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6799.

Abstract 5685: MiR-345-5p attenuates pancreatic cancer metastasis through KLK7/E-Cad/β-Catenin/Rap1 axis

Abstract Background: Pancreatic cancer (PC) is a highly lethal malignancy with a 5-year survival rate of 10.8%, primarily attributed to early invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) plays a crucial role in the metastatic progression of PC. Existing therapies, such as Gemcitabine, Abraxane, and FOLFIRINOX, have limited efficacy, emphasizing the need for identifying molecular targets to enhance PC patient survival. Recent advancements in miRNA therapies show promise, but a detailed understanding of miRNAs regulating PC growth and metastasis remains elusive. This study aims to uncover the role of miR-345-5p in regulating PC metastasis. Methods: A miRNA microarray analysis of KC mouse data identified miR-345-5P as one of the significantly down-regulated miRNA, targeting multiple metastasis-related genes. Expression of miR-345-5p in PC was assessed using published databases, validated through qPCR and ISH in PC cell lines, serum samples, and patient tissue. In vitro overexpression and in vivo assays elucidated the functional roles of miR-345-5p in PC metastasis. A computational approach and dual-luciferase assays were used to identify the target of miR-345-5p. Western blots, immunohistochemistry, and immunofluorescence were utilized to decipher miR-345-5p mediated molecular mechanisms. Results: MiR-345-5p expression was downregulated in PC tissue, serum, and cell lines compared to non-tumor tissues. Restoring miR-345-5p reduced PC cell proliferation, migration, and invasion in vitro and in vivo. KLK 7 was confirmed as a direct target, and functional experiments revealed miR-345-5p's acting as a tumor-suppressor by inhibiting the E-Cad/B-catenin/Rap1 axis signaling pathway. MiR-345-5p overexpression stabilized the E-Cadherin-β-catenin complex, leading to reduced β-catenin accumulation in the nucleus and thus leading to declined transcription of crucial genes (C-myc, cyclin D1 & MMP7) responsible for PC progression. Given the pivotal role of the c-myc gene in metabolism, we performed a metabolomic analysis. The findings unveiled significant metabolic rewiring, marked by reductions in glucose and amino acid metabolism, attributable to decreased C-myc expression due to restoration of miR-345 in PC cells. Conclusion: MiR-345-5p overexpression negatively regulates PC proliferation and EMT by directly targeting KLK7/E-Cad/β-catenin/Rap1 axis. Restoring miR-345-5p with standard care therapies could be a promising strategy for improving survival in metastatic PC patients. Citation Format: Nagabhishek Sirpu Natesh, Susheel Kumar Nethi, Brianna M White, Jussuf T. Kaifi, Surinder K. Batra, Surya Mallapragada, Satyanarayana Rachagani. MiR-345-5p attenuates pancreatic cancer metastasis through KLK7/E-Cad/β-Catenin/Rap1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5685.

PTTG1 induces pancreatic cancer cell proliferation and promotes aerobic glycolysis by regulating c-myc.

This study aimed to clarify the role of pituitary tumor-transforming gene 1 (PTTG1) in proliferation, migration, invasion, and aerobic glycolysis of pancreatic cancer cells, and evaluate the potential of PTTG1 as a therapeutic target. PTTG1 expression in pancreatic cancers was analyzed using the GEPIA databank. In the Panc1 cell with the PTTG1 knockdown or Mia-PaCa2 cells with PTTG1 overexpression, the cell proliferation was evaluated using cell viability curves and colony formation, and wound heal assay and transwell assay were performed to evaluate the migration and invasion, respectively. Furthermore, a western blot was performed to evaluate the expressions of PTTG1, proliferating cell nuclear antigen, E-cadherin, N-cadherin, and c-myc. Meanwhile, the glucose uptake, extracellular acidification rates (ECAR), and oxygen consumption rates (OCR) were analyzed. Our results showed that PTTG1 expression is upregulated in pancreatic cancer, which promoted cell proliferation. Low PTTG1 contributed to higher disease-free survival and overall survival. In Panc1 cell, PTTG1 knockdown resulted in reduced cell viability and colony formation. The migration and invasion abilities of the cells were also reduced in Panc1 with PTTG1 knockdown. Correspondingly, PTTG1 knockdown decreased c-myc expression, glucose uptake, ECAR, and OCR in Panc1 cells. In Mia-PaCa2 cells, PTTG1 overexpression promoted cell proliferation, aerobic glycolysis, and translocation of β-catenin to the nucleus by regulating c-myc. In conclusion, PTTG1 induces proliferation, migration, and invasion, and promotes aerobic glycolysis in pancreatic cancer cells via regulating c-myc, demonstrating the potential of PTTG1 as a therapeutic target.

Lipid-Mediated Modulation of DNA Damage Signaling As a Prognostic and Therapeutic Strategy Against Multiple Myeloma

Multiple myeloma (MM), characterized by the accumulation of tumor plasma cells in the bone marrow, is the second most common hematologic cancer. Occurrence of chemoresistance invariably leads patients to relapse and results in loss of clinical control over the disease. Cancer cells show a deregulation of different pathways, including lipid metabolism alterations and high genomic instability. Yet, no functional relationship between these two features has been identified. We have shown in a previous work that the metabolism of lipids impacts in how DNA damage is detected, signaled and repaired (Ovejero et al 2023 EMBO J). We reported that breaks in the DNA trigger the birth of lipid droplets (LD), a central lipid-storage organelle, and that this event is key for cells to tolerate this specific DNA damage. Given that MM cells are particularly dependent on DNA repair pathways for survival, we postulated that new therapeutic perspectives would emerge by targeting the formation of LD. Analysis of the LD formation gene expression profile of a cohort of newly diagnosed MM patients (UAMS-TT2 MM cohort, n = 345) allowed the establishment of a LD score comprising 13 genes associated with a significant prognostic value after multiple testing correction. The LD score gene expression-based risk score enabled the identification of MM patients with a poor outcome. The prognostic value of the LD score was validated in two other independent cohorts of newly diagnosed patients (HM cohort, n = 206; UAMS-TT3 MM cohort, n = 158) and in a cohort of patients at relapse treated with anti-CD38 MoAb (Mtp-Dara, n = 49). We subsequently studied the presence of LD in primary MM cells obtained from 34 patients. Importantly, we observed a significant positive correlation between the percentage of LD-positive cells and the value of the LD score (p = 0,02). This prompted us to investigate the therapeutic interest of interfering with LD formation to target MM cells. To this end, we treated 26 Human Myeloma Cell Lines (HMCLs) with drugs selected on an educated-guess basis and identified positive hits. These results were validated employing a shRNA strategy in selected HCMLs. Flow cytometry analyses revealed that this regime triggered a cell cycle halt, where cells accumulated in the G 1 phase at the expense of S and G 2. This was accompanied by a decrease in c-MYC expression, as well as an induction of cell proliferation arrest markers such as the G 1-checkpoint CDK inhibitors p21 and p27. Mechanistically, we observed a significant increase in Chk1 and Chk2 phosphorylations, two downstream events of DNA damage signaling despite that basal DNA damage levels remained unchanged. Together, these data convey the notion that limiting LD formation in MM cell lines boosts the detection of endogenously present DNA damage, thus enforcing a more robust DNA damage signaling which culminates in a drastic loss of proliferative capacity and viability. Of major importance, we validated the therapeutic interest of this strategy in primary MM cells from patients (n = 19). A significant higher toxicity in MM cells was found compared to normal cells from the microenvironment (tumoral: ****, p < 0.0001; normal: ***, p = 0,0006). Moreover, we tested the therapeutic interest of combining this approach with conventional treatments used in MM. Interestingly, we identified a synergistic effect when combined with melphalan, a DNA-alkylating agent used in MM treatment. This synergy was observed in HCMLs and in primary MM cells of patients (n = 7). Furthermore, significant synergistic effects were found in HMCLs by combining with the proteasome inhibitors bortezomib and carfilzomib. Altogether, these data unveil both the LD score and LD-bearing cell quantification as new prognostic factors in MM, and open new therapeutic perspectives to improve the treatment of MM patients by targeting LD metabolism.

In Silico, In Vitro, and In Vivo Investigations on Adapalene as Repurposed Third Generation Retinoid against Multiple Myeloma and Leukemia.

The majority of hematopoietic cancers in adults are incurable and exhibit unpredictable remitting-relapsing patterns in response to various therapies. The proto-oncogene c-MYC has been associated with tumorigenesis, especially in hematological neoplasms. Therefore, targeting c-MYC is crucial to find effective, novel treatments for blood malignancies. To date, there are no clinically approved c-MYC inhibitors. In this study, we virtually screened 1578 Food and Drug Administration (FDA)-approved drugs from the ZINC15 database against c-MYC. The top 117 compounds from PyRx-based screening with the best binding affinities to c-MYC were subjected to molecular docking studies with AutoDock 4.2.6. Retinoids consist of synthetic and natural vitamin A derivatives. All-trans-retinoic acid (ATRA) were highly effective in hematological malignancies. In this study, adapalene, a third-generation retinoid usually used to treat acne vulgaris, was selected as a potent c-MYC inhibitor as it robustly bound to c-MYC with a lowest binding energy (LBE) of -7.27 kcal/mol, a predicted inhibition constant (pKi) of 4.69 µM, and a dissociation constant (Kd value) of 3.05 µM. Thus, we examined its impact on multiple myeloma (MM) cells in vitro and evaluated its efficiency in vivo using a xenograft tumor zebrafish model. We demonstrated that adapalene exerted substantial cytotoxicity against a panel of nine MM and two leukemic cell lines, with AMO1 cells being the most susceptible one (IC50 = 1.76 ± 0.39 µM) and, hence, the focus of this work. Adapalene (0.5 × IC50, 1 × IC50, 2 × IC50) decreased c-MYC expression and transcriptional activity in AMO1 cells in a dose-dependent manner. An examination of the cell cycle revealed that adapalene halted the cells in the G2/M phase and increased the portion of cells in the sub-G0/G1 phase after 48 and 72 h, indicating that cells failed to initiate mitosis, and consequently, cell death was triggered. Adapalene also increased the number of p-H3(Ser10) positive AMO1 cells, which is a further proof of its ability to prevent mitotic exit. Confocal imaging demonstrated that adapalene destroyed the tubulin network of U2OS cells stably transfected with a cDNA coding for α-tubulin-GFP, refraining the migration of malignant cells. Furthermore, adapalene induced DNA damage in AMO1 cells. It also induced apoptosis and autophagy, as demonstrated by flow cytometry and western blotting. Finally, adapalene impeded tumor growth in a xenograft tumor zebrafish model. In summary, the discovery of the vitamin A derivative adapalene as a c-MYC inhibitor reveals its potential as an avant-garde treatment for MM.

Calcineurin-mediated dephosphorylation enhances the stability and transactivation of c-Myc

c-Myc, a transcription factor, induces cell proliferation and is often aberrantly or highly expressed in cancers. However, molecular mechanisms underlying this aberrantly high expression remain unclear. Here, we found that intracellular Ca2+ concentration regulates c-Myc oncoprotein stability. We identified that calcineurin, a Ca2+-dependent protein phosphatase, is a positive regulator of c-Myc expression. Calcineurin depletion suppresses c-Myc targeted gene expression and c-Myc degradation. Calcineurin directly dephosphorylates Thr58 and Ser62 in c-Myc, which inhibit binding to the ubiquitin ligase Fbxw7. Mutations within the autoinhibitory domain of calcineurin, most frequently observed in cancer, may increase phosphatase activity, increasing c-Myc transcriptional activity in turn. Notably, calcineurin inhibition with FK506 decreased c-Myc expression with enhanced Thr58 and Ser62 phosphorylation in a mouse xenograft model. Thus, calcineurin can stabilize c-Myc, promoting tumor progression. Therefore, we propose that Ca2+ signaling dysfunction affects cancer-cell proliferation via increased c-Myc stability and that calcineurin inhibition could be a new therapeutic target of c-Myc-overexpressing cancers.

Overexpression of c-MYC Promoter Binding Protein-1 Enhances Proliferation and Glucose Metabolism of Melanoma Cells Lines.

c-MYC promoter binding protein (MBP-1) is a product of alternatively translated mRNA encoding alpha-enolase (ENO1). In contrast to ENO1, MBP-1 possesses no enzymatic activity but acts as a transcriptional repressor of c-MYC. Ectopic over-expression of MBP-1 in tumor cells was shown to reduce cell proliferation and tumorigenicity, thus making it an attractive target for anticancer strategies. This study aimed to assess the effects of MBP-1 over-expression on human cutaneous melanoma cell lines. We overexpressed the full-length MBP-1 or its C-terminal truncated variant (MBP-1ΔC), in two human melanoma cell lines (A375, WM9) and assessed their subcellular localization. qPCR was then used to quantitate c-MYC transcription. Further, 5-ethynyl-2'-deoxyuridine incorporation assay was used to measure cell proliferation and a lactate assay was performed to measure the glycolysis rate of cells in normoxia and hypoxia. Finally, an in vitro wound-healing assay was performed to evaluate cell migration. The overexpressed MBP-1 variants predominantly localized in the cytoplasm and barely decreased c-MYC expression. Unexpectedly, the proliferation rate of MBP-1- transduced cells increased in comparison to controls, as did the rate of glucose metabolism in hypoxia. Furthermore, over-expression of MBP-1, but not MBP-1ΔC, led to a substantial decrease in the cell migration capacity of metastatic WM9 cells but not A375 cells from the primary tumor lesion. Misslocalization of over-expressed MBP-1 in the cytoplasm of two melanoma cell lines resulted in an unexpected tumor promoting activity by increasing cell proliferation and glycolysis rates in hypoxia.

DNA-PKcs as an upstream mediator of OCT4-induced MYC activation in small cell lung cancer

Small cell lung cancer (SCLC) is a neuroendocrine tumor noted for the rapid development of both metastases and resistance to chemotherapy. High mutation burden, ubiquitous loss of TP53 and RB1, and a mutually exclusive amplification of MYC gene family members contribute to genomic instability and make the development of new targeted agents a challenge. Previously, we reported a novel OCT4-induced MYC transcriptional activation pathway involving c-MYC, pOCT4S111, and MAPKAPK2 in progressive neuroblastoma, also a neuroendocrine tumor. Using tumor microarray analysis of clinical samples and preclinical models, we now report a correlation in expression between these proteins in SCLC. In correlating c-MYC protein expression with genomic amplification, we determined that some SCLC cell lines exhibited high c-MYC without genomic amplification, implying amplification-independent MYC activation. We then confirmed direct interaction between OCT4 and DNA-PKcs and identified specific OCT4 and DNA-PKcs binding sites. Knock-down of both POU5F1 (encoding OCT4) and PRKDC (encoding DNA-PKcs) resulted in decreased c-MYC expression. Further, we confirmed binding of OCT4 to the promoter/enhancer region of MYC. Together, these data establish the presence of a DNA-PKcs/OCT4/c-MYC pathway in SCLCs. We then disruptively targeted this pathway and demonstrated anticancer activity in SCLC cell lines and xenografts using both DNA-PKcs inhibitors and a protein-protein interaction inhibitor of DNA-PKcs and OCT4. In conclusion, we demonstrate here that DNA-PKcs can mediate high c-MYC expression in SCLCs, and that this pathway may represent a new therapeutic target for SCLCs with high c-MYC expression.

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.

Abstract P6-11-16: Withaferin A induces metabolic crisis in breast cancer cell lines via decreasing c-myc expression: potential therapeutic implication

Abstract Background: Breast cancer is the most diagnosed cancer and the leading cause of mortality among women in India and worldwide. Reprogrammed glucose metabolism is considered as the hallmark of the cancer with immense therapeutic relevance. Withaferin A (WA), a phytocompound isolated from the plant Withania somnifera, commonly known as Ashwagandha has the remarkable anticancer role. However, the mechanism of action of WA in breast cancer metabolism is still unelucidated. Breast cancer cells have metabolic vulnerability and high glucose dependency. Thus targeting glycolysis could be the better therapeutic approach. Chemotherapy and radiotherapy have the side effects, to overcome this natural products can be used to treat cancer. The potential of natural compounds in targeting metabolic vulnerabilities of cancer and highlighting prospective therapeutic benefits that can be determined by improving our understanding of this field. Aims and Objective: To explore the therapeutic effect of WA in breast cancer cell lines and to identify the role of WA in targeting Warburg effect via c-myc. Cancer cells exhibit upregulated Warburg effect, to fulfill the bioenergetics and biosynthetic demands of the rapidly growing cancer cells. Thus targeting the Warburg effect could be the better therapeutic approach. Materials and methods: Breast cancer cell lines (MBA-MB-231, MBA-MB-468, MBA-MB-453 and MCF-7) were procured from NCCS Pune and maintained in DMEM media supplemented with 10% FBS. SRB dye was used for cell viability as it binds to the protein of the cells. Further Colony formation assay (using crystal violet dye) was done to evaluate the effect of WA on colony formation. Metabolic assays \ (lactate production, glucose uptake and ATP generation) were performed using kits (Eton Bioscience). Transient silencing using si-RNA of c-myc was done with lipofectamine 3000. Silencing of c-myc was done for Warburg effect and protein expression. RNA was isolated from breast cancer cells using Qaigen kit and RT-PCR was performed to evaluate the glycolytic gene expression before and after the treatment of the WA in breast cancer cell lines. To check the expression of glycolytic genes at protein level we had done the western blot. Protein isolation was done in RIPA lysis buffer and western blot was performed using primary antibodies of GLUT1, HK2, PKM2, c-myc. Statistical analysis was analyzed in graph pad prism. Breast cancer patient METABRIC data was analyzed and pathway deregulation score was calculated from Pathifier algorithm. Results: Withaferin A decreased the glucose uptake, lactate production and ATP production in different breast cancer cell lines. Further, WA induced suppression of key glycolytic enzymes via c-myc, decreased cell proliferation, biomass and colony formation ability of the breast cancer cells. Silencing of c-myc gene also showed the similar results to WA such as decrease in Warburg effect and reduction in glycolytic proteins expression. Through the LC-MS analysis WA decreases the key glycolytic metabolites and other pathway metabolites, induce metabolic catastrophe in breast cancer cells. Clinical relevance of our experiments was validated in dataset of ~2000 breast tumors (METABRIC) using Pathifier algorithm wherein we calculated deregulation score of glycolysis pathways in each of the tumor and normal sample. Importantly, higher deregulation of glycolysis was observed in breast tumor compared to normal tissues and found to be associated with poor prognosis. Conclusion: Our results highlight the anti- carcinogenic effect of Withaferin A in modulating breast cancer metabolism and the clinical significance of glycolysis in general. WA decreases the glucose metabolism and its flux through key metabolic pathways associated with the glycolytic intermediates. Therefore it could be argued that therapeutic targeting of breast cancer metabolism by WA may improve clinical outcome. Citation Format: Asifa Khan, Shumaila Siddiqui, Sheersh Massey, Daman Saluja, Syed Akhtar Husain, Mohammad Askandar Iqbal. Withaferin A induces metabolic crisis in breast cancer cell lines via decreasing c-myc expression: potential therapeutic implication [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-16.

AXL upregulates c‑Myc expression through AKT and ERK signaling pathways in breast cancers.

Breast cancer (BC) is common worldwide. c-Myc and AXL are both overexpressed in BC, promoting its progression. The present study aimed to investigate the role of AXL in c-Myc expression in BC. Overexpression of AXL increased c-Myc expression while knockdown of AXL decreased c-Myc expression as determined by western blot analysis. Pharmaceutical inhibition of AXL also suppressed c-Myc expression. AKT and ERK inhibitor LY294002 and U0126 suppressed c-Myc expression, respectively. AXL overexpression which activates AKT and ERK signaling, upregulates c-Myc expression, while kinase-dead AXL which cannot activate AKT and ERK signaling, does not upregulate c-Myc expression, emphasizing the important role of these two signaling pathways in c-Myc upregulation. Finally, expression data of BC tissues from The Cancer Proteome Atlas displayed an association between AXL and c-Myc. Taken together, the present study revealed that AXL upregulates c-Myc expression through AKT and ERK signaling pathways in BC.

Epstein-Barr Virus Synergizes with BRD7 to Conquer c-Myc-Mediated Viral Latency Maintenance via Chromatin Remodeling.

Epstein-Barr virus (EBV) switches between latent and lytic phases in hosts, which is important in the development of related diseases. However, the underlying mechanism of controlling the viral biphasic life cycle and how EBV mediates this regulation remain largely unknown. This study identified bromodomain-containing protein 7 (BRD7) as a crucial host protein in EBV latent infection. Based on the chromatin immunoprecipitation (ChIP) sequencing of endogenous BRD7 in Burkitt lymphoma cells, we found that EBV drove BRD7 to regulate cellular and viral genomic loci, including the transcriptional activation of c-Myc, a recently reported regulator of EBV latency. Additionally, EBV-mediated BRD7 signals were enriched around the FUSE (far-upstream sequence element) site in chromosome 8 and the enhancer LOC108348026 in the lgH locus, which might activate the c-Myc alleles. Mechanically, EBV-encoded nuclear antigen 1 (EBNA1) bound to BRD7 and colocalized at promoter regions of the related genes, thus serving as cofactors for the maintenance of viral latency. Moreover, the disruption of BRD7 decreased the c-Myc expression, induced the BZLF1 expression, and reactivated the lytic cycle. Our findings reveal the unique role of BRD7 to synergize with EBV in maintaining the viral latency state via chromatin remodeling. This study paves the way for understanding the new molecular mechanism of EBV-induced chromatin remodeling and latent-lytic switch, providing novel therapeutic candidate targets for EBV persistent infection. IMPORTANCE When establishing persistent infection in most human hosts, EBV is usually latent. How the viral latency is maintained in cells remains largely unknown. c-Myc was recently reported to act as a controller of the lytic switch, while whether and how EBV regulates it remain to be explored. Here, we identified that BRD7 is involved in controlling EBV latency. We found that EBV-mediated BRD7 was enriched in both the normal promoter regions and the translocation alleles of c-Myc, and disruption of BRD7 decreased c-Myc expression to reactivate the lytic cycle. We also demonstrated that EBV-encoded EBNA1 bound to and regulated BRD7. Therefore, we reveal a novel mechanism by which EBV can regulate its infection state by coordinating with host BRD7 to target c-Myc. Our findings will help future therapeutic intervention strategies for EBV infection and pathogenesis.

1,25(OH)2 D3 induced vitamin D receptor signaling negatively regulates endoplasmic reticulum-associated degradation (ERAD) and androgen receptor signaling in human prostate cancer cells

Steroid hormone signaling is critical in the tumor progression and the regulation of physiological mechanisms such as endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) in prostate cancer. 1,25(OH)2 D3 is an active metabolite of vitamin D classified as a steroid hormone. It exhibits anti-tumor effects, including angiogenesis and suppression of cell cycle progression. Moreover, progressively reducing expression levels of vitamin D receptor (VDR) are observed in many cancer types, including the prostate. In the present study, we investigated the molecular action of 1,25(OH)2 D3 on ERAD, UPR and androgenic signaling. We found that 1,25(OH)2 D3 negatively regulated the expression level of ERAD components and divergently controlled the inositol-requiring enzyme 1⍺ (IRE1⍺) and protein kinase RNA-like ER kinase (PERK) branches of UPR in LNCaP human prostate cancer cells. Also, similar results were obtained with another human prostate cancer cell line, 22Rv1. More strikingly, we found that androgenic signaling is negatively regulated by VDR signaling. Also, molecular docking supported the inhibitory effect of 1,25(OH)2 D3 on AR signaling. Moreover, we found VDR signaling suppressed tumor progression by decreasing c-Myc expression and reducing the epithelial-mesenchymal transition (EMT). Additionally, 1,25(OH)2 D3 treatment significantly inhibited the 3D-tumor formation of LNCaP cells. Our results suggest that further molecular characterization of the action of VDR signaling in other cancer types such as estrogenic signal in breast cancer will provide important contributions to a better understanding of the roles of steroid hormone receptors in carcinogenesis processes.

LPLUNC1 reduces glycolysis in nasopharyngeal carcinoma cells through the PHB1-p53/c-Myc axis.

Cancer cells prefer glycolysis to support their proliferation. Our previous studies have shown that the long palate, lung, and nasal epithelial cell clone 1 (LPLUNC1) can upregulate prohibitin 1 (PHB1) expression to inhibit the proliferation of nasopharyngeal carcinoma (NPC) cells. Given that PHB1 is an important regulator of cell energy metabolism, we explored whether and how LPLUNC1 regulated glucose glycolysis in NPC cells. LPLUNC1 or PHB1 overexpression decreased glycolysis and increased oxidative phosphorylation (OXPHOS)-related protein expression in NPC cells, promoting phosphorylated PHB1 nuclear translocation through 14-3-3σ. LPLUNC1 overexpression also increased p53 but decreased c-Myc expression in NPC cells, which were crucial for the decrease in glycolysis and increase in OXPHOS-related protein expression induced by LPLUNC1 overexpression. Finally, we found that treatment with all-trans retinoic acid (ATRA) reduced the viability and clonogenicity of NPC cells, decreased glycolysis, and increased OXPHOS-related protein expression by enhancing LPLUNC1 expression in NPC cells. Therefore, the LPLUNC1-PHB1-p53/c-Myc axis decreased glycolysis in NPC cells, and ATRA upregulated LPLUNC1 expression, ATRA maybe a promising drug for the treatment of NPC.

CSIG-32. ABL1 AND 2 DRIVE MEDULLOBLASTOMA PROLIFERATION AND ADHESION IN LEPTOMENINGEAL DISSEMINATION

Abstract INTRODUCTION The molecular mechanisms that drive leptomeningeal dissemination (LD) of medulloblastoma (MB) are largely unknown. The Abelson (ABL) family kinases, originally identified as oncogenes in leukemia, play an important role in lung cancer cell migration, invasion, cell adhesion, and chemotherapy resistance. The objective of this work was to elucidate the role of ABL kinases in MB LD. METHODS ABL1 and ABL2 mRNA expression of patient specimens was analyzed. shRNA knockdowns of ABL1/2 and pharmacologic inhibition of ABL1/2 were used for in vitro and in vivo analyses of MB LD. RESULTS ABL1 and ABL2 levels were significantly higher in MB patients with LD than without (p=2.6x10-10 and 7.2x10-7, respectively) and overall survival was significantly reduced in patients with high ABL1 and 2 expression (p=0.019 and 0.00027, respectively). In vitro pharmacologic inhibition and genetic knockdown of ABL1 and ABL2 in Group 3/4 MB resulted in statistically significant cell death, decreased adhesion to vitronectin (a key leptomeningeal extracellular matrix [ECM] protein), decreased migration through ECM extract, and decreased c-myc expression in multiple MB cell types. In mouse models of MB LD, ABL1/2 knockdown significantly decreased LD as determined by bioluminescence and improved overall survival (p=0.0044). Pharmacologic inhibition of ABL1/2 using nilotinib resulted in improved median overall survival (64 vs. 76.5 days). CONCLUSIONS ABL1 and ABL2 are preferentially expressed in patients with MB LD at diagnosis and are associated with poor survival. Both genetic inactivation and pharmacologic inhibition of ABL1 and 2 decreased MB LD in vitro and in vivo, likely via reduction of c-myc signaling. This preliminary work suggests a key role for ABL1/2 in driving MB LD.

Abstract 5367: DNA-PKcs inhibitors impair OCT4-mediated MYC transcriptional activation in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a disease found almost exclusively in smokers and is a highly aggressive cancer with incredibly poor outcomes. Initially, most patients diagnosed with SCLC respond to platinum + etoposide (PE)-based therapy, but relapse occurs in nearly all patients and is fatal. SCLC is a cancer with a heavy mutational burden, and loss-of-function mutations of the tumor suppressor genes RB1 and TP53 are nearly ubiquitous in SCLC tumors. Amplification and overexpression of the MYC of oncogene occurs in a subset of SCLC tumors that are associated with tumor progression, treatment resistance, and poor outcomes, therefore we pursued the inhibition of the MYC activation. The purpose of this study is to inhibit a novel pathway of MYC transcriptional activation via DNA-PKcs-mediated phosphorylation of OCT4 at its Ser93 residue in SCLC, a MYC transcriptional activation pathway that we discovered. We identified OCT4 being a transcription factor that primarily induce MYC activation. Subsequently, Serine 93 residue of OCT4, a new OCT-4 binding site, was phosphorylated by DNA-PKcs that was a critical step for OCT4-induced MYC activation. In a panel of 19 small cell lung cancer, DNA-PKcs, phosphorylation of OCT4 at Ser93, and c-MYC were positively correlated. We confirmed the binding of DNA-PKcs to OCT4 and identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by DNA-PKcs, and DNA-PKcs or OCT4 knockdown decreased c-MYC expression. DNA-PKcs inhibitors also reduced phosphorylation of OCT4 at Ser93 leading to decreases in c-MYC and showed cytotoxic effects via inducing apoptosis by DNA-PKcs inhibitors. DNA-PKcs inhibitors augments antitumor activity of Bcl-2 inhibitors cell lines and xenograft models of small cell lung cancer. In conclusion, report here that DNA-PKcs-mediated phosphorylation of OCT4 at Ser93 is a novel mechanism for activating the MYC oncogene in SCLC, and that DNA-PKcs is a viable target to decrease c-MYC expression in these highly aggressive tumors. Citation Format: Inhyoung Yang, Ismail Mohiuddin, Sung-Jen Wei, Martinez Gloria, Min H. Kang. DNA-PKcs inhibitors impair OCT4-mediated MYC transcriptional activation in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5367.

1,25-Dihydroxyvitamin D3 suppresses CD4+ T-cell effector functionality by inhibition of glycolysis.

In CD4+ T helper cells, the active form of vitamin D3 , 1,25-dihydroxyvitamin D3 (1,25D) suppresses production of inflammatory cytokines, including interferon-gamma (IFN-γ), but the mechanisms for this are not yet fully defined. In innate immune cells, response to 1,25D has been linked to metabolic reprogramming. It is unclear whether 1,25D has similar effects on CD4+ T cells, although it is known that antigen stimulation of these cells promotes an anabolic metabolic phenotype, characterized by high rates of aerobic glycolysis to support clonal expansion and effector cytokine expression. Here, we performed in-depth analysis of metabolic capacity and pathway usage, employing extracellular flux and stable isotope-based tracing approaches, in CD4+ T cells treated with 1,25D. We report that 1,25D significantly decreases rates of aerobic glycolysis in activated CD4+ T cells, whilst exerting a lesser effect on mitochondrial glucose oxidation. This is associated with transcriptional repression of Myc, but not repression of mTOR activity under these conditions. Consistent with the modest effect of 1,25D on mitochondrial activity, it also did not impact CD4+ T-cell mitochondrial mass or membrane potential. Finally, we demonstrate that inhibition of aerobic glycolysis by 1,25D substantially contributes to its immune-regulatory capacity in CD4+ T cells, since the suppression of IFN-γ expression was significantly blunted in the absence of aerobic glycolysis. 1,25-Dihydroxyvitamin D3 (1,25D) suppresses the production of inflammatory cytokines such as interferon-gamma (IFN-γ) by CD4+ T cells, but the underpinning mechanisms are not yet fully defined. Here, we identify that 1,25D inhibits aerobic glycolysis in activated CD4+ T cells, associated with decreased c-Myc expression. This mechanism appears to substantially contribute to the suppression of IFN-γ by 1,25D, since this is significantly blunted in the absence of aerobic glycolysis.

Exosome-Mediated miR-4792 Transfer Promotes Bladder Cancer Cell Proliferation via Enhanced FOXC1/c-Myc Signaling and Warburg Effect.

Bladder cancer is the second-most common malignancy in the urogenital system and the most common in men. However, our understanding of the driving mechanisms of bladder cancer remains incomplete. The forkhead box (FOX) family of transcription factors is implicated in urogenital development and bladder malignancies. Many exosomal microRNAs have been identified as regulators and mediators of the expression of FOX, including the expression of FOXC1. miR-4792 has been known as a tumor miRNA suppressor. However, the function of miR-4792/FOXC1 signaling in bladder cancer development remains unknown. Here, we studied the role of miR-4792/FOXC1 signaling in bladder cancer by using multiple bladder cancer cell lines and bladder cancer mouse models through in vitro and in vivo approaches. We showed that FOXC1 is highly expressed in multiple bladder cancer cell lines and bladder tumor tissues. The knockdown of FOXC1 expression in bladder cancer cell lines decreases c-Myc expression levels, retards cell growth, and reduces aerobic glycolysis (also known as the Warburg effect) and lactic acid content. By contrast, the overexpression of FOXC1 elicits the opposite effects. FOXC1-downregulated bladder cancer cells form significantly smaller tumors in vivo. The inhibition of c-Myc reverses the effects of FOXC1 overexpression and leads to reduced cell proliferation, aerobic glycolysis, and lactic acid content. miR-4792 expression is downregulated in bladder tumor tissues. miR-4792 exposure to bladder cancer cells reduces the expression levels of FOXC1 and c-Myc, slows down cell growth, and decreases aerobic glycolysis and lactic acid content. However, the enhanced miR-4792 expression elicits opposite effects. These findings provided the first evidence that the exosome-mediated delivery of miR-4792 could play an important role in bladder cancer development through the downregulation of FOXC1 and c-Myc, which further inhibited aerobic glycolysis and lactic acid content.