N-myc Downstream-regulated Gene 1 Knockdown Research Articles

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Role of KLF4/NDRG1/DRP1 axis in hypoxia-induced pulmonary hypertension

N-myc downstream regulated gene 1 (NDRG1) has recently drawn increasing attention because of its involvement in angiogenesis, cell proliferation, and differentiation. We used in vitro [human pulmonary artery smooth muscle cells (hPASMCs)] and in vivo (rat) models under hypoxic conditions and found a vital role of NDRG1 in reducing apoptosis and increasing proliferation and migration by overexpressing and knocking down NDRG1. We also proved that hypoxia induced the protein expression of dynamin-related protein 1 (DRP1) and stimulated The phosphatidylinositol-3-kinase (PI3K)/ Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathways, and these effects were reversed by NDRG1 knockdown. The relationship between NDRG1 and DRP1 and the PI3K/Akt/mTOR pathway was further evaluated by adding mdivi-1 (DRP1 inhibitor) or LY294002 (PI3K inhibitor). NDRG1 was found to regulate the proliferation, apoptosis, and migration of hypoxia-treated hPASMCs via DRP1 and PI3K/Akt/mTOR signaling pathways. We explored the upstream regulators of NDRG1 using in vivo and in vitro hypoxia models. Hypoxia was found to upregulate and downregulate KLF transcription factor 4 (KLF4) protein expression in the cytoplasm and nucleus, respectively. Further, we showed that KLF4 regulated the proliferation and migration of hypoxia-treated hPASMCs via NDRG1. These results indicated a link between KLF4, NDRG1, and DRP1 for the first time, providing new ideas for treating hypoxic pulmonary hypertension.

NDRG1 facilitates lytic replication of Kaposi's sarcoma-associated herpesvirus by maintaining the stability of the KSHV helicase.

The presumed DNA helicase encoded by ORF44 of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays a crucial role in unwinding viral double-stranded DNA and initiating DNA replication during lytic reactivation. However, the regulatory mechanism of KSHV ORF44 has not been fully elucidated. In a previous study, we identified that N-Myc downstream regulated gene 1 (NDRG1), a host scaffold protein, facilitates viral genome replication by interacting with proliferating cell nuclear antigen (PCNA) and the latent viral protein latency-associated nuclear antigen (LANA) during viral latency. In the present study, we further demonstrated that NDRG1 can interact with KSHV ORF44 during viral lytic replication. We also found that the mRNA and protein levels of NDRG1 were significantly increased by KSHV ORF50-encoded replication and transcription activator (RTA). Remarkably, knockdown of NDRG1 greatly decreased the protein level of ORF44 and impaired viral lytic replication. Interestingly, NDRG1 enhanced the stability of ORF44 and inhibited its ubiquitin-proteasome-mediated degradation by reducing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44. In summary, NDRG1 is a novel binding partner of ORF44 and facilitates viral lytic replication by maintaining the stability of ORF44. This study provides new insight into the mechanisms underlying KSHV lytic replication.

NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity.

N-myc downstream regulated gene-1 (NDRG1) has been identified as a putative metastasis suppressor gene and proved to be a key player in cancer spreading and proliferation in our previous work. However, the effects of NDRG1 on tumor invasion and the mechanisms behind it are rarely understood. Here we provided in silico evidence that NDRG1 plays a crucial role in actin reorganization in colorectal cancer (CRC). Through in vitro experiments, we next observed filopodia formation was altered in NDRG1-modified cell lines, while cell division cycle-42 (CDC42) displayed excessive activation in NDRG1-silenced cells. Mechanistically, NDRG1 loss disrupts the binding between RhoGDIα and CDC42 and triggers the activation of CDC42 and the downstream cascades PAK1/Cofilin, thereby promotes the formation of filopodia and invasiveness of CRC. The knockdown of NDRG1 led to enhanced dissemination of CRC cells in vivo and correlates with active CDC42 expression. Using clinical sample analysis, we found an elevated level of active CDC42 in patients with advanced T stage, and it was negatively related to NDRG1 expression. In sum, these results uncover a mechanism utilized by NDRG1 to regulate CDC42 activity in coordinating cytoskeleton reorganization, which was crucial in cancer invasion.

Increased NDRG1 expression suppresses angiogenesis via PI3K/AKT pathway in human placental cells

To observe whether and how N-myc downstream-regulated gene 1 (NDRG1) regulates placental angiogenesis via JEG-3 placental-derived cells. Expression of NDRG1 in stably transfected JEG-3 cells was detected using western blot and real-time quantitative polymerase chain reaction. Angiogenesis was examined by tube formation assay. The levels of placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) were examined using enzyme-linked immunosorbent assay. The expression of vascular endothelial growth factor (VEGF), PI3K, and AKT was examined by western blot. The relationship between PI3K and NDRG1 was detected by co-immunoprecipitation. NDRG1 was significantly down-regulated at both the mRNA and protein level by lentivirus (Lv)-NDRG1-shRNA (P<0.001), whereas it was significantly up-regulated by Lv-NDRG1 (P<0.001). NDRG1 knockdown significantly increase the expression of PLGF and VEGF in JEG-3 cells (P<0.001), while NDRG1 knockdown significantly reduced the secretion of sFlt-1 (P<0.001). NDRG1 was specific bound to PI3K, and NDRG1 knockdown significantly up-regulated the expressions of PI3K and AKT in JEG-3 cells (P<0.001). NDRG1 suppresses angiogenesis in preeclampsia, and the PI3K/AKT signaling pathway may be involved in the regulation of angiogenesis by NDRG1.

Hsa_circ_0003159 inhibits gastric cancer progression by regulating miR-223-3p/NDRG1 axis

BackgroundAbnormally expressed circular RNAs (circRNAs) are implicated in the development and treatment of gastric cancer (GC). Previous study has reported that hsa_circ_0003159 is expressed in GC. However, the role and mechanism of hsa_circ_0003159 in GC progression remain unclear.MethodsGC tissues and normal tissues were harvested from 55 patients in this study. The levels of hsa_circ_0003159, microRNA (miR)-223-3p and N-myc downstream regulated gene 1 (NDRG1) were measured by quantitative real-time polymerase chain reaction or western blot. Cell proliferation, migration, invasion and apoptosis were determined by cell counting kit (CCK)-8, transwell assay, flow cytometry and western blot, respectively. The target association of miR-223-3p-hsa_circ_0003159 and miR-223-3p-NDRG1 was explored by dual-luciferase reporter assay. Xenograft model was established to assess the roles of hsa_circ_0003159 in GC in vivo.ResultsHsa_circ_0003159 was lowly expressed in GC tissues and cells and mainly presented in the cytoplasm. Low expression of hsa_circ_0003159 was associated with lower overall survival and disease-free survival. Hsa_circ_0003159 overexpression inhibited proliferation, migration and invasion but induced apoptosis in GC cells. MiR-223-3p was a target of hsa_circ_0003159 and abated the effect of hsa_circ_0003159 on proliferation, migration, invasion and apoptosis in GC cells. Hsa_circ_0003159 promoted NDRG1 expression by competitively sponging miR-223-3p. Knockdown of NDRG1 reversed the suppressive effect of hsa_circ_0003159 on GC progression. Besides, hsa_circ_0003159 decreased GC cell xenograft tumor growth by regulating miR-223-3p and NDRG1.ConclusionHsa_circ_0003159 suppressed proliferation, migration, invasion and xenograft tumor growth but promoted apoptosis by decreasing miR-223-3p and increasing NDRG1 in GC, indicating a novel target for treatment of GC.

Abstract P3-01-10: Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis

Abstract Background: Inflammatory breast cancer (IBC) is a rare (1-2%) but lethal form of primary breast cancer responsible for 10% of breast cancer-related deaths in the United States. IBC tumors grow aggressively and have a high risk of metastasis, particularly to the brain. Unraveling the pathways involved in IBC aggressiveness and metastasis could reveal new therapeutic targets. We recently identified NDRG1 (N-Myc downstream regulated gene1) in a newly isolated subline of MDA-IBC3 cells as a top candidate protein associated with enhanced propensity for brain metastasis. Although the metastasis suppressor properties of NDRG1 are widely described, its function in the pathobiology of IBC is unknown. Here we investigated the function of NDRG1 in tumorigenesis and metastatic progression to brain in IBC. Methods: NDRG1 gene expression was analyzed in large publically available breast cancer clinical datasets and the IBC Consortium dataset, and median expression level was used to stratify patients into NDRG1-high and NDRG1-low groups. Tissue microarrays of IBC patient samples were immunostained with anti-NDRG1 antibody. Stable knockdown (KD) of NDRG1 in IBC cell lines (HER2+: MDA-IBC3; TNBC: SUM149, BCX010) was obtained by lentiviral gene transfer. For in vivo studies, control and NDRG1 KD IBC cell lines were transplanted into cleared mammary fat pads of SCID/Beige mice and tumor growth monitored via caliper measurements. Reverse phase protein array analysis was used for proteomic profiling. Results: In several independent cohorts of patients with breast cancer (including The Cancer Genome Atlas), NDRG1 mRNA expression was significantly higher in ER-negative and high-grade breast tumors (p&amp;lt;0.0001) and was associated with poor overall survival (p=0.0003), relapse-free survival (p&amp;lt;0.0001), and metastasis-free survival (p=0.0012). NDRG1 expression was higher in more aggressive, basal-like and HER2+ subtypes than in hormone receptor-positive subtypes (p&amp;lt;0.0001), and was also higher in IBC vs. non-IBC tumors (p=0.007, IBC consortium dataset). Immunostaining showed NDRG1 was frequently expressed in IBC patient tumors (83%) and was associated with worse overall survival (OS, p=0.0129) and disease-specific survival (DSS, p=0.013). On multivariate analysis, NDRG1 was an independent prognostic factor for poor OS [HR 2.5 (95%CI, 1.3-4.7), p=0.0047] and DSS [HR 2.6 (95%CI 1.3-5.1), p=0.006]. Notably, NDRG1 expression was higher in patients with brain metastasis vs. primary tumors (GSE43837, p=0.018) and correlated with shorter time to development of brain metastasis (GSE2034, p=0.026). NDRG1-high sublines had enhanced propensity for brain metastasis as shown by higher incidence (100% vs. 44% for NDRG1-low sublines, p=0.01) and increased brain metastasis burden (p=0.0008). Invitro, silencing NDRG1 in IBC cell lines significantly reduced colony formation, migration, invasion, and cancer stem-like properties. In vivo, silencing of NDRG1 inhibited primary tumor growth of SUM149 (p&amp;lt;0.0001) and MDA-IBC3 (p=0.0426) xenografts. Proteomics analysis showed that NDRG1 correlated positively with activation of the EGFR/STAT3 signaling pathway. Mechanistically, NDRG1 knockdown in IBC cell lines reduced EGFR expression and suppressed phosphorylation of EGFR and STAT3, whereas NDRG1 overexpression increased EGFR expression and enhanced EGFR-STAT3 activation. Conclusions: We showed that high NDRG1 was associated with worse clinical outcomes in patients with IBC and that NDRG1 drives tumor progression in IBC potentially via activation of the EGFR-STAT3 signaling pathway. Targeting the NDRG1-EGFR axis may be a novel therapeutic approach in IBC and other aggressive breast cancers. Additional investigations are underway to determine the detailed mechanisms of NDRG1-mediated IBC tumorigenesis and brain metastasis Citation Format: Emilly Schlee Villodre, Xiaoding Hu, Richard Larson, Bedrich L Eckhardt, Yun Gong, Lei Huo, Juhee Song, Savitri Krishnamurthy, Nuhad Ibrahim, Naoto T Ueno, Debu Tripathy, Wendy A Woodward, Bisrat G Debeb. Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-10.

146 N-myc downstream regulated gene 1 (NDRG1) promotes adipocyte differentiation in infantile hemangioma endothelial cells

Infantile hemangioma (IH) is the most common vascular tumor of infancy, characterized by a rapid growth phase followed by a spontaneous involution to adipose and fibrous tissue over time. However, the mechanism of pathogenesis in IH involution is still unknown. N-myc downstream regulated gene 1(NDRG1) is recently known to regulate adipocyte differentiation through down regulation of suppressor genes TGF-β, as well as induction of terminal differentiation markers. Here, we evaluated the role of NDRG1 during differentiation from hemangioma endothelial cells (HemEC) to adipocytes. We found that NDRG1 expression in HemEC derived from IH tissues was upregulated during proliferating phase and downregulated during involution. NDRG1 was identified as a significant regulator of HemEC differentiation. Loss-of function analysis using siRNA mediated knockdown of NDRG1 demonstrated inhibition of adipogenesis. NDRG1 knockdown decreased stem-like properties of HemEC in IH including induced pluripotent stem cell factors such as C-MYC. Taken together, we showed a functional role for NDRG1 in promotion of multipotent hemangioma derived endothelial cells to the adipocytes

Upregulation of NDRG1 predicts poor outcome and facilitates disease progression by influencing the EMT process in bladder cancer

N-myc downstream regulated gene 1 (NDRG1) is an intracellular protein involved in cell differentiation and was recently reported to exert various effects in several cancers. However, its expression and role in bladder cancer remain unclear. Our study enrolled 100 bladder cancer patients to detect NDRG1 expression in tumour tissues by immunohistochemistry. Correlations between NDRG1 expression and clinical factors were analysed. An NDRG1 overexpression plasmid and NDRG1 siRNAs were transfected into bladder cancer cell lines. Cell biological behaviours were assessed by CCK-8, flow cytometry, wound healing and Transwell assays. Additionally, the influence of NDRG1 on epithelial-mesenchymal transition (EMT) was investigated by western blotting and real-time PCR. NDRG1 expression in urine from bladder cancer patients was examined by ELISA. NDRG1 protein levels were significantly increased in bladder cancer patients and correlated with tumour stage (p = 0.025), lymph node metastasis (p = 0.034) and overall survival (p = 0.016). Patients with high NDRG1 expression had poorer outcomes than those with low NDRG1 expression. NDRG1 overexpression was associated with increased cell proliferation, migration, and invasion and decreased apoptotic cell numbers; NDRG1 knockdown resulted in the inverse effects. Moreover, upregulated NDRG1 expression was associated with downregulated Cytokeratin 7 and Claudin-1 expression and upregulated N-cad, β-catenin and slug expression. Downregulated NDRG1 expression was associated with the inverse effects. Urine protein levels could distinguish bladder cancer patients from healthy controls, with an area under the curve of 0.909. NDRG1 promoted EMT in bladder cancer and could be an effective diagnostic and prognostic biomarker in bladder cancer patients.

NDRG1 negatively regulates proliferation and Milk bio-synthesis of bovine epithelial cells via the mTOR signaling pathway

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with diverse processes such as cell growth and differentiation, lipid synthesis, stress and immune responses. Here we explored the role of NDRG1 expression in bovine mammary tissue and epithelial cells under an inflammatory condition. Results showed that NDRG1 expression was elevated in bovine mammary tissue with mastitis and mammary epithelial cells treated by heat inactivated Escherichia coli and Staphylococcus aureus compared to normal tissue and untreated cells. Overexpression of NDRG1 significantly inhibited cell proliferation and migration, β-casein secretion, gene expressions of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6 and IL-8, and activation of mTOR signal pathway of mammary epithelial cells, and vice versa by NDRG1 knockdown. These findings suggest that NDRG1 has immense potential in the regulation of properties in bovine mammary epithelial cells under an inflammatory condition.

NDRG1 facilitates the replication and persistence of Kaposi's sarcoma-associated herpesvirus by interacting with the DNA polymerase clamp PCNA.

Kaposi’s sarcoma-associated herpesvirus (KSHV) latently infects host cells and establishes lifelong persistence as an extra-chromosomal episome in the nucleus. To persist in proliferating cells, the viral genome typically replicates once per cell cycle and is distributed into daughter cells. This process involves host machinery utilized by KSHV, however the underlying mechanisms are not fully elucidated. In present study, we found that N-Myc downstream regulated gene 1 (NDRG1), a cellular gene known to be non-detectable in primary B cells and endothelial cells which are the major cell types for KSHV infection in vivo, was highly upregulated by KSHV in these cells. We further demonstrated that the high expression of NDRG1 was regulated by latency-associated nuclear antigen (LANA), the major viral latent protein which tethers the viral genome to host chromosome and plays an essential role in viral genome maintenance. Surprisingly, knockdown of NDRG1 in KSHV latently infected cells resulted in a significant decrease of viral genome copy number in these cells. Interestingly, NDRG1 can directly interact with proliferating cell nuclear antigen (PCNA), a cellular protein which functions as a DNA polymerase clamp during DNA replication. Intriguingly, we found that NDRG1 forms a complex with LANA and PCNA and serves as a scaffold protein bridging these two proteins. We further demonstrated that NDRG1 is critical for mediating LANA to recruit PCNA onto terminal repeat (TR) of KSHV genome, and facilitates viral DNA replication and episome persistence. Taken together, our findings suggest that NDRG1 plays an important role in KSHV viral genome replication, and provide new clues for understanding of KSHV persistence.

Caffeic Acid Phenethyl Ester Induces N-myc Downstream Regulated Gene 1 to Inhibit Cell Proliferation and Invasion of Human Nasopharyngeal Cancer Cells.

Caffeic acid phenethyl ester (CAPE), a bioactive component extracted from propolis, is widely studied due to its anti-cancer effect. Nasopharyngeal carcinoma (NPC) is distinct from other head and neck carcinomas and has a high risk of distant metastases. N-myc downstream regulated gene 1 (NDRG1) is demonstrated as a tumor suppressor gene in several cancers. Our result showed that CAPE treatment could repress NPC cell growth, through induction of S phase cell cycle arrest, and invasion. CAPE treatment stimulated NDRG1 expression in NPC cells. NDRG1 knockdown increased NPC cell proliferation and invasion and rendered NPC cells less responsive to CAPE growth-inhibiting effect, indicating CAPE repressed NPC cell growth partly through NDRG1indcution. CAPE treatment increased phosphorylation of ERK, JNK, and p38 in a dose- and time-dependent manner. Pre-treatments by inhibitors of ERK (PD0325901), JNK (SP600125), or p38 (SB201290), respectively, all could partly inhibit the CAPE effect on NDRG1 induction in NPC cells. Further, STAT3 activity was also repressed by CAPE in NPC cells. In summary, CAPE attenuates NPC cell proliferation and invasion by upregulating NDRG1 expression via MAPK pathway and by inhibiting phosphorylation of STAT3. Considering the poor prognosis of NPC patients with metastasis, CAPE could be a promising agent against NPC.

N-myc downstream-regulated gene 1 facilitates influenza A virus replication by suppressing canonical NF-κB signaling

The highly pathogenic avian influenza (HPAI) A/H5N1 virus hijacks host cellular machinery to complete its life cycle; identification of the host factors involved in viral replication may facilitate antiviral drug development. Here, we first characterize a metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), and showed that it plays a crucial role in H5N1 viral replication. We found that H5N1 infection upregulated NDRG1 mRNA and protein expression. Overexpression of NDRG1 released approximately 4-fold more virions compared to the control group, whereas knockdown of NDRG1 resulted in a drop in viral RNA and protein production. Further investigation revealed that NDRG1 facilitated HPAI A/H5N1 viral replication by suppressing the canonical NF-κB signaling pathway. Furthermore, our results also showed that the NDRG1 mRNA level was mainly stimulated by M1 and PB1 viral proteins. Overall, our results suggest that NDRG1 plays a positive role in HPAI replication by suppressing the canonical NF-κB signaling pathway.

NDRG1 inhibition sensitizes osteosarcoma cells to combretastatin A-4 through targeting autophagy

Combretastatin A-4 (CA-4), a tubulin-depolymerizing agent, shows promising antitumor efficacy and has been under several clinical trials in solid tumors for 10 years. Autophagy has an important pro-survival role in cancer therapy, thus targeting autophagy may improve the efficacy of antitumor agents. N-myc downstream-regulated gene 1 (NDRG1) is a significant stress regulatory gene, which mediates cell survival and chemoresistance. Here we reported that CA-4 could induce cell-protective autophagy, and combination treatment of CA-4 and autophagy inhibitor chloroquine (CQ) exerted synergistic cytotoxic effect on human osteosarcoma (OS) cells. Meanwhile, CA-4 or CQ could increase the expression of NDRG1 independently. We further performed mechanistic study to explore how CA-4 and CQ regulate the expression of NDRG1. Using luciferase reporter assay, we found that CA-4 transcriptionally upregulated NDRG1 expression, whereas CQ triggered colocalization of NDRG1 and lysosome, which subsequently prevented lysosome-dependent degradation of NDRG1. Further, we showed that knockdown of NDRG1 caused the defect of lysosomal function, which accumulated LC3-positive autophagosomes by decreasing their fusion with lysosomes. Moreover, NDRG1 inhibition increased apoptosis in response to combination treatment with CA-4 and CQ. Taken together, our study revealed abrogation of NDRG1 expression sensitizes OS cells to CA-4 by suppression of autophagosome–lysosome fusion. These results provide clues for developing more effective cancer therapeutic strategies by the concomitant treatment with CA-4 and clinical available autophagy inhibitors.

P01.32 NDRG1 induced downregulation of CCL2 influences the migration of macrophages and impacts the tumor microenvironement

AbstractBACKGROUND: Malignant gliomas belong to the most aggressive neoplasms in humans. The interactions and intercellular communication between malignant and non-malignant cells in the tumor microenvironment are tumor-promoting and critically to improve the understanding of the disease. N-myc downstream regulated gene 1 (NDRG1) is a stress inducible gene and key determinant of resistance towards alkylating chemotherapy in glioblastoma. NDRG1 promotes invasive growth and acts antiapoptotic in hepatocellular carcinoma. In human pancreatic cancer cells NDRG1 suppresses chemokine expression, decreases macrophage infiltration and microvascular density. Further understanding the influence of NDRG1 on the tumor microenvironment may lead to new therapeutic approaches in glioma. METHODS: To analyze the NDRG1 effects on the glioma microenvironment, we used human and murine NDRG1 knockdown (KD) cells. In orthotopic xenograft experiments control and KD microenvironments were compared for infiltrating immune cells dependent on NDRG1. Interaction and signaling mechanisms were evaluated in vitro using western blots, cytokine and migration assays and treatment with the NF-kB inhibitor JSH-23 as well as SGK1 inhibitor EMD638683 to assess the influence of NDRG1. Results: Orthotopic NDRG1 KD tumors were significantly larger compared to control tumors. Ex vivo flow cytometry analyses of the tumor microenvironment revealed a significant increase in peripheral macrophages, dendritic cells and monocytic myeloid derived suppressor cells in NDRG1 KD tumors. In vitro cytokine array analyses showed an increase of the chemokine CCL2 in NDRG1 KD cells compared to control cells. This was confirmed by ELISA. Macrophages showed an increased migration rate in the NDRG1 knockdown environment. To analyze molecular mechanisms CCL2 secretion was blocked with a NF-κB inhibitor (JSH-23) and stimulated with a SGK1 inhibitor (EMD638683). We found that NDRG1 induced NF-κB signaling attenuation is mediated through decreases in IKK α expression and IκBα phosphorylation. CONCLUSION: We identified CCL2 as a NDRG1-induced NF-κB target gene that triggers macrophage recruitment. Our findings support an immunomodulatory effect of NDRG1 on the tumor microenvironment and provide novel insights into the biological relevance of the tumor stroma and the function of NDRG1 with important implications for the development of cancer therapeutics.

NDRG1 is important to maintain the integrity of airway epithelial barrier through claudin-9 expression.

Impairment of epithelial barrier integrity caused by environmental triggers is associated with the pathogenesis of airway inflammation. Using human airway epithelial cells, we attempted to identify molecule(s) that promote airway epithelial barrier integrity. Microarray analyses were conducted using the Affimetrix human whole genome gene chip, and we identified the N-myc downstream-regulated gene 1 (NDRG1) gene, which was induced during the development of the epithelial cell barrier. Immunohistochemical analysis revealed strong NDRG1 expression in ciliated epithelial cells in nasal tissues sampled from patients with chronic rhinosinusitis (CRS), and the low expression of NDRG1 was observed in goblet cells or damaged epithelial cells. NDRG1 gene knockdown with its specific siRNA decreased the transepithelial electrical resistance and increased the dextran permeability. Immunocytochemistry revealed that NDRG1 knockdown disrupted tight junctions of airway epithelial cells. Next, we analyzed the effects of NDRG1 knockdown on the expression of tight and adhesion junction molecules. NDRG1 knockdown significantly decreased only claudin-9 expression, but did not decrease other claudin family molecules, such as E-cadherin, and ZO-1, -2, or -3. Knockdown of claudin-9 markedly impaired the barrier function in airway epithelial cells. These results suggest that NDRG1 is important for the barrier integrity in airway epithelial cells.

Downregulation of the N-myc downstream regulated gene 1 is related to enhanced proliferation, invasion and migration of pancreatic cancer.

The N-myc downstream regulated gene1 (NDRG1) is differently expressed in human malignancies according to the tumor type. We investigated the expression of NDRG1 in pancreatic cancer tissues and cell lines as well as how it affects tumor growth, invasion and migration in pancreatic cancer cells. Experimental groups included NDRG1 overexpression and knockdown pancreatic cancer cell lines. Lentivirus-based empty vector transfected cells (NC group) were considered control groups. Proliferation, invasion and migration related proteins such as STAT3, MMPs, PTEN, PI3K/AKT were assessed by CCK-8, Transwell assay and western blotting. Efficient NDRG1 overexpression results in reduced cell proliferation, invasion and migration. Inversely, downregulation of NDRG1 promoted proliferation, invasion and migration. We also found NDRG1 could deactivate p-STAT3, PI3K, p-AKT, MMP2, MMP9 and activate PTEN. NDRG1 is a potential anti-oncogene. Its upregulation significantly decreases pancreatic cancer tumorigenesis, likely by inhibiting STAT3 and the PI3K/AKT signaling pathway.

NDRG1 overexpressing gliomas are characterized by reduced tumor vascularization and resistance to antiangiogenic treatment

Hypoxia-regulated molecules play an important role in vascular resistance to antiangiogenic treatment. N-myc downstream-regulated-gene 1 (NDRG1) is significantly upregulated during hypoxia in glioma. It was the aim of the present study to analyze the role of NDRG1 on glioma angiogenesis and on antiangiogenic treatment. Orthotopically implanted NDRG1 glioma showed reduced tumor growth and vessel density compared to controls. RT-PCR gene array analysis revealed a 30-fold TNFSF15 increase in NDRG1 tumors. Consequently, the supernatant from NDRG1 transfected U87MG glioma cells resulted in reduced HUVEC proliferation, migration and angiogenic response in tube formation assays in vitro. This effect was provoked by increased TNFSF15 promoter activity in NDRG1 cells. Mutations in NF-κB and AP-1 promoter response elements suppressed TNFSF15 promoter activity. Moreover, U87MG glioma NDRG1 knockdown supernatant contained multiple proangiogenic proteins and increased HUVEC spheroid sprouting. Sunitinib treatment of orhotopically implanted mice reduced tumor volume and vessel density in controls; in NDRG1 overexpressing cells no reduction of tumor volume or vessel density was observed. NDRG1 overexpression leads to reduced tumor growth and angiogenesis in experimental glioma via upregulation of TNFSF15. In NDRG1 overexpressing glioma antiangiogenic treatment does not yield a therapeutic response.

Abstract 1983: Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells

Abstract Hypoxia has been intensively investigated over past decades based on the observations that hypoxic tumors were more resistant to therapy and had a worse prognosis. Previously, we reported that N-myc downstream-regulated gene 1 (NDRG1) was strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuation of oxygen concentrations. However, the regulatory mechanism of NDRG1 under hypoxia remains elusive. Therefore, the purpose of this study is to identify the novel transcription factors that regulate NDRG1 and to investigate the functional roles of NDRG1 in hypoxia. In silico analysis predicted binding sites of aryl hydrocarbon receptor (AHR) existing in the NDRG1 promoter. Western blotting showed that nuclear AHR was up-regulated in the presence of cobalt and hypoxia. Immunofluorescence staining and chromatin immunoprecipitation assays showed that AHR translocated to nuclei and bound to NDRG1 promoter in hypoxia. Luciferase reporter assays further showed that the binding site of AHR at -412 ∼ -388 bp played a crucial role in regulating NDRG1 under hypoxia-mimicking conditions. Moreover, hypoxia-mimetic induction of NDRG1 by was attenuated by knockdown of AHR expression using short interfering RNA. Also, overexpression of AHR facilitated cell proliferation and migration via up-regulation of NDRG1, whereas shRNA knockdown of NDRG1 reduced cell growth and motility. In summary, these results showed for the first time that AHR positively regulates NDRG1 transcription through a AHR binding site by hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regime to prevent tumor malignancy under hypoxia. Citation Format: En-Yu Li, Wei-Yung Huang, Mong-Hsun Tsai, Eric Y. Chuang, Liang-Chuan Lai. Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1983. doi:10.1158/1538-7445.AM2015-1983

NDRG1 attenuates epithelial–mesenchymal transition of nasopharyngeal cancer cells via blocking Smad2 signaling

N-myc downstream-regulated gene 1 (NDRG1) has been implicated in tumorigenesis and metastasis in different cancers. However, its role in nasopharyngeal carcinoma remains unknown. We found that NDRG1 expression level was high in nasopharyngeal cancer 5-8F cells but low in 5-8F-LN cells with lymphatic metastasis potential. Knockdown of NDRG1 by shRNA promoted 5-8F cell proliferation, migration, and invasion in vitro and its tumorigenesis in vivo. Moreover, NDRG1 deficiency induced an epithelial–mesenchymal transition (EMT) of 5-8F cells as shown by an attenuation of E-cadherin and an induction of N-cadherin and vimentin expression. NDRG1 knockdown also enhanced Smad2 expression and phosphorylation. Smad2 signaling was attenuated in 5-8F cells but was significantly activated in 5-8F-LN cells. Knockdown of Smad2 restored E-cadherin but attenuated N-cadherin expression in NDRG1-deficient 5-8F cells, suggesting a reduction of EMT. Consistently, blockade of Smad2 in 5-8F-LN cells increased E-cadherin while diminishing N-cadherin and vimentin expression. These data indicate that Smad2 mediates the NDRG1 deficiency-induced EMT of 5-8F cells. In tumors derived from NDRG1-deficient 5-8F cells, E-cadherin expression was inhibited while vimentin and Smad2 were increased in a large number of cancer cells. Most importantly, NDRG1 expression was attenuated in human nasopharyngeal carcinoma tissues, resulted in a lower survival rate in patients. The NDRG1 was further decreased in the detached nasopharyngeal cancer cells, which was associated with a further reduced survival rate in patients with lymphatic metastasis. Taken together, these results demonstrated that NDRG1 prevents nasopharyngeal tumorigenesis and metastasis via inhibiting Smad2-mediated EMT of nasopharyngeal cells.