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TGF?-Induced Lung Cancer Cell Migration Is NR4A1-Dependent.

ABSTRACT: TGF? induces migration of lung cancer cells (A549, H460, and H1299), dependent on activation of c-Jun N-terminal kinase (JNK1), and is inhibited by the JNK1 inhibitor SP600125. Moreover, TGF?-induced migration of the cells is also blocked by the nuclear export inhibitor leptomycin B (LMB) and the orphan nuclear receptor 4A1 (NR4A1) ligand 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), which retains NR4A1 in the nucleus. Subsequent analysis showed that the TGF?/TGF? receptor/PKA/MKK4 and -7/JNK pathway cascade phosphorylates and induces nuclear export of NR4A1, which in turn forms an active complex with Axin2, Arkadia (RNF111), and RNF12 (RLIM) to induce proteasome-dependent degradation of SMAD7 and enhance lung cancer cell migration. Thus, NR4A1 also plays an integral role in mediating TGF?-induced lung cancer invasion, and the NR4A1 ligand CDIM8, which binds nuclear NR4A1, represents a novel therapeutic approach for TGF?-induced blocking of lung cancer migration/invasion. IMPLICATIONS: Effective treatment of TGF?-induced lung cancer progression could involve a number of agents including the CDIM/NR4A1 antagonists that block not only TGF?-induced migration, but several other NR4A1-regulated prooncogenic genes/pathways in lung cancer cell lines.

SUBMITTER: Hedrick E

PROVIDER: S-EPMC6343492 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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TGFβ-Induced Lung Cancer Cell Migration Is NR4A1-Dependent.

Hedrick Erik E Mohankumar Kumaravel K Safe Stephen S

Molecular cancer research : MCR 20180802 12

TGFβ induces migration of lung cancer cells (A549, H460, and H1299), dependent on activation of c-Jun N-terminal kinase (JNK1), and is inhibited by the JNK1 inhibitor SP600125. Moreover, TGFβ-induced migration of the cells is also blocked by the nuclear export inhibitor leptomycin B (LMB) and the orphan nuclear receptor 4A1 (NR4A1) ligand 1,1-bis(3'-indolyl)-1-(<i>p</i>-hydroxyphenyl)methane (CDIM8), which retains NR4A1 in the nucleus. Subsequent analysis showed that the TGFβ/TGFβ receptor/PKA/M ...[more]

PMID: 30072581

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Project description:Breast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology.NR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting.Breast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins.NR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.

Project description:Resveratrol is a polyphenolic phytochemical found in fruits, nuts and vegetables that contributes to the remarkable dietary effects of polyphenolic as inhibitors aging and multiple aging related diseases. In addition, resveratrol has been extensively investigated as an inhibitor of inflammatory diseases including cancer, however, the underlying mechanisms of these chemotherapeutic effects of resveratrol are not completely understood. In cancer cells resveratrol inhibits cell growth, survival, migration and invasion, and many of the effects of resveratrol resemble those observed for bis-indole derived (CDIM) compounds that bind the pro-oncogenic nuclear receptor 4A1 (NR4A1, Nur77) and act as receptor antagonists. Using an isothermal titration calorimetry binding assay, we observed that resveratrol bound to the ligand binding domain of NR4A1 with a KD value of 2.4 µM and a ΔG of -32.2 kJ/mol. Resveratrol also inhibited NR4A1-dependent transactivation in H460 and H1299 lung cancer cells suggesting that resveratrol is an NR4A1 antagonist. This observation was confirmed in a series of functional (cell proliferation, survival, migration and invasion) and gene expression assays in H460 and H1299 cells showing that treatment with resveratrol mimicked the effects of NR4A1 knockdown and were similar to results of previous studies using CDIM/NR4A1 antagonists. These data indicate that applications of resveratrol may be more effective in patients that overexpress NR4A1 which is a negative prognostic factor for patients with some solid tumor-derived cancers. Significance Statement We have examined the mechanism of action of resveratrol and show binding to NR4A1 (KD = 2.4 µM) and inhibition of NR4A1-dependent transactivation in lung cancer cells. Treatment of H460 and H1299 lung cancer cells with resveratrol inhibits cell growth, survival, migration/invasion and related genes, and acts as an NR4A1 antagonist. Resveratrol can now be used more effectively in cancer chemotherapy by targeting patients that overexpress NR4A1 in lung cancer.

Project description:Background Smac mimetics are a type of drug that can induce apoptosis by antagonizing IAP family members in cancer treatment. However, a recent study showed that Smac mimetics can trigger cell invasion and migration in cancer cells by activating the NF-?B pathway. Methods We assessed lung cancer cell elongation, invasion and migration under treatment with the Smac mimetic LCL161. Functional analyses (in vitro and in vivo) were performed to detect the contribution of NIK and OTUD7B to LCL161-induced cell invasion and migration. The role of OTUD7B in regulation of the TRAF3/NIK/NF-?B pathway under LCL161 treatment was analysed by immunoblotting, immunoprecipitation, luciferase and ubiquitin assays, shRNA silencing and plasmid overexpression. Expression levels of OTUD7B, NIK and TRAF3 in tissue samples from lung cancer patients were examined by immunohistochemistry. Results We found that LCL161 stimulates lung cancer cell elongation, invasion and migration at non-toxic concentrations. Mechanistically, LCL161 results in NIK accumulation and activates the non-canonical rather than the canonical NF-?B pathway to enhance the transcription of target genes, such as IL-2 and MMP-9. Importantly, knockdown of NIK dramatically suppresses LCL161-induced cell invasion and migration by reducing the proteolytic processing of p100 to p52 and target gene transcription. Interestingly, we discovered that OTUD7B increases TRAF3 and decreases NIK to inhibit the non-canonical NF-?B pathway and that overexpression of OTUD7B suppresses LCL161-induced cell invasion and migration. Notably, OTUD7B directly binds to TRAF3 rather than to NIK and deubiquitinates TRAF3, thereby inhibiting TRAF3 proteolysis and preventing NIK accumulation and NF-?B pathway activation. Furthermore, the OTU domain of OTUD7B is required for the inhibition of LCL161-induced cell invasion and migration, as demonstrated by transfection of the C194S/H358R(CH) mutant OTUD7B. Finally, we investigated whether OTUD7B inhibits LCL161-induced lung cancer cell intrapulmonary metastasis in vivo, and our analysis of clinical samples was consistent with the above findings. Conclusions Our study highlights the importance of OTUD7B in the suppression of LCL161-induced lung cancer cell invasion and migration, and the results are meaningful for selecting lung cancer patients suitable for LCL161 treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-020-01751-3.

Dataset Information

TGF?-Induced Lung Cancer Cell Migration Is NR4A1-Dependent.

Publications

TGFβ-Induced Lung Cancer Cell Migration Is NR4A1-Dependent.

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