Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

Download Full-text

Coordination of RNA and protein condensation by the P granule protein MEG-3

10.1101/2020.10.15.340570 ◽

2020 ◽

Author(s):

Helen Schmidt ◽

Andrea Putnam ◽

Dominique Rasoloson ◽

Geraldine Seydoux

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Protein ◽

Protein Protein Interactions ◽

C Elegans ◽

Intrinsically Disordered ◽

Disordered Protein ◽

C Terminus ◽

Necessary And Sufficient

ABSTRACTGerm granules are RNA-protein condensates in germ cells. The mechanisms that drive germ granule assembly are not fully understood. MEG-3 is an intrinsically-disordered protein required for germ (P) granule assembly in C. elegans. MEG-3 forms gel-like condensates on liquid condensates assembled by PGL proteins. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). Using in vitro and in vivo experiments, we find the MEG-3 C-terminus is necessary and sufficient to build MEG-3/PGL co-condensates independent of RNA. The HMGL domain is required for high affinity MEG-3/PGL binding in vitro and for assembly of MEG-3/PGL co-condensates in vivo. The MEG-3 IDR binds RNA in vitro and is required but not sufficient to recruit RNA to P granules. Our findings suggest that P granule assembly depends in part on protein-protein interactions that drive condensation independent of RNA.

Download Full-text

Twist, Snail, and Sox9 form an allosterically regulated complex, the EMTosome, on a bipartite E-box site

10.1101/2020.02.03.932962 ◽

2020 ◽

Author(s):

Daniel S McCracken ◽

Hongzhuang Peng ◽

Kasirajan Ayyanathan ◽

Yike Lindy Jiang ◽

Sarah Welsh ◽

...

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Breast Carcinoma Cell Line ◽

Protein Protein Interactions ◽

Mesenchymal Transition ◽

E Box ◽

Additional Protein ◽

E Boxes

AbstractEpithelial-Mesenchymal transition (EMT) of primary tumor cells is a critical trans-differentiation event that contributes to dissemination and metastasis. The process of EMT is controlled by specific DNA-binding transcription factors (TFs) that reprogram the tumor transcriptome. In particular, the canonical EMT-TFs Twist and Snail can induce an EMT program when overexpressed in cancer cells, and both are found upregulated in metastatic cancers. Twist and Snail bind DNA directly, by recognition to variants of the E-Box sequence CANNTG. However, it is unclear how this binding is regulated. We have used a biochemical approach to dissect DNA binding and protein-protein interactions that occur amongst these proteins. We find that Twist preferentially recognizes a dyad repeat of E-boxes that are not directly bound by Snail. Our data suggest that Twist use its WR domain to recruit Snail into a binding complex through the Snail zinc-finger motifs. We analyzed Twist-Snail complexes in the breast carcinoma cell line SUM1315 and found evidence that it contains an additional protein partner, Sox9. Notably, we report that a native Twist complex can be displaced from its dyad binding site by consensus DNA binding sites for Snail and Sox9 even though these proteins do not contact the Twist dyad site. Taken together, our findings suggest that Snail and Sox9 interact with Twist to regulate its DNA binding ability via protein-protein interactions, thereby allosterically regulating Twist DNA binding. We designate this ternary complex EMTosome. These results may inform efforts to therapeutically target the EMT program in order to target cancer metastasis.

Download Full-text

Upregulation of FBXW7 Suppresses Renal Cancer Metastasis and Epithelial Mesenchymal Transition

Disease Markers ◽

10.1155/2017/8276939 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 6

Author(s):

Yangke Cai ◽

Meng Zhang ◽

Xiaofu Qiu ◽

Bingwei Wang ◽

Yu Fu ◽

...

Keyword(s):

Cell Lines ◽

Renal Cell ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Coding Region ◽

Mesenchymal Transition ◽

Renal Cell Line ◽

Emt Markers

Background and Objective. FBXW7, known as a general tumor suppressor, is commonly lowly expressed in metastatic malignancies. We aim to investigate the potential influence of FBXW7 overexpression on renal cell carcinoma (RCC) metastasis. Methods. We employed quantitative real-time PCR (qRT-PCR) and Western blotting (WB) to quantify the FBXW7 expression in RCC cell lines. Upregulation of FBXW7 was performed in vitro on RCC cells using the lentivirus covering coding region FBXW7 cDNA sequence, and functional tests were performed to verify FBXW7 overexpression on migration and invasion of RCC cells. Moreover, WB was employed to determine the expressions of MMP-2, MMP-9, and MMP-13, as well as EMT markers in the transfected RCC cells. Results. FBXW7 was significantly downregulated in RCC cell lines, dominated by 786-O and ACHN, when compared to normal renal cell line HK-2. Moreover, upregulation of FBXW7 in 786-O and ACHN cell lines significantly inhibited cell migration and invasion, as well as EMT. Present study also showed that FBXW7 was involved in the migration and invasion of RCC cells via regulating the expressions of MMP-2, MMP-9, and MMP-13. Conclusion. Our findings demonstrate that upregulation of FBXW7 inhibits RCC metastasis and EMT. FBXW7 is a potential therapeutic target for RCC patients.

Download Full-text

Knockdown of ARK5 Expression Suppresses Invasion and Metastasis of Gastric Cancer

Cellular Physiology and Biochemistry ◽

10.1159/000478685 ◽

2017 ◽

Vol 42 (3) ◽

pp. 1025-1036 ◽

Cited By ~ 12

Author(s):

Dehu Chen ◽

Guiyuan Liu ◽

Ning Xu ◽

Xiaolan You ◽

Haihua Zhou ◽

...

Keyword(s):

Gastric Cancer ◽

Western Blot ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Invasion And Metastasis ◽

Ags Cells ◽

Mesenchymal Transition

Background/Aims: Gastric cancer (GC) is a common and lethal malignancy, and AMP-activated protein kinase-related kinase 5 (ARK5) has been discovered to promote cancer metastasis in certain types of cancer. In this study, we explored the role of ARK5 in GC invasion and metastasis. Methods: ARK5 and epithelial-mesenchymal transition (EMT)-related markers were determined by immunohistochemistry and western blot in GC specimens. Other methods including stably transfected against ARK5 into SGC7901 and AGS cells, western blot, migration and invasion assays in vitro and nude mice tumorigenicity in vivo were also employed. Results: The results demonstrated that ARK5 expression was increased and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with GC. Knockdown of ARK5 expression remarkably suppressed GC cells invasion and metastasis via regulating EMT, rather than proliferation in vitro and in vivo. And knockdown of ARK5 expression in GC cells resulted in the down-regulation of the mTOR/p70S6k signals, Slug and SIP1. Conclusion: The elevated ARK5 expression was closely associated with cancer metastasis and patient survival, and it seemed to function in GC cells migration and invasion via EMT alteration, together with the alteration of the mTOR/p70S6k signals, Slug and SIP1, thus providing a potential therapeutic target for GC.

Download Full-text

Intrinsic Disorder in Tetratricopeptide Repeat Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms21103709 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3709 ◽

Cited By ~ 1

Author(s):

Nathan W. Van Bibber ◽

Cornelia Haerle ◽

Roy Khalife ◽

Bin Xue ◽

Vladimir N. Uversky

Keyword(s):

Protein Interactions ◽

Posttranslational Modifications ◽

Tandem Repeats ◽

Intrinsically Disordered Protein ◽

Intrinsic Disorder ◽

Protein Protein Interactions ◽

Systematic Analysis ◽

Tetratricopeptide Repeats ◽

Intrinsically Disordered ◽

Tetratricopeptide Repeat Proteins

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications.

Download Full-text

CD36 promotes the epithelial–mesenchymal transition and metastasis in cervical cancer by interacting with TGF-β

Journal of Translational Medicine ◽

10.1186/s12967-019-2098-6 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 8

Author(s):

Min Deng ◽

Xiaodong Cai ◽

Ling Long ◽

Linying Xie ◽

Hongmei Ma ◽

...

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Mesenchymal Transition ◽

Expression Levels ◽

Cd36 Expression ◽

Cervical Cancer Cells

Abstract Background Accumulating evidence indicates that CD36 initiates metastasis and correlates with an unfavorable prognosis in cancers. However, there are few reports regarding the roles of CD36 in initiation and metastasis of cervical cancer. Methods Using immunohistochemistry, we analyzed 133 cervical cancer samples for CD36 protein expression levels, and then investigated the correlation between changes in its expression and clinicopathologic parameters. The effect of CD36 expression on the epithelial–mesenchymal transition (EMT) in cervical cancer cells was evaluated by Western immunoblotting analysis. In vitro invasion and in vivo metastasis assays were also used to evaluate the role of CD36 in cervical cancer metastasis. Results In the present study, we confirmed that CD36 was highly expressed in cervical cancer samples relative to normal cervical tissues. Moreover, overexpression of CD36 promoted invasiveness and metastasis of cervical cancer cells in vitro and in vivo, while CD36 knockdown suppressed proliferation, migration, and invasiveness. We demonstrated that TGF-β treatment attenuated E-cadherin expression and enhanced the expression levels of CD36, vimentin, slug, snail, and twist in si-SiHa, si-HeLa, and C33a–CD36 cells, suggesting that TGF-β synergized with CD36 on EMT via active CD36 expression. We also observed that the expression levels of TGF-β in si-SiHa cells and si-HeLa cells were down-regulated, whereas the expression levels of TGF-β were up-regulated in C33a–CD36 cells. These results imply that CD36 and TGF-β interact with each other to promote the EMT in cervical cancer. Conclusions Our findings suggest that CD36 is likely to be an effective target for guiding individualized clinical therapy of cervical cancer.

Download Full-text

Inhibitor of DNA-Binding Protein 4 Suppresses Cancer Metastasis through the Regulation of Epithelial Mesenchymal Transition in Lung Adenocarcinoma

Cancers ◽

10.3390/cancers11122021 ◽

2019 ◽

Vol 11 (12) ◽

pp. 2021 ◽

Cited By ~ 1

Author(s):

Chi-Chung Wang ◽

Yuan-Ling Hsu ◽

Chi-Jen Chang ◽

Chia-Jen Wang ◽

Tzu-Hung Hsiao ◽

...

Keyword(s):

Lung Cancer ◽

Dna Binding ◽

Lung Adenocarcinoma ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Inhibitor Of Dna Binding ◽

E Cadherin

Metastasis is a predominant cause of cancer death and the major challenge in treating lung adenocarcinoma (LADC). Therefore, exploring new metastasis-related genes and their action mechanisms may provide new insights for developing a new combative approach to treat lung cancer. Previously, our research team discovered that the expression of the inhibitor of DNA binding 4 (Id4) was inversely related to cell invasiveness in LADC cells by cDNA microarray screening. However, the functional role of Id4 and its mechanism of action in lung cancer metastasis remain unclear. In this study, we report that the expression of Id4 could attenuate cell migration and invasion in vitro and cancer metastasis in vivo. Detailed analyses indicated that Id4 could promote E-cadherin expression through the binding of Slug, cause the occurrence of mesenchymal-epithelial transition (MET), and inhibit cancer metastasis. Moreover, the examination of the gene expression database (GSE31210) also revealed that high-level expression of Id4/E-cadherin and low-level expression of Slug were associated with a better clinical outcome in LADC patients. In summary, Id4 may act as a metastatic suppressor, which could not only be used as an independent predictor but also serve as a potential therapeutic for LADC treatment.

Download Full-text

NRF2 activates a partial epithelial-mesenchymal transition and is maximally present in a hybrid epithelial/mesenchymal phenotype

Integrative Biology ◽

10.1093/intbio/zyz021 ◽

2019 ◽

Vol 11 (6) ◽

pp. 251-263 ◽

Cited By ~ 28

Author(s):

Federico Bocci ◽

Satyendra C Tripathi ◽

Samuel A Vilchez Mercedes ◽

Jason T George ◽

Julian P Casabar ◽

...

Keyword(s):

Cancer Cells ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Therapy Resistance ◽

Collective Cell Migration ◽

Mesenchymal Transition ◽

Regulatory Circuit ◽

Clinical Records ◽

E Cadherin

Abstract The epithelial-mesenchymal transition (EMT) is a key process implicated in cancer metastasis and therapy resistance. Recent studies have emphasized that cells can undergo partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype – a cornerstone of tumour aggressiveness and poor prognosis. These cells can have enhanced tumour-initiation potential as compared to purely epithelial or mesenchymal ones and can integrate the properties of cell-cell adhesion and motility that facilitates collective cell migration leading to clusters of circulating tumour cells (CTCs) – the prevalent mode of metastasis. Thus, identifying the molecular players that can enable cells to maintain a hybrid E/M phenotype is crucial to curb the metastatic load. Using an integrated computational-experimental approach, we show that the transcription factor NRF2 can prevent a complete EMT and instead stabilize a hybrid E/M phenotype. Knockdown of NRF2 in hybrid E/M non-small cell lung cancer cells H1975 and bladder cancer cells RT4 destabilized a hybrid E/M phenotype and compromised the ability to collectively migrate to close a wound in vitro. Notably, while NRF2 knockout simultaneously downregulated E-cadherin and ZEB-1, overexpression of NRF2 enriched for a hybrid E/M phenotype by simultaneously upregulating both E-cadherin and ZEB-1 in individual RT4 cells. Further, we predict that NRF2 is maximally expressed in hybrid E/M phenotype(s) and demonstrate that this biphasic dynamic arises from the interconnections among NRF2 and the EMT regulatory circuit. Finally, clinical records from multiple datasets suggest a correlation between a hybrid E/M phenotype, high levels of NRF2 and its targets and poor survival, further strengthening the emerging notion that hybrid E/M phenotype(s) may occupy the ‘metastatic sweet spot’.

Download Full-text

MicroRNA-26a and -26b inhibit lens fibrosis and cataract by negatively regulating Jagged-1/Notch signaling pathway

Cell Death and Differentiation ◽

10.1038/cdd.2016.152 ◽

2017 ◽

Vol 24 (8) ◽

pp. 1431-1442 ◽

Cited By ~ 20

Author(s):

Xiaoyun Chen ◽

Wei Xiao ◽

Weirong Chen ◽

Xialin Liu ◽

Mingxing Wu ◽

...

Keyword(s):

Epithelial Cells ◽

Notch Signaling ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Notch Pathway ◽

Lens Epithelial Cells ◽

Mesenchymal Transition ◽

Fibrotic Diseases

Abstract Fibrosis is a chronic process involving development and progression of multiple diseases in various organs and is responsible for almost half of all known deaths. Epithelial–mesenchymal transition (EMT) is the vital process in organ fibrosis. Lens is an elegant biological tool to investigate the fibrosis process because of its unique biological properties. Using gain- and loss-of-function assays, and different lens fibrosis models, here we demonstrated that microRNA (miR)-26a and miR-26b, members of the miR-26 family have key roles in EMT and fibrosis. They can significantly inhibit proliferation, migration, EMT of lens epithelial cells and lens fibrosis in vitro and in vivo. Interestingly, we revealed that the mechanisms of anti-EMT effects of miR-26a and -26b are via directly targeting Jagged-1 and suppressing Jagged-1/Notch signaling. Furthermore, we provided in vitro and in vivo evidence that Jagged-1/Notch signaling is activated in TGFβ2-stimulated EMT, and blockade of Notch signaling can reverse lens epithelial cells (LECs) EMT and lens fibrosis. Given the general involvement of EMT in most fibrotic diseases, cancer metastasis and recurrence, miR-26 family and Notch pathway may have therapeutic uses in treating fibrotic diseases and cancers.

Download Full-text