Structural characterization of the DNA binding mechanism of retinoic acid-related orphan receptor gamma.

Retinoic acid-related orphan receptor gamma (RORγ) plays critical roles in regulating various biological processes and has been linked to immunodeficiency disorders and cancers. DNA recognition is essential for RORγ to exert its functions. However, the underlying mechanism of the DNA binding by RORγ remains unclear. In this study, we present the crystal structure of the complex of RORγ1 DNA-binding domain (RORγ1-DBD)/direct repeat DNA element DR2 at 2.3 Å resolution. We demonstrate that RORγ1-DBD binds the DR2 motif as a homodimer, with the C-terminal extension (CTE) region of RORγ1-DBD contributing to the DNA recognition and the formation of dimeric interface. Further studies reveal that REV-ERB-DBD and RXR-DBD, also bind the DR2 site as a homodimer, while NR4A2-DBD binds DR2 as a monomer. Our research uncovers a binding mechanism of RORγ1 to the DR2 site and provides insights into the biological functions of RORγ1 and the broader RORs subfamily.

Amyloid aggregates induced by the p53-R280T mutation lead to loss of p53 function in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor that is highly prevalent in Southeast Asia, especially in South China. The pathogenesis of NPC is complex, and genetic alterations of tumor suppressors and proto-oncogenes play important roles in NPC carcinogenesis. p53 is unexpectedly highly expressed in NPC and possesses an uncommon mutation of R280T, which is different from a high frequency of hotspot mutations or low expression in other tumors. However, the mechanism of p53 loss of function and its correlation with R280T in NPC are still unclear. In this study, p53 amyloid aggregates were found to be widespread in NPC and can be mainly induced by the R280T mutation. Aggregated p53-R280T impeded its entry into the nucleus and was unable to initiate the transcription of downstream target genes, resulting in decreased NPC cell cycle arrest and apoptosis. In addition, NPC cells with p53-R280T amyloid aggregates also contributed aggressively to tumor growth in vivo. Transcriptome analysis suggested that p53 amyloid aggregation dysregulated major signaling pathways associated with the cell cycle, proliferation, apoptosis, and unfolded protein response (UPR). Further studies revealed that Hsp90, as a key molecular chaperone in p53 folding, was upregulated in NPC cells with p53-R280T aggregation, and the upregulated Hsp90 facilitated p53 aggregation in turn, forming positive feedback. Therefore, Hsp90 inhibitors could dissociate p53-R280T aggregation and restore the suppressor function of p53 in vitro and in vivo. In conclusion, our study demonstrated that p53-R280T may misfold to form aggregates with the help of Hsp90, resulting in the inability of sequestered p53 to initiate the transcription of downstream target genes. These results revealed a new mechanism for the loss of p53 function in NPC and provided novel mechanistic insight into NPC pathogenesis.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Structural insight into the macrocyclic inhibitor TPX-0022 of c-Met and c-Src.

c-Met has been an attractive target of prognostic and therapeutic studies in various cancers. TPX-0022 is a macrocyclic inhibitor of c-Met, c-Src and CSF1R kinases and is currently in phase I/II clinical trials in patients with advanced solid tumors harboring MET gene alterations. In this study, we determined the co-crystal structures of the c-Met/TPX-0022 and c-Src/TPX-0022 complexes to help elucidate the binding mechanism. TPX-0022 binds to the ATP pocket of c-Met and c-Src in a local minimum energy conformation and is stabilized by hydrophobic and hydrogen bond interactions. In addition, TPX-0022 exhibited potent activity against the resistance-relevant c-Met L1195F mutant and moderate activity against the c-Met G1163R, F1200I and Y1230H mutants but weak activity against the c-Met D1228N and Y1230C mutants. Overall, our study reveals the structural mechanism underlying the potency and selectivity of TPX-0022 and the ability to overcome acquire resistance mutations and provides insight into the development of selective c-Met macrocyclic inhibitors.

Structures of p53/BCL-2 complex suggest a mechanism for p53 to antagonize BCL-2 activity.

Mitochondrial apoptosis is strictly controlled by BCL-2 family proteins through a subtle network of protein interactions. The tumor suppressor protein p53 triggers transcription-independent apoptosis through direct interactions with BCL-2 family proteins, but the molecular mechanism is not well understood. In this study, we present three crystal structures of p53-DBD in complex with the anti-apoptotic protein BCL-2 at resolutions of 2.3-2.7 Å. The structures show that two loops of p53-DBD penetrate directly into the BH3-binding pocket of BCL-2. Structure-based mutations at the interface impair the p53/BCL-2 interaction. Specifically, the binding sites for p53 and the pro-apoptotic protein Bax in the BCL-2 pocket are mostly identical. In addition, formation of the p53/BCL-2 complex is negatively correlated with the formation of BCL-2 complexes with pro-apoptotic BCL-2 family members. Defects in the p53/BCL-2 interaction attenuate p53-mediated cell apoptosis. Overall, our study provides a structural basis for the interaction between p53 and BCL-2, and suggests a molecular mechanism by which p53 regulates transcription-independent apoptosis by antagonizing the interaction of BCL-2 with pro-apoptotic BCL-2 family members.

Structural basis of the farnesoid X receptor/retinoid X receptor heterodimer on inverted repeat DNA.

Farnesoid X receptor (FXR) is a ligand-activated transcription factor known as bile acid receptor (BAR). FXR plays critical roles in various biological processes, including metabolism, immune inflammation, liver regeneration and liver carcinogenesis. FXR forms a heterodimer with the retinoid X receptor (RXR) and binds to diverse FXR response elements (FXREs) to exert its various biological functions. However, the mechanism by which the FXR/RXR heterodimer binds the DNA elements remains unclear. In this study, we aimed to use structural, biochemical and bioinformatics analyses to study the mechanism of FXR binding to the typical FXRE, such as the IR1 site, and the heterodimer interactions in the FXR-DBD/RXR-DBD complex. Further biochemical assays showed that RAR, THR and NR4A2 do not form heterodimers with RXR when bound to the IR1 sites, which indicates that IR1 may be a unique binding site for the FXR/RXR heterodimer. Our studies may provide a further understanding of the dimerization specificity of nuclear receptors.

Proteomics Profiling Reveals the Molecular Signatures and Potential Therapeutic Targets of Human Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a malignant tumor distinctly characterized by ethnic and geographic distribution, is highly prevalent in Southern China and Southeast Asia. However, the molecular mechanisms of NPC have not been fully revealed at the proteomic level. In this study, 30 primary NPC samples and 22 normal nasopharyngeal epithelial tissues were collected for proteomics analysis, and a relatively complete proteomics landscape of NPC was depicted for the first time. By combining differential expression analysis, differential co-expression analysis, and network analysis, potential biomarkers and therapeutic targets were identified. Some identified targets were verified by biological experiments. We found that 17-AAG, a specific inhibitor of the identified target heat shock protein 90 (HSP90), could be a potential therapeutic drug for NPC. Finally, consensus clustering identified two NPC subtypes with specific molecular features. The subtypes and the related molecules were verified by an independent data set and may have different progression-free survival. The results of this study provide a comprehensive understanding of the proteomics molecular signatures of NPC and provide new perspectives and inspiration for prognostic determination and treatment of NPC.

CapG promoted nasopharyngeal carcinoma cell motility involving Rho motility pathway independent of ROCK.

BACKGROUND: Gelsolin-like capping actin protein (CapG) modulates actin dynamics and actin-based motility with a debatable role in tumorigenic progression. The motility-associated functions and potential molecular mechanisms of CapG in nasopharyngeal carcinoma (NPC) remain unclear. METHODS: CapG expression was detected by immunohistochemistry in a cohort of NPC tissue specimens and by Western blotting assay in a variety of NPC cell lines. Loss of function and gain of function of CapG in scratch wound-healing and transwell assays were performed. Inactivation of Rac1 and ROCK with the specific small molecular inhibitors was applied to evaluate CapG's role in NPC cell motility. GTP-bound Rac1 and phosphorylated-myosin light chain 2 (p-MLC2) were measured in the ectopic CapG overexpressing cells. Finally, CapG-related gene set enrichment analysis was conducted to figure out the significant CapG-associated pathways in NPC. RESULTS: CapG disclosed increased level in the poorly differentiated NPC tissues and highly metastatic cells. Knockdown of CapG reduced NPC cell migration and invasion in vitro, while ectopic CapG overexpression showed the opposite effect. Ectopic overexpression of CapG compensated for the cell motility loss caused by simultaneous inactivation of ROCK and Rac1 or inactivation of ROCK alone. GTP-bound Rac1 weakened, and p-MLC2 increased in the CapG overexpressing cells. Bioinformatics analysis validated a positive correlation of CapG with Rho motility signaling, while Rac1 motility pathway showed no significant relationship. CONCLUSIONS: The present findings highlight the contribution of CapG to NPC cell motility independent of ROCK and Rac1. CapG promotes NPC cell motility at least partly through MLC2 phosphorylation and contradicts with Rac1 activation.

p53 amyloid aggregation in cancer: function, mechanism, and therapy.

Similar to neurodegenerative diseases, the concept that tumors are prion like diseases has been proposed in recent years. p53, the most well-known tumor suppressor, has been extensively studied for its expression, mutation, and function in various tumors. Currently, an interesting phenomenon of p53 prion-like aggregation has been found in several tumors, and studies have found that its pathological aggregation may lead to functional alterations and ultimately affect tumor progression. It has been demonstrated that the mechanism of p53 aggregation involves its mutation, domains, isoform, etc. In addition to p53 itself, some other factors, including Zn2+ concentration, pH, temperature and chaperone abnormalities, can also contribute to p53 aggregation. Although there are some studies about the mechanism and role of p53 aggregation and amyloidosis in tumors, there still exist some controversies. In this paper, we review the mechanism of p53 amyloid fibril structure and discuss the characteristics and effects of p53 amyloid aggregation, as well as the pathogenic mechanism leading to the occurrence of aggregation in tumors. Finally, we summarize the various inhibitors targeting p53 aggregation and prion-like behavior. In conclusion, a comprehensive understanding of p53 aggregation can expand our understanding of the causes leading its loss of physiological function and that targeting p53 aggregation might be a promising therapeutic strategy for tumor therapy.

Suberoylanilide Hydroxamic Acid (SAHA) Treatment Reveals Crosstalk Among Proteome, Phosphoproteome, and Acetylome in Nasopharyngeal Carcinoma Cells.

Suberoylanilide hydroxamic acid (SAHA), a famous histone deacetylase (HDAC) inhibitor, has been utilized in clinical treatment for cutaneous T-cell lymphoma. Previously, the mechanisms underlying SAHA anti-tumor activity mainly focused on acetylome. However, the characteristics of SAHA in terms of other protein posttranslational modifications (PTMs) and the crosstalk between various modifications are poorly understood. Our previous work revealed that SAHA had anti-tumor activity in nasopharyngeal carcinoma (NPC) cells as well. Here, we reported the profiles of global proteome, acetylome, and phosphoproteome of 5-8 F cells upon SAHA induction and the crosstalk between these data sets. Overall, we detected and quantified 6,491 proteins, 2,456 phosphorylated proteins, and 228 acetylated proteins in response to SAHA treatment in 5-8 F cells. In addition, we identified 46 proteins exhibiting both acetylation and phosphorylation, such as WSTF and LMNA. With the aid of intensive bioinformatics analyses, multiple cellular processes and signaling pathways involved in tumorigenesis were clustered, including glycolysis, EGFR signaling, and Myc signaling pathways. Taken together, this study highlighted the interconnectivity of acetylation and phosphorylation signaling networks and suggested that SAHA-mediated HDAC inhibition may alter both acetylation and phosphorylation of viral proteins. Subsequently, cellular signaling pathways were reprogrammed and contributed to anti-tumor effects of SAHA in NPC cells.

Characterization of the modification of Kelch-like ECH-associated protein 1 by different fumarates.

Fumarates (fumaric acid esters), primarily dimethyl fumarate (DMF) and monoethyl fumarate (MEF) and its salts, are orally administered systemic agents used for the treatment of psoriasis and multiple sclerosis. It is widely believed that the pharmaceutical activities of fumarates are exerted through the Keap1-Nrf2 pathway. Although it has been revealed that DMF and MEF differentially modify specific Keap1 cysteine residues and result in the differential activation of Nrf2, how the modification of DMF and MEF impacts the biochemical properties of Keap1 has not been well characterized. Here, we found that both DMF and MEF can only modify the BTB domain of Keap1 and that only C151 is accessible for covalent binding in vitro. Dynamic fluorescence scanning (DSF) assays showed that the modification of DMF to Keap1 BTB increased its thermal stability, while the modification of MEF dramatically decreased its thermal stability. Further crystal structures revealed no significant conformational variation between the DMF-modified and MEF-modified BTBs. Overall, our biochemical and structural study provides a better understanding of the covalent modification of fumarates to Keap1 and may suggest fundamentally different mechanisms adopted by fumarates in regulating the Keap1-Nrf2 pathway.

Structural study of ponatinib in inhibiting SRC kinase.

Ponatinib is a multi-target tyrosine kinase inhibitor that targets ABL, SRC, FGFR, and so on. It was designed to overcome the resistance of BCR-ABL mutation to imatinib, especially the gatekeeper mutation ABLT315I. The molecular mechanism by which ponatinib overcomes mutations of BCR-ABL and some other targets has been explained, but little information is known about the characteristics of ponatinib binding to SRC. Here, we showed that ponatinib inhibited wild type SRC kinase but failed to inhibit SRC gatekeeper mutants in both biochemical and cellular assays. We determined the crystal structure of ponatinib in complex with the SRC kinase domain. In addition, by structural analysis, we provided a possible explanation for why ponatinib showed different effects on SRC and other kinases with gatekeeper mutations. The resistance mechanism of SRC gatekeeper mutations to ponatinib may provide meaningful information for designing inhibitors against SRC family kinases in the future.

Structural insights into the potency and selectivity of covalent pan-FGFR inhibitors.

FIIN-2, TAS-120 (Futibatinib) and PRN1371 are highly potent pan-FGFR covalent inhibitors targeting the p-loop cysteine of FGFR proteins, of which TAS-120 and PRN1371 are currently in clinical trials. It is critical to analyze their target selectivity and their abilities to overcome gatekeeper mutations. In this study, we demonstrate that FIIN-2 and TAS-120 form covalent adducts with SRC, while PRN1371 does not. FIIN-2 and TAS-120 inhibit SRC and YES activities, while PRN1371 does not. Moreover, FIIN-2, TAS-120 and PRN1371 exhibit different potencies against different FGFR gatekeeper mutants. In addition, the co-crystal structures of SRC/FIIN-2, SRC/TAS-120 and FGFR4/PRN1371 complexes reveal structural basis for kinase targeting and gatekeeper mutations. Taken together, our study not only provides insight into the potency and selectivity of covalent pan-FGFR inhibitors, but also sheds light on the development of next-generation FGFR covalent inhibitors with high potency, high selectivity, and stronger ability to overcome gatekeeper mutations.

Afatinib Reverses EMT via Inhibiting CD44-Stat3 Axis to Promote Radiosensitivity in Nasopharyngeal Carcinoma.

BACKGROUND: Afatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC. METHODS: Quantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo. RESULTS: In the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis. CONCLUSION: Collectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.

Coupling of Cell Surface Biotinylation and SILAC-Based Quantitative Proteomics Identified Myoferlin as a Potential Therapeutic Target for Nasopharyngeal Carcinoma Metastasis.

Distant metastasis is a major cause of treatment failure in nasopharyngeal carcinoma (NPC) patients. Cell surface proteins represent attractive targets for cancer diagnosis or therapy. However, the cell surface proteins associated with NPC metastasis are poorly understood. To identify potential therapeutic targets for NPC metastasis, we isolated cell surface proteins from two isogenic NPC cell lines, 6-10B (low metastatic) and 5-8F (highly metastatic), through cell surface biotinylation. Stable isotope labeling by amino acids in cell culture (SILAC) based proteomics was applied to comprehensively characterize the cell surface proteins related with the metastatic phenotype. We identified 294 differentially expressed cell surface proteins, including the most upregulated protein myoferlin (MYOF), two receptor tyrosine kinases(RTKs) epidermal growth factor receptor (EGFR) and ephrin type-A receptor 2 (EPHA2) and several integrin family molecules. These differentially expressed proteins are enriched in multiple biological pathways such as the FAK-PI3K-mTOR pathway, focal adhesions, and integrin-mediated cell adhesion. The knockdown of MYOF effectively suppresses the proliferation, migration and invasion of NPC cells. Immunohistochemistry analysis also showed that MYOF is associated with NPC metastasis. We experimentally confirmed, for the first time, that MYOF can interact with EGFR and EPHA2. Moreover, MYOF knockdown could influence not only EGFR activity and its downstream epithelial-mesenchymal transition (EMT), but also EPHA2 ligand-independent activity. These findings suggest that MYOF might be an attractive potential therapeutic target that has double effects of simultaneously influencing EGFR and EPHA2 signaling pathway. In conclusion, this is the first study to profile the cell surface proteins associated with NPC metastasis and provide valuable resource for future researches.

Structural insight into the molecular mechanism of p53-mediated mitochondrial apoptosis.

The tumor suppressor p53 is mutated in approximately half of all human cancers. p53 can induce apoptosis through mitochondrial membrane permeabilization by interacting with and antagonizing the anti-apoptotic proteins BCL-xL and BCL-2. However, the mechanisms by which p53 induces mitochondrial apoptosis remain elusive. Here, we report a 2.5 Å crystal structure of human p53/BCL-xL complex. In this structure, two p53 molecules interact as a homodimer, and bind one BCL-xL molecule to form a ternary complex with a 2:1 stoichiometry. Mutations at the p53 dimer interface or p53/BCL-xL interface disrupt p53/BCL-xL interaction and p53-mediated apoptosis. Overall, our current findings of the bona fide structure of p53/BCL-xL complex reveal the molecular basis of the interaction between p53 and BCL-xL, and provide insight into p53-mediated mitochondrial apoptosis.

Discovery of a novel AR/HDAC6 dual inhibitor for prostate cancer treatment.

Androgen receptor (AR) and histone deacetylase 6 (HDAC6) are important targets for cancer therapy. Given that both AR antagonists and HDAC6 inhibitors modulate AR signaling, a novel AR/HDAC6 dual inhibitor is investigated for its anticancer effects in castration-resistant prostate cancer (CRPC). Zeta55 inhibits nuclear translocation of AR and suppresses androgen-induced PSA and TMPRSS2 expression. Meanwhile, Zeta55 selectively inhibits HDAC6 activity, leading to AR degradation. Zeta55 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a CRPC xenograft model. These results provide preclinical proof of principle for Zeta55 as a promising therapeutic in prostate cancer treatment.

Mechanism of forkhead transcription factors binding to a novel palindromic DNA site.

Forkhead transcription factors bind a canonical consensus DNA motif, RYAAAYA (R = A/G, Y = C/T), as a monomer. However, the molecular mechanisms by which forkhead transcription factors bind DNA as a dimer are not well understood. In this study, we show that FOXO1 recognizes a palindromic DNA element DIV2, and mediates transcriptional regulation. The crystal structure of FOXO1/DIV2 reveals that the FOXO1 DNA binding domain (DBD) binds the DIV2 site as a homodimer. The wing1 region of FOXO1 mediates the dimerization, which enhances FOXO1 DNA binding affinity and complex stability. Further biochemical assays show that FOXO3, FOXM1 and FOXI1 also bind the DIV2 site as homodimer, while FOXC2 can only bind this site as a monomer. Our structural, biochemical and bioinformatics analyses not only provide a novel mechanism by which FOXO1 binds DNA as a homodimer, but also shed light on the target selection of forkhead transcription factors.

Characterization of ibrutinib as a non-covalent inhibitor of SRC-family kinases.

Ibrutinib is a BTK-targeted irreversible inhibitor. In this study, we demonstrate that ibrutinib potently inhibits SRC activity in a non-covalent manner via mass spectrometry and crystallography. The S345C mutation renders SRC to bind covalently with ibrutinib, and restores the potency of ibrutinib against the gatekeeper mutant. The co-crystal structure of ibrutinib/SRC shows Ser345 of SRC did not form covalent bond with ibrutinib, leading to a decrease of potency and loss of the ability to overcome the gatekeeper mutation of SRC. The X-ray crystallographic studies also provide structural insight into why ibrutinib behaves differently against gatekeeper mutants of different kinases.