Steroid receptor coactivator 1 promotes human hepatocellular carcinoma invasiveness through enhancing MMP-9.

SRC-1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC-1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC-1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta-catenin signalling. In this study, we uncover a previously unknown role of SRC-1 in HCC metastasis. Our findings reveal that SRC-1 promotes HCC metastasis through the augmentation of MMP-9 expression. The knockdown of SRC-1 effectively mitigated HCC cell metastasis both in vitro and in vivo by suppressing MMP-9 expression. Furthermore, we observed a positive correlation between SRC-1 mRNA levels and MMP-9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC-1 operates as a coactivator for NF-κB and AP-1, enhancing MMP-9 promoter activity in HCC cells. Higher levels of SRC-1 and MMP-9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC-1 expression, significantly decreased MMP-9 expression and inhibited HCC metastasis in both in vitro and in vivo settings. Our results demonstrated the pivotal role of SRC-1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.

The Src1-PGC1α-AP1 complex-dependent secretion of substance P induces inflammation and apoptosis in encephalomyocarditis virus-infected mice.

Substance P (SP), a neuropeptide consisting of 11 amino acid residues, is involved in the pathogenesis of encephalomyocarditis virus (EMCV)-induced myocarditis by stimulating the production of proinflammatory cytokines. However, the underlying mechanism that regulates SP production is still unknown. In this study, we report the transcriptional regulation of the Tachykinin Precursor 1 (TAC1) gene that encodes SP by a transcriptional complex composed of Steroid Receptor Coactivator 1 (Src1), Peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1α), and Activator Protein 1 (AP1) transcription factor. Infection of mice with EMCV induced the accumulation of PGC1α and increased TAC1 expression, thereby promoting the secretion of SP, initiating apoptosis, and elevating proinflammatory cytokine levels. In vitro overexpression of the Src1-PGC1α-AP1 members also induced TAC1 expression, increased the SP concentration, initiated apoptosis, and elevated proinflammatory cytokine concentrations. Depletion or inhibition of the Src1-PGC1α-AP1 complex reversed these effects. The administration of gossypol, an Src1 inhibitor, or SR1892, a PGC1α inhibitor, to EMCV-infected mice attenuated myocarditis. Taken together, our results reveal that the upregulation of TAC1 and the secretion of SP in EMCV-induced myocarditis are dependent on the Src1-PGC1α-AP1 complex. Targeting the Src1-PGC1α-AP1 complex may represent a new therapeutic strategy for myocarditis.

Dynamic interplay between corticosteroid treatment and the role of SRC-1 gene dysregulation in the progression of WHO-Grade 4 Astrocytoma.

BACKGROUND: Corticosteroid is commonly used before surgery to control cerebral oedema in brain tumours and is frequently continued throughout treatment. Its long-term effect of on the recurrence of WHO-Grade 4 astrocytoma remains controversial. The interaction between corticosteroid, SRC-1 gene and cytotoxic T-cells has never been investigated. METHODS: A retrospective cohort of 36 patients with WHO-Grade 4 astrocytoma were examined for CD8 + T-cell and SRC-1 gene expressions through IHC and qRT-PCR. The impact of corticosteroid on CD8+T-cells infiltration, SRC-1 expression, and tumour recurrence was analyzed. RESULTS: The mean patients age was 47-years, with a male to female ratio 1.2. About 78% [n = 28] of the cases showed reduced or no CD8+T-cell expression while 22% [n = 8] of cases have showed medium to high CD8+T-cell expression. SRC-1 gene was upregulated in 5 cases [14%] and 31 cases [86%] showed SRC-1 downregulation. The average of total days and doses of administered corticosteroid from the preoperative period to the postoperative period was at range of 14-106 days and 41-5028 mg, respectively. There was no significant statistical difference in RFI among tumours expressing high or low CD8+T-cells when corticosteroid was administered in recommended or exceeded doses [p-value = 0.640]. There was a significant statistical difference in RFI between CD8+T-Cell expression and SRC-1 gene dysregulation [p-value = 002]. Tumours with high CD8+T T-cell expression and SRC-1 gene downregulation had late recurrence. CONCLUSIONS: Corticosteroid treatment can directly affect the SRC-1 gene regulation but does not directly influence cytotoxic T-cells infiltration or tumor progression. However, SRC-1 gene downregulation can facilitate late tumor recurrence.

The NCOA1-CBP-NF-κB transcriptional complex induces inflammation response and triggers endotoxin-induced myocardial dysfunction.

Inflammatory pathways represented by TLR4/NF-κB (Toll-like receptor 4/Nuclear factor-κB) axis signaling are activated in the pathogenesis of endotoxin-induced myocardial dysfunction (EIMD). However, the underlying mechanism by which NF-κB coordinates with other transcriptional coactivators/corepressors to regulate the expression of proinflammatory cytokine genes remains unclear. We established an EIMD-mouse model by intraperitoneal injection of lipopolysaccharides (LPS), and we discovered that NCOA1 (nuclear receptor coactivator 1) assembled with CBP (CREB binding protein) and NF-κB subunits to form a transcriptional complex that specifically bound to promoters of proinflammatory cytokine genes to activate their expression. LPS treatment also inhibited DNMT1 (DNA methyltransferase 1) expression, thereby decreasing DNA methylation of a CpG island located on the promoter of NCOA1 and causing NCOA1 overexpression. Screening small molecules that abolished NCOA1-CBP interaction in a yeast system identified a compound PSSM2126 that effectively blocked the NCOA1-CBP interaction in vitro and in vivo. Administration of PSSM2126 to EIMD mice significantly alleviated the inflammation response and improved cardiac function. Collectively, our results reveal that an NCOA1-dependent transactivation mechanism can regulate proinflammatory cytokine expression, thereby improving our understanding of the activation of NF-κB targets. The promising inhibition of the NCOA1-CBP interaction by PSSM2126 may provide a new therapeutic option for EIMD.

SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites.

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.

An acetylation-enhanced interaction between transcription factor Sox2 and the steroid receptor coactivators facilitates Sox2 transcriptional activity and function.

SRY-box 2 (Sox2) is a transcription factor with critical roles in maintaining embryonic stem (ES) cell and adult stem cell functions and in tumorigenesis. However, how Sox2 exerts its transcriptional function remains unclear. Here, we used an in vitro protein-protein interaction assay to discover transcriptional regulators for ES cell core transcription factors (Oct4, Sox2, Klf4, and c-Myc) and identified members of the steroid receptor coactivators (SRCs) as Sox2-specific interacting proteins. The SRC family coactivators have broad roles in transcriptional regulation, but it is unknown whether they also serve as Sox2 coactivators. We demonstrated that these proteins facilitate Sox2 transcriptional activity and act synergistically with p300. Furthermore, we uncovered an acetylation-enhanced interaction between Sox2 and SRC-2/3, but not SRC-1, demonstrating it is Sox2 acetylation that promotes the interaction. We identified putative Sox2 acetylation sites required for acetylation-enhanced interaction between Sox2 and SRC-3 and demonstrated that acetylation on these sites contributes to Sox2 transcriptional activity and recruitment of SRC-3. We showed that activation domains 1 and 2 of SRC-3 both display a preferential binding to acetylated Sox2. Finally, functional analyses in mouse ES cells demonstrated that knockdown of SRC-2/3 but not SRC-1 in mouse ES cells significantly downregulates the transcriptional activities of various Sox2 target genes and impairs ES cell stemness. Taken together, we identify specific SRC family proteins as novel Sox2 coactivators and uncover the role of Sox2 acetylation in promoting coactivator recruitment and Sox2 transcriptional function.

Heterodimer formation with retinoic acid receptor RXRα modulates coactivator recruitment by peroxisome proliferator-activated receptor PPARγ.

Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET-based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.

Steroid Receptor Coactivator-1 (SRC-1) Promoted Cell Metastasis of Gastric Cancer via VEGFC Activator by NF-κB.

The regulatory mechanism and function of steroid receptor coactivator-1 (SRC-1) was determined in vitro and the role played in gastric cancer was investigated. The study collected 64 patients with gastric cancer tissue and paracancerous tissue to investigate the clinical patterns of SRC-1 expression in gastric cancer. Quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, and immunofluorescence staining were used in this study. In patients with gastric cancer, SRC-1 serum expression levels were up-regulated. Over-expression of SRC-1 promoted cell growth and cell metastasis in vitro model of gastric cancer. However, down-regulation of SRC-1 reduced cell growth and cell metastasis in vitro model of gastric cancer. SRC-1 over-expression induced vascular endothelial growth factor C (VEGFC) protein expressions in vitro model by activation of nuclear factor-kappa B (NF-kB) expression. The inhibition of NF-κB reduced the pro-cancer effects of SRC-1 on cell growth and cell metastasis in vitro model of gastric cancer through inhibition of VEGFC expression. These results suggest that SRC-1 promoted cell metastasis of gastric cancer via VEGFC activator by NF-κB. These novel findings may shed further light on the pathogenesis of gastric cancer and on potential precursor markers.

BRCA2 represses the transcriptional activity of pS2 by E2-ERα.

Germline mutations to the breast cancer 2 (BRCA2) gene have been associated with hereditary breast cancer. In addition to estrogen uptake, BRCA2 expression increases in the S phase of the cell cycle and largely contributes to DNA damage repair associated with DNA replication. However, the role of BRCA2 in estrogen induction remains unclear. An expression plasmid was created to induce BRCA2 activation upon the addition of estradiol by introducing mutations to the binding sequences for the transcription factors USF1, E2F1, and NF-κB within the promoter region of BRCA2. Then, the estrogen receptor (ER) sites of the proteins that interact with BRCA2 upon the addition of estradiol were identified. Both proteins were bound by the helical domain of BRCA2 and activation function-2 of the ER, suggesting that this binding may regulate the transcriptional activity of pS2, a target gene of the estradiol-ER, by suppressing the binding of SRC-1, a coactivator required for activation of the transcription factor.

Pigmented PRRX1::NCOA1-rearranged fibroblastic tumor: A rare morphologic variant of an emerging mesenchymal tumor.

PRRX::NCOAx-rearranged fibroblastic tumor is a recently described, morphologically distinctive subcutaneous fibroblastic tumor with benign behavior. To date, 12 cases have been reported. Here, we report a new case of PRRX::NCOAx-rearranged fibroblastic tumor showing a prominent pigmented component. The lesion occurred on the shoulder of a 23-year-old male. It was an at least 2.5 cm subcutaneous tumor with a multinodular and plexiform appearance. Morphologically, the tumor was characterized by a variably cellular proliferation of uniform oval to spindle cells arranged in fascicles and cords within a myxocollagenous stroma. Irregular, elongated, dilated vessels were prominent at the periphery of tumor nodules. In addition, nests and clusters of pigment-laden epithelioid and dendritic cells were present. Immunohistochemically, the non-pigmented tumor cells showed patchy positivity for factor XIIIa and focal positivity for S100 protein. The pigmented cells were positive for S100 protein, SOX10, MITF, and a pan-melanocytic cocktail (Melan-A, HMB-45, and tyrosinase). Next-generation RNA sequencing identified an in-frame PRRX1::NCOA1 fusion. In summary, this case highlights a rare pigmented variant of PRRX::NCOAx-rearranged fibroblastic tumor, expanding the morphologic spectrum of this newly described mesenchymal tumor.

Modification of ASC1 by UFM1 is crucial for ERα transactivation and breast cancer development.

Biological roles for UFM1, a ubiquitin-like protein, are largely unknown, and therefore we screened for targets of ufmylation. Here we show that ufmylation of the nuclear receptor coactivator ASC1 is a key step for ERα transactivation in response to 17ß-estradiol (E2). In the absence of E2, the UFM1-specific protease UfSP2 was bound to ASC1, which maintains ASC1 in a nonufmylated state. In the presence of E2, ERα bound ASC1 and displaced UfSP2, leading to ASC1 ufmylation. Polyufmylation of ASC1 enhanced association of p300, SRC1, and ASC1 at promoters of ERα target genes. ASC1 overexpression or UfSP2 knockdown promoted ERα-mediated tumor formation in vivo, which could be abrogated by treatment with the anti-breast cancer drug tamoxifen. In contrast, expression of ufmylation-deficient ASC1 mutant or knockdown of the UFM1-activating E1 enzyme UBA5 prevented tumor growth. These findings establish a role for ASC1 ufmylation in breast cancer development by promoting ERα transactivation.

Steroid receptor coactivator-1 enhances the stemness of glioblastoma by activating long noncoding RNA XIST/miR-152/KLF4 pathway.

Glioblastoma (GBM) recurrence is attributed to the presence of therapy-resistant glioblastoma stem cells. Steroid receptor coactivator-1 (SRC-1) acts as an oncogenic regulator in many human tumors. The relationship between SRC-1 and GBM has not yet been studied. Herein, we investigate the role of SRC-1 in GBM. In this study, we found that SRC-1 expression is positively correlated with grades of glioma and inversely correlated with glioma patient's prognosis. Steroid receptor coactivator-1 promotes the proliferation, migration, and tumor growth of GBM cells. Notably, SRC-1 knockdown suppresses the stemness of GBM cells. Mechanistically, long noncoding RNA X-inactive specific transcript (XIST) is regulated by SRC-1 at the posttranscriptional level and mediates the function of SRC-1 in promoting stemness-like properties of GBM. Steroid receptor coactivator-1 can promote the expression of Kruppel-like factor 4 (KLF4) through the XIST/microRNA (miR)-152 axis. Additionally, arenobufagin and bufalin, SRC small molecule inhibitors, can reduce the proliferation and stemness of GBM cells. This study reveals SRC-1 promotes the stemness of GBM by activating the long noncoding RNA XIST/miR-152/KLF4 pathway and provides novel markers for diagnosis and therapy of GBM.

Connexin32 activates necroptosis through Src-mediated inhibition of caspase 8 in hepatocellular carcinoma.

Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.

Recurrent MEIS1-NCOA2/1 fusions in a subset of low-grade spindle cell sarcomas frequently involving the genitourinary and gynecologic tracts.

Sarcomas with MEIS1-NCOA2 fusions have been so far reported in 2 cases each of primitive renal sarcomas and intraosseous pelvic rhabdomyosarcomas. Their histologic spectrum, anatomic distribution, and clinical behavior remain poorly defined. In this study, we report 6 additional spindle cell sarcomas with MEIS1-NCOA2 or NCOA1 fusions that fall into the same disease spectrum with the previously reported renal sarcomas. The patients' age range was wide (20-76 years, mean 46) and all except one were female. The tumors arose in the kidney (n = 2), and one each in the uterine corpus, vagina, scrotum, and para-rectal region. The consistent morphology was that of monomorphic spindle to ovoid cells in a storiform, whorling, or solid pattern. Alternating cellularity, myxoid stroma, and microcystic changes were seen in some cases. Mitotic activity varied greatly (<1-33/10 high power fields). The immunophenotype was nonspecific, with most cases expressing variable degrees of TLE1, WT1, cyclin D1, CD56, and CD10. Using various platforms of RNA-based targeted sequencing, MEIS1-NCOA2 fusions were recurrently identified in 5 cases, and a novel MEIS1-NCOA1 fusion was found in one renal tumor. The gene fusions were validated by fluorescence in situ hybridization using custom BAC probes. Of the 5 patients with available follow-up (5 months to 8 years), all experienced local recurrences, but no distant spread or death from disease. Our results expand the clinicopathologic spectrum of sarcomas with MEIS1-NCOA2/1 fusions, providing evidence of an undifferentiated spindle cell phenotype with nonspecific immunoprofile and low-grade clinical behavior.

PRRX1-NCOA1-rearranged fibroblastic tumour: a clinicopathological, immunohistochemical and molecular genetic study of six cases of a potentially under-recognised, distinctive mesenchymal tumour.

AIMS: PRRX1-NCOA1-rearranged fibroblastic tumour is a recently described, rare mesenchymal tumour. Only four cases have been previously reported. The aim of this article is to report six additional cases of this unusual mesenchymal neoplasm, with an emphasis on its differential diagnosis. METHODS AND RESULTS: The six cases were from three females and three males (age, 20-49 years; median, 42 years). Three tumours were located on the abdominal wall; two from the shoulder/axillary areas, and one on the lateral hip. All presented as slow-growing subcutaneous nodules, ranging from 26 to 55 mm (median, 40 mm). The tumours consisted of circumscribed, variably cellular nodules composed of relatively bland plump spindled to epithelioid cells arranged singly, in cords, and occasionally in nests, embedded in hyalinised and collagenous stroma. Small hypocellular myxoid zones with ropey collagen fibres were present, as were irregularly dilated, gaping, crescent-shaped or staghorn-like thin-walled vessels, best appreciated at the periphery. Immunohistochemistry for CD34, S100, MUC4 and STAT6 was consistently negative. RNA-sequencing revealed PRRX1-NCOA1 fusions in all cases. Of the four cases with limited follow-up (1.5-4 months), none recurred following local surgical excision. CONCLUSIONS: The morphological features of PRRX1-NCOA1-rearranged fibroblastic tumour overlap with those of RB1-deficient soft-tissue tumours, solitary fibrous tumour, and low-grade fibromyxoid sarcoma/sclerosing epithelioid fibrosarcoma. This differential diagnosis can be resolved with a combination of careful morphological study and the application of a panel of immunostains, although molecular genetic study is most definitive. The natural history of PRRX1-NCOA1-rearranged fibroblastic tumour appears to be quite favourable, although longer-term study of a larger number of cases is warranted.

SRC-1 Deficiency Increases Susceptibility of Mice to Depressive-Like Behavior After Exposure to CUMS.

Steroid receptor coactivator 1 (SRC-1) is one of the coactivators recruited by the nuclear receptors (NRs) when NRs are activated by steroid hormones, such as glucocorticoid. SRC-1 is abundant in hippocampus and hypothalamus and is also related to some major risk factors for depression, implicated by its reduced expression after stress and its effect on hypothalamus-pituitary-adrenal gland axis function. However, whether SRC-1 is involved in the formation of depression remains unclear. In this study, we firstly established chronic unpredictable stress (CUS) to induce depressive-like behaviors in mice and found that SRC-1 expression was reduced by CUS. A large number of studies have shown that neuroinflammation is associated with stress-induced depression and lipopolysaccharide (LPS) injection can lead to neuroinflammation and depressive-like behaviors in mice. Our result indicated that LPS treatment also decreased SRC-1 expression in mouse brain, implying the involvement of SRC-1 in the process of inflammation and depression. Next, we showed that the chronic unpredictable mild stress (CUMS) failed to elicit the depressive-like behaviors and dramatically promoted the expression of SRC-1 in brain of wild type mice. What's more, the SRC-1 knockout mice were more susceptible to CUMS to develop depressive-like behaviors and presented the changed expression of glucocorticoid receptor. However, SRC-1 deficiency did not affect the microglia activation induced by CUMS. Altogether, these results indicate a correlation between SRC-1 level and depressive-like behaviors, suggesting that SRC-1 might be involved in the development of depression induced by stress.

PAX3-NCOA1 alveolar rhabdomyosarcoma of the tongue: A rare entity with challenging diagnosis and management.

Alveolar rhabdomyosarcoma (ARMS) is associated with PAX3/PAX7-FOXO1 fusion, which confers specific clinic and biologic characteristics with inferior outcomes. A minority of tumors still histologically classified as "true" ARMS lack the canonical PAX-FOXO1 fusion but have new molecular alterations. We present the first case of PAX3-NCOA1 ARMS with clinical data and follow-up in a two-year-old girl with ARMS of the tongue and nodal extension, treated with chemotherapy, hemi glossectomy, lymph node dissection, and brachytherapy to conserve oral function and limit long-term sequelae. Given the rarity of such variant fusion in ARMS, international collaboration is required to evaluate its prognostic value.

Expression, Purification and Characterization of CAR/NCOA-1 Tethered Protein in E. coli Using Maltose-Binding Protein Fusion Tag and Gelatinized Corn Starch.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate RORγt activity in a PKC-θ-dependent manner.

Th17 cells are major players in multiple autoimmune diseases and are developmentally contingent on reciprocal functionality between the transcription factor Retineic acid receptor-related orphan nuclear receptor gamma (RORγt) and Forkhead box protein P3 (Foxp3). Here we deciphered a previously unappreciated role of Steroid receptor coactivator 1 (SRC1) in defining the lineage decision for the development of Th17 versus induced T-regulatory (iTreg) cells. We demonstrate that SRC1 functions as a critical coactivator for RORγt in vivo to promote the functional dominance of RORγt over Foxp3 and thus establishing an unopposed Th17 differentiation program. In the absence of SRC1, T cell polarization resulted in decreased IL-17+ and increased Foxp3+ cells during both in vitro differentiation and in vivo development of experimental autoimmune encephalomyelitis. Mechanistically, T cell receptor (TCR) signaling molecule protein kinase C theta (PKC-θ)-mediated phosphorylation of SRC1 is important for inducing enhanced RORγt-SRC1 interaction, stable DNA binding, and resultant IL-17A transcription. Furthermore, phospho-SRC1-mediated recruitment of CARM1 induced prominent asymmetric dimethylation of H3R17 while preventing repressive H3K9 trimethylation and hence further modifying the IL-17 locus for optimal transcription. Moreover, binding of phospho-SRC1 to RORγt displaced bound Foxp3, leading to prompt degradation of the dissociated Foxp3 via a ubiquitin-proteosomal pathway and hence reversing the inhibitory action of Foxp3 on RORγt activity. Thus, SRC1 acts as a crucial molecular mediator to integrate positive PKC-θ-dependent TCR signals to induce peak RORγt activity and establish phenotypic dominance of Th17 over the iTreg pathway.

Establishment and Characterization of a Cell Line (S-RMS1) Derived from an Infantile Spindle Cell Rhabdomyosarcoma with SRF-NCOA2 Fusion Transcript.

Background: Spindle cell rhabdomyosarcoma (S-RMS) is a rare tumor that was previously considered as an uncommon variant of embryonal RMS (ERMS) and recently reclassified as a distinct RMS subtype with NCOA2, NCOA1, and VGLL2 fusion genes. In this study, we established a cell line (S-RMS1) derived from a four-month-old boy with infantile spindle cell RMS harboring SRF-NCOA2 gene fusion. Methods: Morphological and molecular characteristics of S-RMS1 were analyzed and compared with two RMS cell lines, RH30 and RD18. Whole genome sequencing of S-RMS1 and clinical exome sequencing of genomic DNA were performed. Results: S-RMS1 showed cells small in size, with a fibroblast-like morphology and positivity for MyoD-1, myogenin, desmin, and smooth muscle actin. The population doubling time was 3.7 days. Whole genome sequencing demonstrated that S-RMS1 retained the same genetic profile of the tumor at diagnosis. A Western blot analysis showed downregulation of AKT-p and YAP-p while RT-qPCR showed upregulation of endoglin and GATA6 as well as downregulation of TGFßR1 and Mef2C transcripts. Conclusion: This is the first report of the establishment of a cell line from an infantile spindle cell RMS with SRF-NCOA2 gene fusion. S-RMS1 should represent a useful tool for the molecular characterization of this rare and almost unknown tumor.