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High frequent metastasis is the major cause of breast cancer (BC) mortality among women. However, the molecular mechanisms underlying BC metastasis remain largely unknown. Here, we identified six hub BC metastasis driver genes (BEND5, HSD11B1, NEDD9, SAA2, SH2D2A and TNFSF4) through bioinformatics analysis, among which BEND5 is the most significant gene. Low BEND5 expression predicted advanced stage and shorter overall survival in BC patients. Functional experiments showed that BEND5 could suppress BC growth and metastasis

Objective To establish the eukaryotic expression vector of Y-box-binding protein 1 (YB-1) with FLAG-tagged and transfect it into hepatocellular carcinoma HepG2 cells to identify the effects of YB-1 on the proliferation and migration. Methods Human YB-1 gene was amplified from the human ovary library by PCR. YB-1 fraction was double enzyme digested and connected with pcDNA3.0-FLAG vector to construct eukaryotic expression vector pcDNA3.0-FlAG-YB-1, which was transfected into HepG2 cells. The expression of YB-1 was detected by Western blotting, and the effect of YB-1 on the proliferation of HepG2 cells was determined by CCK-8 assay and clone formation. The effect of YB-1 on the migration of HepG2 cells was analyzed by wound healing assays. Results The eukaryotic expression vector pcDNA3.0-FLAG-YB-1 was successfully established. YB-1 protein can be expressed in HepG2 cells, and YB-1 promoted the proliferation and migration of HepG2 cells. Conclusion YB-1 promotes the proliferation and migration of HepG2 cells.

Lung adenocarcinoma (LUAD) is a common type of lung cancer with high frequent metastasis and a high death rate. However, genes responsible for LUAD metastasis are still largely unknown. Here, we identify an important role of ras homolog family member V (RHOV) in LUAD metastasis using a combination of bioinformatic analysis and functional experiments. Bioinformatic analysis shows five hub LUAD metastasis driver genes (RHOV, ZIC5, CYP4B1, GPR18 and TCP10L2), among which RHOV is the most significant gene associated with LUAD metastasis. High RHOV expression predicted shorter overall survival in LUAD patients. RHOV overexpression promotes proliferation, migration, and invasion of LUAD cells, whereas RHOV knockdown inhibits these biological behaviors. Moreover, knockdown of RHOV suppresses LUAD tumor growth and metastasis in nude mice. Mechanistically, RHOV activates Jun N-terminal Kinase (JNK)/c-Jun signalling pathway, an important pathway in lung cancer development and progression, and regulates the expression of markers of epithelial-to-mesenchymal transition, a process involved in cancer cell migration, invasion and metastasis. RHOV-induced malignant biological behaviors are inhibited by pyrazolanthrone, a JNK inhibitor. Our findings indicate a critical role of RHOV in LUAD metastasis and may provide a biomarker for prognostic prediction and a target for LUAD therapy.

Triple-negative breast cancer (TNBC) is known for its aggressive phenotype with limited treatment modalities and poor prognosis. The Warburg effect (aerobic glycolysis) is a hallmark of cancer that serves as a promising target for diagnosis and therapy. However, how aerobic glycolysis regulates TNBC remains largely unknown. Here, we show that the glucose transporter (GLUT) family member GLUT12 promotes TNBC tumor growth and metastasis in vitro and in vivo through regulating aerobic glycolysis. MicroRNA let-7a-5p, a tumor suppressor, inhibited GLUT12 expression by targeting its 3'-untranslated region, and suppressed GLUT12-mediated TNBC tumor growth, metastasis, and glycolytic function, including alterations of glucose uptake, lactate production, ATP generation, extracellular acidification rate, and oxygen consumption rate. Inhibiting aerobic glycolysis abolished the ability of let-7a-5p and GLUT12 to regulate TNBC cell proliferation, migration and invasion. In TNBC patients, GLUT12 was significantly upregulated, and let-7a-5p expression was inversely correlated with GLUT12 expression. High expression of let-7a-5p and GLUT12 predicted better and worse clinical outcomes, respectively. Taken together, our results indicate that the let-7a-5p/GLUT12 axis plays key roles in TNBC tumor growth and metastasis, and aerobic glycolysis, and is a potential target for TNBC treatment.

Breast cancer is one of the most common malignancies that seriously threaten women's health. In the process of the malignant transformation of breast cancer, metabolic reprogramming and immune evasion represent the two main fascinating characteristics of cancer and facilitate cancer cell proliferation. Breast cancer cells generate energy through increased glucose metabolism. Lipid metabolism contributes to biological signal pathways and forms cell membranes except energy generation. Amino acids act as basic protein units and metabolic regulators in supporting cell growth. For tumor-associated immunity, poor immunogenicity and heightened immunosuppression cause breast cancer cells to evade the host's immune system. For the past few years, the complex mechanisms of metabolic reprogramming and immune evasion are deeply investigated, and the genes involved in these processes are used as clinical therapeutic targets for breast cancer. Here, we review the recent findings related to abnormal metabolism and immune characteristics, regulatory mechanisms, their links, and relevant therapeutic strategies.

Clear cell renal cell carcinoma (ccRCC) patients are highly angiogenic and treated by targeted therapies against VEGFA/VEGFR signaling pathway. However, tumors with such targeted therapies remain a significant clinic challenge. Understanding the underlying mechanism against angiogenesis is highly desired. Here, we demonstrated that the lncRNA DMDRMR serves as a sponge of miR-378a-5p to increase EZH2 and SMURF1 expression, thus promoting EZH2-mediated transcriptional repression of DAB2IP and SMURF1-mediated degradation of DAB2IP. Consequently, this axis activates VEGFA/VEGFR2 signaling pathway, resulting in angiogenesis and resistance of tumor cells to sunitinib in ccRCC. Moreover, the competing endogenous RNA regulatory axis of DMDRMR is clinically relevant to ccRCC pathogenesis and prognosis of patients with ccRCC. Our results support that the DMDRMR/miR-378a-5p/DAB2IP axis may serve as a novel target for combination diagnosis or therapy of ccRCC patients. Our findings may have highly clinical relevance for future translation to develop the targeted therapies for patients with ccRCC.

Paclitaxol is a first-line treatment for triple-negative breast cancer (TNBC). The molecular mechanisms underlying paclitaxol resistance in TNBC remain largely unclear. In this study, differential expressed genes (DEGs) between TNBC cells and paclitaxol-resistant (taxol-R) TNBC cells were screened by bioinformatics analysis. Among these DEGs, USP18 mRNA expression was significantly increased in taxol-R TNBC cells. USP18 overexpression reduced paclitaxol sensitivity by decreasing paclitaxol-induced apoptosis and cell cycle arrest in TNBC cells. In contrast, USP18 knockdown increased paclitaxol mediated anticancer activity in taxol-R TNBC cells in vitro and in vivo. Mechanistically, USP18 induced autophagy, an important pathway in chemotherapy resistance. The autophagy inhibitor leupeptin could effectively reverse the effect of USP18 on paclitaxol resistance phenotype. These findings suggested that USP18 may be a promising target for overcoming paclitaxol resistance in TNBC.

Aristolochic acids (AAs) are known to be the potent genotoxic carcinogens, of which aristolochic acid I (AAI) and aristolochic acid II (AAII) are the two representative compounds. As the carcinogenic risk of herbs containing AAs is a global health issue, quantitative evaluation of genotoxicity is needed for the risk assessment of AAs. γ-H2AX, which is an acknowledged attractive bifunctional biomarker for simultaneously reflect the DNA damage response and repair, was used to quantitatively determine the DNA damage and repair properties of AAI and AAII in human cell lines, based on our previously developed mass spectrometry method. Results indicated that both AAI and AAII could increase the level of γ-H2AX in cells in a concentration-dependent manner, and the increased level of γ-H2AX induced by AAI was relatively higher than that induced by AAII. Time-effect curves showed that the change tendency of the proportion of γ-H2AX was obviously different in the later period, particularly afterwards 8 h post exposure. Additionally, AAI and AAII induced an opposite change of expression levels of DNA damage repair-associated proteins (ERCC1 and p53) in HepG2 cells, revealing their distinct molecular mechanisms. Findings of the present study are helpful for understanding the genotoxicity mechanism of AAI and AAII.

The epidermal growth factor receptor (EGFR) family members EGFR and HER2 play pivotal roles in oncogenesis and tumor progression. Anticancer drugs targeting EGFR and HER2 have been developed. Long noncoding RNAs (lncRNAs) have been reported to regulate cancer development and progression through signaling pathways. However, lncRNAs that regulate EGFR and HER2 expression remain unknown. Here, we show that lncRNA myosin light chain kinase-antisense RNA 1 (MYLK-AS1) promotes EGFR and HER2 expression and activates their downstream signaling pathway. MYLK-AS1 increases hepatocellular carcinoma (HCC) cell proliferation, migration, and invasion in vitro. Consistently, MYLK-AS1 knockdown hinders tumor growth in vivo. Mechanistically, MYLK-AS1 enhances HCC cell proliferation, migration, and invasion through stimulating the EGFR/HER2-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In addition, MYLK-AS1 is overexpressed in HCC patients and negatively correlated with HCC prognosis. Thus, MYLK-AS1 is an upstream regulator of EGFR/HER2, and acts as an oncogene, suggesting an additional target for cancer therapeutics.

Non-alcoholic steatohepatitis (NASH) is a type of nonalcoholic fatty liver disease and has become a major risk factor for hepatocellular carcinoma (HCC). However, the underlying pathophysiological mechanisms are still elusive. Here, we identify hyaluronan-mediated motility receptor (HMMR) as a critical gene associated with NASH/HCC by combination of bioinformatic analysis and functional experiments. Analysis of differentially expressed genes (DEGs) between normal controls and NASH/HCC identified 5 hub genes (HMMR, UBE2T, TYMS, PTTG1 and GINS2). Based on the common DEGs, analyses of univariate and multivariate Cox regression and the area under the curve (AUC) value of the receiver operating characteristic (ROC) indicate that HMMR is the most significant gene associated with NASH/HCC among five hub genes. Oleate acid (OA), one of fatty acids that induce cellular adipogenesis, stimulates HMMR expression via CCAAT/enhancer-binding protein α (CEBPα). CEBPα increases the expression of HMMR through binding to its promoter. HMMR promotes HCC cell proliferation in vitro via activation of G1/S and G2/M checkpoint transitions, concomitant with a marked increase of the positive cell cycle regulators, including cyclin D1, cyclin E, and cyclin B1. Knockdown of HMMR suppresses HCC tumor growth in nude mice. Our study identifies an important role of HMMR in NASH/HCC, and suggests that HMMR may be a useful target for therapy and prognostic prediction of NASH/HCC patients.

Background Telomerase is a ribonucleoprotein enzyme responsible for maintenance of telomere length which expressed in more than 85% of cancer cells but undetectable in most normal tissue cells. Therefore, telomerase serves as a diagnostic marker of cancers. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. A safe, low‐cost and reliable telomerase activity detection method is still lacking. Method We modified DTA method using biotin‐labeled primers (Biotin‐DTA) and optimized the method by adjusting cell culture temperature and KCl concentration. The sensitivity of the method was confirmed to detect endogenous telomerase activity. The reliability was verified by detection of telomerase activity of published telomerase regulators. The stability was confirmed by comparing the method with TRAP method. Results Cells cultured in 32°C and KCl concentration at 200 mM or 250 mM resulted in robust Biotin‐DTA signal. Endogenous telomerase activity can be detected, which suggested an similar sensitivity as DTA using radioactive isotope markers. Knockdown of telomerase assembly regulator PES1 and DKC1 efficiently reduced telomerase activity. Compared with TRAP method, Biotin‐DTA assay offers greater signal stability over a range of analyte protein amounts. Conclusion Biotin‐labeled, PCR‐free, and nonradioactive direct telomerase assay is a promising new method for the easy, low‐cost, and quantitative detection of telomerase activity., Telomerase activity can be used as a marker to assist in the early diagnosis of tumors, to determine the prognosis of patients, and to detect recurrence. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. Herein, we developed a modified direct telomerase activity detection method that employs neither isotope markers nor PCR amplification. It was based on immunoprecipitation and streptavidin–biotin system. Finally, it was proved to be a promising new method for the easy, low‐cost, and quantitative detection of telomerase activity.

Hepatocellular carcinoma (HCC), which is characterized by reprogrammed lipid metabolism, is a highly malignant tumor with a high incidence and mortality rate. While lipid metabolism is a promising target for HCC therapy, the regulation of lipid metabolism is not well elucidated. Through CRISPR/Cas9 screening, we show that miR-4310 inhibits lipid synthesis by targeting fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). In patients with HCC, miR-4310 is significantly downregulated, and its expression is negatively correlated with expressions of FASN and SCD1. Furthermore, low expression of miR-4310 is associated with poor prognosis. By suppressing SCD1-and FASN-mediated lipid synthesis, miR-4310 inhibits HCC cell proliferation, migration, and invasion in vitro and suppresses HCC tumor growth and metastasis in vivo. Our data indicate that miR-4310 plays an important role in HCC tumor growth and metastasis by regulating the FASN- and SCD1-mediated lipid synthesis pathways. Targeting the miR-4310-FASN/SCD pathway may provide a novel strategy for HCC treatment.

A further understanding of tumor angiogenesis is urgently needed due to the limited therapeutic efficacy of anti-angiogenesis agents. However, the origin of endothelial cells (EC) in tumors remains widely elusive and controversial. Snail has been thoroughly elucidated as a master regulator of the epithelial–mesenchymal transition (EMT), but its role in endothelium generation is not yet established. In this study, we reported a new and unexpected function of Snail in endothelium generation by breast cancer cells. We showed that high Snail-expressing breast cancer cells isolated from patients showed more endothelium generated from these cells. Expression of Snail was positively correlated with endothelial markers in breast cancer patients. The ectopic expression of Snail induced endothelial marker expression, tube formation and DiI-AcLDL uptake of breast cancer cells in vitro, and enhanced tumor growth and microvessel density in vivo. Snail-mediated endothelium generation depended on VEGF and Sox2. Mechanistically, Snail promoted the expression of VEGF and Sox2 through recruiting the p300 activator complex to these promoters. We showed the dual function of Snail in tumor initiation and angiogenesis in vivo and in vitro through activation of Sox2 and VEGF, suggesting Snail may be an ideal target for cancer therapy.

Osteoarthritis (OA) has been recognized as the most common chronic age-related disease. Cartilage degeneration influences OA therapy. Here we report that hematopoietic pre-B cell leukemia transcription factor-interacting protein (HPIP) is essential for OA development. Elevated HPIP levels are found in OA patients. Col2a1-CreERT2/HPIPf/f mice exhibit obvious skeletal abnormalities compared with their HPIPf/f littermates. HPIP deficiency in mice protects against developing OA. Moreover, intra-articular injection of adeno-associated virus carrying HPIP-specific short hairpin RNA in vivo attenuates OA histological signs. Notably, in vitro RNA-sequencing and chromatin immunoprecipitation sequencing profiles identify that HPIP modulates OA cartilage degeneration through transcriptional activation of Wnt target genes. Mechanistically, HPIP promotes the transcription of Wnt targets by interacting with lymphoid enhancer binding factor 1 (LEF1). Furthermore, HPIP potentiates the transcriptional activity of LEF1 and acetylates histone H3 lysine 56 in the promoters of Wnt targets, suggesting that HPIP is an attractive target in OA regulatory network., Osteoarthritis (OA) is associated with cartilage degeneration, and no effective therapy exists. Here, the authors show that the HPIP protein modulates OA progression by regulating Wnt signaling, and that its knockdown in joints via AAV-mediated gene silencing attentuates pathology.

The programmed cell death 1 (PD-1) receptor on the surface of immune cells is an immune checkpoint molecule that mediates the immune escape of tumor cells. Consequently, antibodies targeting PD-1 have shown efficacy in enhancing the antitumor activity of T cells in some types of cancers. However, the potential effects of PD-1 on tumor cells remain largely unknown. Here, we show that PD-1 is expressed across a broad range of tumor cells. The silencing of PD-1 or its ligand, PD-1 ligand 1 (PD-L1), promotes cell proliferation and colony formation in vitro and tumor growth in vivo. Conversely, overexpression of PD-1 or PD-L1 inhibits tumor cell proliferation and colony formation. Moreover, blocking antibodies targeting PD-1 or PD-L1 promote tumor growth in cell cultures and xenografts. Mechanistically, the coordination of PD-1 and PD-L1 activates its major downstream signaling pathways including the AKT and ERK1/2 pathways, thus enhancing tumor cell growth. This study demonstrates that PD-1/PD-L1 is a potential tumor suppressor and potentially regulates the response to anti-PD-1/PD-L1 treatments, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

Epidermal growth factor receptor (EGFR) status is the major determinant of non-small cell lung cancer (NSCLC) therapy selection. Studies have hinted that EGFR antibodies or tyrosine kinase inhibitors were beneficial in patients with EGFR mutation-negative but EGFR-overexpressing of NSCLC. However, the mechanisms underlying EGFR amplification and overexpression in NSCLC remain largely unknown. Here, we report that rs884225, a single nucleotide polymorphism in the EGFR 3'-terminal untranslated region, was significantly associated with EGFR expression level and contributed to NSCLC susceptibility. Mechanistically, the rs884225 C allele enhanced EGFR expression by altering the miR-103a-3p binding site, thus impairing miR-103a-3p's anti-tumourigenic function. As a tumour suppressor gene, miR-103a-3p expression correlated with overall and recurrence-free survival in NSCLC patients. Furthermore, miR-103a-3p inhibited growth and metastasis via effects on the KRAS pathway and epithelial-to-mesenchymal transition in EGFR wild-type NSCLC cell lines, respectively, which substantially reduced EGFR expression and activity. Thus, rs884225 may be a biomarker for NSCLC susceptibility, and miR-103a-3p may be a potential therapeutic target in NSCLC.

Wang et al. unveil a new mechanism by which Src may function as an oncogene. Phosphorylation of FHL1 by Src triggers the translocation of FHL1 to the nucleus, where it regulates the activity of the transcription factor BCLAF1 to promote tumor cell growth and loses the ability to regulate cell adhesion and suppress growth., FHL1 has been recognized for a long time as a tumor suppressor protein that associates with both the actin cytoskeleton and the transcriptional machinery. We present in this study a paradigm that phosphorylated FHL1 functions as an oncogenic protein by promoting tumor cell proliferation. The cytosolic tyrosine kinase Src interacts with and phosphorylates FHL1 at Y149 and Y272, which switches FHL1 from a tumor suppressor to a cell growth accelerator. Phosphorylated FHL1 translocates into the nucleus, where it binds to the transcription factor BCLAF1 and promotes tumor cell growth. Importantly, the phosphorylation of FHL1 is increased in tissues from lung adenocarcinoma patients despite the down-regulation of total FHL1 expression. Kindlin-2 was found to interact with FHL1 and recruit FHL1 to focal adhesions. Kindlin-2 competes with Src for binding to FHL1 and suppresses Src-mediated FHL1 phosphorylation. Collectively, we demonstrate that FHL1 can either suppress or promote tumor cell growth depending on the status of the sites for phosphorylation by Src.

PES1 is a new component of telomerase assembly that regulates telomerase activity, telomere length, and cellular senescence., Telomerase defers the onset of telomere shortening and cellular senescence by adding telomeric repeat DNA to chromosome ends, and its activation contributes to carcinogenesis. Telomerase minimally consists of the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR). However, how telomerase assembles is largely unknown. Here, we demonstrate that PES1 (Pescadillo), a protein overexpressed in many cancers, forms a complex with TERT and TR through direct interaction with TERT, regulating telomerase activity, telomere length maintenance, and senescence. PES1 does not interact with the previously reported telomerase components Reptin, Pontin, p23, and Hsp90. PES1 facilitates telomerase assembly by promoting direct interaction between TERT and TR without affecting TERT and TR levels. PES1 expression correlates positively with telomerase activity and negatively with senescence in patients with breast cancer. Thus, we identify a previously unknown telomerase complex, and targeting PES1 may open a new avenue for cancer therapy.

Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2–CDC25 and CDC25–14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity., CDC25 phosphatases are important cell cycle regulators. Here, the authors show that the LIM domain-containing proteins (for example, FHL1) induce inhibitory CDC25 phosphorylation resulting in radioresistance and that a specific peptide can increase tumour radiosensitivity by increasing CDC25 activity.

Unlike estrogen receptor α (ERα) that predominantly promotes hormone-dependent breast tumor growth, ERβ exhibits antitumor effects in a variety of cancer types. We recently identified a phosphotyrosine residue in ERβ, but not ERα, that dictates ERβ transcriptional activity and antitumor function. We show here that this ER isotype-specific phosphotyrosine switch is important for regulating ERβ activity in cell proliferation, migration, and invasion. At the mechanistic level, phosphorylated ERβ, which recruits transcriptional coactivator p300, is in turn targeted by p300 for ubiquitination and proteasome-dependent protein turnover. Furthermore, ERβ-specific agonists such as S-equol enhance ERβ phosphorylation, suggesting a crosstalk between ligand- and posttranslational modification-dependent ERβ activation. Inhibition of xenograft tumor growth by S-equol is associated with reduced tumor Ki-67 expression and elevated ERβ tyrosine phosphorylation. Taken together, our data support the notion that phosphotyrosine-dependent ERβ signaling is an attractive target for anticancer treatment.

The proinflammatory cytokine interleukin-1β (IL-1β) is involved in the initiation and progression of osteoarthritis (OA) by stimulating the expression of matrix-degrading proteinases, such as a disintegrin metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), a key player in OA pathogenesis. However, how IL-1β induces ADAMTS-5 overexpression is poorly understood. We demonstrate that IL-1β regulates ADAMTS-5 expression by suppressing microRNA-30a (miR-30a). Bioinformatics was performed to predict miRNAs targeting ADAMTS-5. miR-30a inhibited ADAMTS-5 expression by directly targeting its 3'-untranslated region. miR-30a expression was downregulated in OA patients and was negatively correlated with ADAMTS-5 expression and positively correlated with Hospital for Special Surgery (HSS) scores. IL-1β suppressed miR-30a expression by recruiting the activator protein (AP-1) transcription factor c-jun/c-fos to the miR-30a promoter. IL-1β-induced c-jun/c-fos expression regulated ADAMTS-5 expression and cartilage matrix degradation via miR-30a in human chondrocytes. These data indicate that the IL-1β/AP-1/miR-30a/ADAMTS-5 pathway contributes to IL-1β-induced cartilage matrix degradation in human OA chondrocytes. miR-30a may act as a pivotal regulator of cartilage homeostasis and a potential diagnostic and therapeutic target for OA.ADAMTS-5 was identified as a novel direct target of miR-30a. IL-1β suppresses miR-30a expression through activation of AP-1 (c-jun/c-fos). AP-1/miR-30a is essential for IL-1β-induced ADAMTS-5 upregulation in OA. Downregulation of miR-30a in OA is negatively correlated with ADAMTS-5 expression.

The DEK oncogene is overexpressed in various cancers and overexpression of DEK correlates with poor clinical outcome. Vascular endothelial growth factor (VEGF) is the most important regulator of tumor angiogenesis, a process essential for tumor growth and metastasis. However, whether DEK enhances tumor angiogenesis remains unclear. Here, we show that DEK is a key regulator of VEGF expression and tumor angiogenesis. Using chromatin immunoprecipitation assay, we found that DEK promoted VEGF transcription in breast cancer cells (MCF7, ZR75-1 and MDA-MB-231) by directly binding to putative DEK-responsive element (DRE) of the VEGF promoter and indirectly binding to hypoxia response element (HRE) upstream of the DRE through its interaction with the transcription factor hypoxia-inducible factor 1α (HIF-1α), a master regulator of tumor angiogenesis and growth. DEK is responsible for recruitment of HIF-1α and the histone acetyltransferase p300 to the VEGF promoter. DEK-enhanced VEGF increases vascular endothelial cell proliferation, migration and tube formation as well as angiogenesis in the chick chorioallantoic membrane. DEK promotes tumor angiogenesis and growth in nude mice in HIF-1α-dependent and -independent manners. Immunohistochemical staining showed that DEK expression positively correlates with the expression of VEGF and microvessel number in 58 breast cancer patients. Our data establish DEK as a sequence-specific binding transcription factor, a novel coactivator for HIF-1α in regulation of VEGF transcription and a novel promoter of angiogenesis.

Cholesterol increases the risk of aggressive prostate cancer and has emerged as a potential therapeutic target for prostate cancer. The functional roles of cholesterol in prostate cancer metastasis are not fully understood. Here, we found that cholesterol induces the epithelial-to-mesenchymal transition (EMT) through extracellular-regulated protein kinases 1/2 pathway activation, which is mediated by EGFR and adipocyte plasma membrane-associated protein (APMAP) accumulation in cholesterol-induced lipid rafts. Mechanistically, APMAP increases the interaction with EGFR substrate 15-related protein (EPS15R) to inhibit the endocytosis of EGFR by cholesterol, thus promoting cholesterol-induced EMT. Both the mRNA and protein levels of APMAP are upregulated in clinical prostate cancer samples. Together, these findings shed light onto an APMAP/EPS15R/EGFR axis that mediates cholesterol-induced EMT of prostate cancer cells. Significance: This study delineates the molecular mechanisms by which cholesterol increases prostate cancer progression and demonstrates that the binding of cholesterol-induced APMAP with EPS15R inhibits EGFR internalization and activates ERK1/2 to promote EMT.

Objective To purify recombinant protein of human von Hippel-Lindau (VHL) and identify its function. Methods VHL gene sequence was amplified from human mammary cDNA using PCR and inserted into the prokaryotic expression vector pGEX-KG. Glutathione S-transferase-VHL (GST-VHL) recombinant plasmid we obtained was converted into BL21(DE3) sensitive bacteria to induce a small amount of GST-VHL protein. The expressed product was detected by SDS-PAGE and Western blot analysis. The recombinant protein was purified by GST beads and its function was verified by GST pull-down assay. Results The obtained recombinant plasmid could be successfully digested by double enzymes. Gene sequencing showed that the VHL sequence was correct and there was no mutation. The recombinant protein with approximately relative molecular mass (M

Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF) is the most important regulator of tumor angiogenesis. However, how transcription factors interact with histone-modifying enzymes to regulate VEGF transcription and tumor angiogenesis remains unclear. Here, we show that transcription factor GATA1 associates with the histone methyltransferase SET7 to promote VEGF transcription and breast tumor angiogenesis. Using chromatin immunoprecipitation assay, we found that GATA1 was required for recruitment of SET7, RNA polymerase II and transcription factor II B to VEGF core promoter. GATA1 enhanced breast cancer cell (MCF7, ZR75-1 and MDA-MB-231)-secreted VEGF via SET7, which promoted vascular endothelial cell (HUVEC) proliferation, migration and tube formation. SET7 was required for GATA1-induced breast tumor angiogenesis and growth in nude mice. Immunohistochemical staining showed that expression of GATA1 and SET7 was upregulated and positively correlated with VEGF expression and microvessel number in 80 breast cancer patients. GATA1 and SET7 are independent poor prognostic factors in breast cancer. Our data provide novel insights into VEGF transcriptional regulation and suggest GATA1/SET7 as cancer therapeutic targets.

Pulsatilla chinensis is one of the 50 famous fundamental herbs used in traditional Chinese medicine. Saponins are the main components of P. chinensis. Although the anti-proliferative function of saponins has been established in plenty types of cancer, the role of saponins on tumor invasion and metastasis has not been reported, and the mechanisms of how saponins exert the anti-tumor functions are still poorly characterized. Here, we demonstrate that, in breast cancer (BC) cells, raddeanoside R13, a component of saponins extracted from P. chinensis, exhibits strong anti-proliferative and anti-metastasis ability, accompanied by cell cycle arrest, apoptosis, autophagy, and reversion of epithelial-mesenchymal transition (EMT). Raddeanoside R13 (R13) inhibits BC cell proliferation via the activation of G1/S checkpoint transitions, concomitant with a marked decrease of the positive cell cycle regulators, including cyclin D1, cyclin A, and cyclin B1. R13 induces BC cell apoptosis accompanied by the increased levels of cleaved PARP and caspase-3. R13 inhibits BC cell migration and invasion and regulates the expression of the markers of EMT, which plays a critical role in cancer cell migration and invasion. Moreover, R13 suppresses BC tumor growth and metastasis in nude mice. These data highlight the important role of R13 in BC cell proliferation and progression and suggest that R13 may be a useful drug for BC therapy.

Hematopoietic pre-B cell leukemia transcription factor interacting protein (HPIP) has been shown to play an important role in the development and progression of some cancers. However, the role of HPIP in gastric cancer (GC) is unclear. Here, we show that HPIP is upregulated in most GC patients and promotes GC cell proliferation, migration, and invasion. In GC patients, HPIP positively associates with tumor size and nodal metastasis, and negatively associates with tumor differentiation. Hematopoietic pre-B cell leukemia transcription factor interacting protein increases GC cell proliferation through activation of G1 /S and G2 /M cell cycle transitions, accompanied by a marked increase of the positive cell cycle regulators, including cyclin D1, cyclin A, and cyclin B1. Hematopoietic pre-B cell leukemia transcription factor interacting protein enhances GC cell migration and invasion, and modulates epithelial-mesenchymal transition, which plays a key role in cancer cell migration and invasion. These data underscore the critical role of HPIP in GC cell proliferation and progression and suggest that HPIP inhibition may be a useful therapeutic strategy for GC treatment.

The transcription factor estrogen receptor β (ERβ) plays roles in the central nervous, endocrine, cardiovascular, and immune systems. ERβ can be SUMOylated. However, the underlying mechanism remains unclear. Here, we show that RSRC1/SRrp53 interacts with ERβ and SUMOylation of RSRC1 is required for regulation of PIAS1-mediated ERβ SUMOylation. RSRC1 promotes ERβ SUMOylation through enhanced interaction between ERβ and PIAS1. RSRC1 represses ERβ transcriptional activity through regulation of ERβ SUMOylation. By establishing RSRC1 as a novel cofactor for SUMOylation, our data provide insight into regulation of ERβ SUMOylation and indicate that SUMOylation of one protein can regulate another protein SUMOylation.

Sulphur mustard (2,2'-dichloroethyl sulfide; SM) is a vesicant chemical warfare agent whose mechanism of acute or chronic action is not known with any certainty and to date there is no effective antidote. SM accumulation in adipose tissue (AT) has been originally verified in our previous study. To evaluate the biological effect caused by the presence of abundant SM in adipocyte and assess the biological role of AT in SM poisoning, in vitro and in vivo experiments were performed. High content analysis revealed multi-cytotoxicity in SM exposed cells in a time and dose dependent manner, and adipocytes showed a relative moderate damage compared with non-adipocytes. Cell co-culture model was established and revealed the adverse effect of SM-exposed adipocyte supernatant on the growth of co-cultured cells. The pathological changes in AT from 10mg/kg SM percutaneously exposed rats were checked and inflammation phenomena were observed. The mRNA and protein levels of inflammation-related adipokines secreted from AT in rats exposed to 1, 3 and 10mg/kg doses of SM were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. The expressions of proinflammatory and anti-inflammatory adipokines together promoted the inflammation development in the body. The positive correlations between AT and serum adipokine levels were explored, which demonstrated a substantial role of AT in systemic inflammation responding to SM exposure. Thus, AT is not only a target of SM but also a modulator in the SM toxicity.

Lactate dehydrogenase A (LDHA), a key enzyme regulating aerobic glycolysis, is overexpressed in many human cancers, and correlates with poor clinical outcomes. Aerobic glycolysis is a hallmark of cancer, and drugs targeting its regulators, including LDHA, are being developed. However, the mechanisms of LDHA inhibition and the physiological significance of the LDHA inhibitors in cancer cells are unclear. Here, we show that microRNA-30a-5p (miR-30a-5p) suppresses LDHA expression by directly targeting its 3'-UTR. Through inhibition of LDHA, miR-30a-5p dampens glycolysis by decreasing glucose uptake, lactate production, ATP generation, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in breast cancer cells. Importantly, glycolysis regulated by miR-30a-5p is critical for its regulating breast tumor growth and metastasis both in vitro and in vivo. In breast cancer patients, miR-30a-5p expression is negatively correlated with LDHA expression. Moreover, patients who had increased glucose uptake in tumors assessed by PET scans showed decreased miR-30a-5p expression and increased expression of LDHA. Our findings provide clues regarding the role of miR-30a-5p as a tumor suppressor in breast cancer through the inhibition of LDHA. Targeting LDHA through miR-30a-5p could be a potential therapeutic strategy in breast cancer.

Dysregulation of the epidermal growth factor receptor (EGFR) promotes cancer cell growth, invasion and metastasis. However, its relevant downstream effectors are still limited. Here, we show that EGFR promotes breast tumor growth and metastasis by downregulating the tumor suppressor micoRNA-338-3p (miR-338-3p) and activating the EYA2 (EYA transcriptional coactivator and phosphatase 2) oncoprotein. EGFR represses miR-338-3p expression largely through HIF1α transcription factor. miR-338-3p inhibits EYA2 expression by binding to the 3′-untranslated region of EYA2. EGFR increases EYA2 expression via HIF1α repression of miR-338-3p. Through the miR-338-3p/EYA2 pathway, EGFR increases breast cancer cell growth, epithelial-to-mesenchymal transition, migration, invasion and lung metastasis in vitro and in a allograft tumor mouse model in vivo. In breast cancer patients, miR-338-3p expression negatively correlates with the expression of EGFR and EYA2, EGFR status positively associates with EYA2 expression, and miR-338-3p and EYA2 predict breast cancer lung metastasis when expressed in primary breast cancers. These data suggest that the miR-338-3p/EYA2 axis contributes to EGFR-mediated tumor growth and lung metastasis and that miR-338-3p activation or EYA2 inhibition or combination therapy targeting EGFR/miR-338-3p/EYA2 axis may be a promising way to treat patients with metastatic cancer.

To identify proteins with a potential role in the interaction of Bifidobacterium longum with intestinal epithelial cells, we profiled the protein response of B. longum NCC2705 following interaction with Caco-2 cells. Thirty-one protein spots, belonging to a total of 23 proteins, which exhibited a change in abundance of at least 3-fold were identified in B. longum NCC2705 following co-culture with Caco-2 cells, and were subsequently identified. Changes in expression were confirmed at the transcriptional level for a selection of these proteins. Enolase (Eno) and elongation factor Tu (EF-Tu) were amongst the proteins that showed the most prominent increase in abundance. Interaction of these proteins with plasminogen (Plg) was analyzed by Plg overlay assays, glutathione S-transferase (GST)-pull down, and western blot analysis. The results suggested that EF-Tu and Eno serve as surface receptors for B. longum NCC2705 binding to human plasminogen. Purified GST–EF-Tu and GST–Eno inhibited adhesion of B. longum NCC2705 to Caco-2 cells. Collectively, our data suggest that Eno and EF-Tu moonlight as adhesions, and are possibly involved in the protective role played by B. longum NCC2705 in defense against enteric pathogens. Biological significance The interaction of bifidobacteria with the human host plasminogen/plasmin system confirms the existence of a new component in the molecular cross-talk between bacteria and the host. Our study analyzed proteins EF-Tu and Eno with Plg binding activity, and they can inhibit adhesion of B. longum NCC2705 to Caco-2 cells, suggesting their role in the bacterial adherent to the enterocyte surface.

Estrogen receptors ERα and ERβ share considerable sequence homology yet exert opposite effects on breast cancer cell proliferation. While the proliferative role of ERα in breast tumors is well characterized, it is not clear whether the antitumor activity of ERβ can be mobilized in breast cancer cells. Here, we have shown that phosphorylation of a tyrosine residue (Y36) present in ERβ, but not in ERα, dictates ERβ-specific activation of transcription and is required for ERβ-dependent inhibition of cancer cell growth in culture and in murine xenografts. Additionally, the c-ABL tyrosine kinase and EYA2 phosphatase directly and diametrically controlled the phosphorylation status of Y36 and subsequent ERβ function. A nonphosphorylatable, transcriptionally active ERβ mutant retained antitumor activity but circumvented control by upstream regulators. Phosphorylation of Y36 was required for ERβ-mediated coactivator recruitment to ERβ target promoters. In human breast cancer samples, elevated phosphorylation of Y36 in ERβ correlated with high levels of c-ABL but low EYA2 levels. Furthermore, compared with total ERβ, the presence of phosphorylated Y36-specific ERβ was strongly associated with both disease-free and overall survival in patients with stage II and III disease. Together, these data identify a signaling circuitry that regulates ERβ-specific antitumor activity and has potential as both a prognostic tool and a molecular target for cancer therapy.

In addition to nuclear estrogen receptor (ER) acting as a transcription factor, extranuclear ER also plays an important role in cancer cell growth regulation through activation of kinase cascades. However, the molecular mechanisms by which extranuclear ER exerts its function are still poorly understood. Here, we report that mediator of ERBB2-driven cell motility (MEMO) regulates extranuclear functions of ER. MEMO physically and functionally interacted with ER. Through its interaction with the growth factor receptors IGF1R and ERBB2, MEMO mediated extranuclear functions of ER, including activation of mitogen-activated protein kinase (MAPK) and protein kinase B/AKT, two important growth regulatory protein kinases, and integration of function with nuclear ER. Activation of MAPK and AKT was responsible for MEMO modulation of ER phosphorylation and estrogen-responsive gene expression. Moreover, MEMO increased anchorage-dependent and -independent growth of ER-positive breast cancer cells in vitro and was required for estrogen-induced breast tumor growth in nude mice. Together, our studies identified MEMO as a new component of extranuclear ER signalosome and suggest an essential role for MEMO in the regulation of ER-positive breast cancer cell growth.

Lung cancer remains the leading cause of cancer-related death worldwide. Paclitaxel, either as monotherapy or combined with other agents, is the standard treatment for advanced non-small cell lung cancer (NSCLC), the most common type of lung cancer. However, both de novo and acquired resistance against paclitaxel frequently occurs and represents a huge clinical problem. The underlying mechanisms remain poorly characterized. Here, by comparing microRNA (miRNA) expression levels using miRNA arrays, we observed differential expression of miR-30a-5p in two independent lung cancer cell pairs (paclitaxel-resistant vs paclitaxel-sensitive A549 cell lines). Overexpression of miR-30a-5p sensitizes NSCLC cells to paclitaxel both in vitro and in vivo. In addition, miR-30a-5p increases paclitaxel sensitivity by promoting chemotherapy-induced apoptosis via downregulating BCL-2, a key apoptosis regulator. High miR-30a-5p expression is positively correlated with enhanced responsiveness to paclitaxel and predicts a more favorable clinical outcome in NSCLC patients. Moreover, miR-30a-5p expression is negatively correlated with BCL-2 expression in NSCLC tissues. These data indicate that miR-30a-5p may be useful to treat paclitaxel-resistant lung cancer and may also provide a biomarker to predict paclitaxel responsiveness in lung cancer.BCL-2 is a novel direct target of miR-30a-5p. miR-30a-5p enhances NSCLC paclitaxel sensitivity in vitro and in vivo. miR-30a-5p sensitizes NSCLC cells to paclitaxel by inducing apoptosis through BCL-2 inhibition. miR-30a-5p negatively correlates with BCL-2 and predicts a favorable clinical outcome in NSCLC patients.

The radiation-induced bystander effect (RIBE) is an important factor in tumor radiation therapy because it may increase the probability of normal cellular injury and the likelihood of secondary cancers after radiotherapy. Here, we identified the role of miR-495 in alleviating RIBEs during radiotherapy. Luciferase reporter assay results confirmed that miR-495 regulated endothelial nitric oxide synthase (eNOS) by targeting the Sp1 3'-untranslated region. Consequently, after radiation, tumor cells expressed less eNOS and Sp1 than controls. In vitro cell irradiation data based on flow-cytometric analysis and enzymed linked immunosorbent assay confirmed that nitric oxide (NO) and its downstream product transforming growth factor β1 (TGF-β1) were critical signaling factors contributing to RIBEs. Fewer normal LO2 liver cells were injured and fewer micronuclei were observed when treated with the medium of the miR-495 overexpressing HepG2 and ZR75-1 tumor cells. Accordingly, treatment with the miR-495 antagomir led to higher NO and TGF-β1 levels and more injured LO2 cells. In vivo experiments indicated that local irradiation of tumors overexpressing miR-495 produced fewer necrotic foci in non-irradiated liver tissue compared with controls. miR-495 was upregulated in clinical cancer tissues compared with adjacent non-cancerous tissues, and radiation significantly reduced the expression level of miR-495 in carcinoma cell lines. In summary, miR-495 may have promise as an adjuvant for tumor radiation therapy to decrease RIBEs involving the Sp1/eNOS pathway.

Telomerase is composed of the catalytic subunit TERT (Telomerase reverse transcriptase),RNA subunit TERC (Telomerase RNA component) and other telomerase associated proteins. These two compartments with other telomerase subunits can assemble a holoenzyme, which maintain the length of telomeres. Telomerase plays important roles in cell senescence and tumor formation. The molecular mechanisms of the regulation of telomerase are very complicated. These processes comprise the regulation of transcription, post-transcription, post-translation and subcellular localization. Trafficking and assemble of TERT and TERC, as well as recruitment to telomeres, are also involved in this process. Here we review the regulation mechanism of telomerase from these aspects, and aim to laid a foundation for telomerase associated research and the drug targeting the telomerase.

This study was initiated to evaluate the clinical significant of HER2 extracellular domain (ECD) in the real-time management of breast cancer patients.Five-hundred forty-six eligible breast cancer patients were divided according to their clinical background. The correlation between ECD, tissue HER2, and clinical outcome of the patients were analyzed.Receiver operating characteristic analysis revealed that ECD measured before receiving neoadjuvant therapy yielded the highest area under the curve (0.9185; P0.0001), indicating that ECD and tissue HER2 levels are consistent in untreated tumor-bearing patients. At cut-off of 15.0ng/ml, the prognostic value of ECD was demonstrated using univariate (HR=1.664, P0.0001) and multivariate (HR=1.547, P=0.011) Cox regression analysis. Kaplan-Meier survival curves revealed that patients with elevated ECD had shorter progression-free survival (PFS) (4.0 vs. 6.1months, P0.0001). Elevated ECD was also an adverse predictor for PFS in response to anti-HER2 therapy (4.3 vs. 10.2months, P=0.0155). In contrast, ≥20%, decreased ECD was associated with longer PFS in patients who received anti-HER2 therapy (10.9 vs. 2.4months, P=0.0164) and overall (10.7 vs. 2.8months, P=0.0034).A patient's clinical history can help determine whether ECD could provide added value for breast cancer management.

Aberrant expression of ubiquitin Protein ligase E3C (UBE3C) has been documented in breast cancer (BC). MicroRNAs (miRNAs) were shown to play an important role in the regulation of tumor properties in BC. However, whether miRNAs contributes to UBE3C expression in BC cells remains poorly understood. In this study, we report that UBE3C was a direct target of miR-30a-5p. Expression of miR-30a-5p in BC cells reduced UBE3C expression. MCF-7 and MDA-MB-453 cells were transfected miR-30a-5p-overexpression, and found that cell proliferation and migration were inhibited. In contrast, when miR-30a-5p inhibitor were transfected into MCF-7 and MDA-MB-453 cells, cell proliferation and migration were promoted. We study demonstrated that upregulation of miR-30a-5p was significantly suppressed levels of cyclin B1, cyclin D1 and c-myc. Moreover, Correlation analysis indicated that expression of miR-30a-5p was highly negatively correlated with UBE3C, which was upregulated in BC specimens. These data highlight the important role of miR-30a-5p/UBE3C axis in BC development and progression. Therefore, miR-30a-5p activation or UBE3C inhibition may be provide a novel strategy for the treatment of BC.

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58(IPK) that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58(IPK) by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58(IPK) in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58(IPK), and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58(IPK) mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.

Four and a half LIM domain protein 2 (FHL2) has been implicated in development and progression of various types of cancers. However, little is known about the biological function of FHL2 in breast cancer. Here, we report that FHL2 physically and functionally interacts with estrogen receptors (ERα and ERβ), important regulators of breast cancer development and progression. The N-terminal half LIM domain or a single LIM domain of FHL2 was sufficient for its interaction with ERα and ERβ. Overexpression of FHL2 reduced ER transcriptional activity in breast cancer cells, whereas reduction of endogenous FHL2 with FHL2 small interfering RNA enhanced ER transactivation. Moreover, FHL2 cooperates with Smad4, a previously known corepressor for ERα, to inhibit ERα transcriptional activity as well as expression of the estrogen-responsive gene cathepsin D. The synergistic inhibition of ERα transcriptional activity by FHL2 and Smad4 may be due to enhanced interaction of Smad4 with ERα by FHL2, because FHL2(1-156), the FHL2 deletion mutant, which showed no synergistic effect, failed to increase such interaction. These data suggested the cooperative regulation of estrogen signaling by FHL2 and Smad4 in breast cancer cells, and might provide a new regulation mechanism underlying breast cancer development and progression.

Summary Aerobic glycolysis (the Warburg effect) facilitates tumor growth, and drugs targeting aerobic glycolysis are being developed. However, how the Warburg effect is directly regulated is largely unknown. Here we show that transcription factor SIX1 directly increases the expression of many glycolytic genes, promoting the Warburg effect and tumor growth in vitro and in vivo . SIX1 regulates glycolysis through HBO1 and AIB1 histone acetyltransferases. Cancer-related SIX1 mutation increases its ability to promote aerobic glycolysis and tumor growth. SIX1 glycolytic function is directly repressed by microRNA-548a-3p, which is downregulated, inversely correlates with SIX1, and is a good predictor of prognosis in breast cancer patients. Thus, the microRNA-548a-3p/SIX1 axis strongly links aerobic glycolysis to carcinogenesis and may become a promising cancer therapeutic target.

Four and a half LIM protein 1 (FHL1) belongs to the Lin-1, Isl-1 and Mec-3 (LIM)-only protein family and plays important roles in muscle growth and carcinogenesis. However, the biological function of FHL1 remains largely unknown. Here, we show that FHL1 physically and functionally interacted with oestrogen receptors (ERs), which are involved in breast cancer development and progression. FHL1 bound specifically to the activation function-1 domain of ER. Physical interaction of FHL1 and ER is required for FHL1 repression of oestrogen-responsive gene transcription. FHL1 affected recruitment of ER to an oestrogen-responsive promoter and ER binding to an oestrogen-responsive element. Overexpression of FHL1 in breast cancer cells decreased expression of oestrogen-responsive proteins, whereas knockdown of endogenous FHL1 with FHL1 small interfering RNA increased the expression of these proteins. Further analysis of 46 breast cancer samples showed that FHL1 expression negatively associated with oestrogen-responsive gene expression in breast cancer cells. FHL1 inhibited anchorage-dependent and -independent breast cancer cell growth. These results suggest that FHL1 may play an important role in ER signalling as well as breast cancer cell growth regulation.

High mobility group box-1 protein, an abundant and conserved constituent of vertebrate nuclei, has recently been reported to be an endogenous immune signal [Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Reports 2004;5:825-30]. High mobility group box-1 protein can trigger the release of interleukin-2 and interleukin-12 from lymphocytes. However, at present the underlying mechanism remains unknown. It has been clarified that nuclear factor of activated T cells-2 transduces most immunological signals in T cells and modulates the production of interleukin-2. So it is natural that we asked whether high mobility group box-1 protein could promote production of interleukin-2 in a nuclear factor of activated T cells-2-dependent way. Our experiments firstly showed that high mobility group box-1 protein could bind to nuclear factor of activated T cells-2 in vivo and in vitro. High mobility group box-1 protein cotransfection markedly upregulated the transcription activity of nuclear factor of activated T cells-2 in promoting interleukin-2 reporter gene transcription, which was demonstrated to be dose-dependent. Cotransfection of high mobility group box-1 protein and nuclear factor of activated T cells-2 induced an 18.4-time increase of interleukin-2 activity in 293T cells and a 117.7-time increase in Hela cells. Moreover, inhibition of either high mobility group box-1 protein or nuclear factor of activated T cells -2 expression by sRNAi led to significant decrease of transcription activity of interleukin-2 reporter gene, suggesting that high mobility group box-1 protein and nuclear factor of activated T cells-2 both take important roles in facilitating interleukin-2 transcription, and high mobility group box-1 protein could act as a coactivator for nuclear factor of activated T cells-2 in enhancing transcription of interleukin-2. This discovery has not been reported elsewhere, and helps to understand the newly highlighted immunological role of high mobility group box-1 protein.

Fibroblast growth factor 2 (FGF2) plays an important role in the development of osteoarthritis (OA) through the regulation of cartilage degradation. However, the molecular mechanism underlying FGF2-induced OA is poorly characterized. MicroRNAs (miRNAs) maintain cartilage homeostasis. To examine whether FGF2 regulates OA through the modulation of miRNA, we screened potential miRNA molecules that could be regulated through FGF2 using microarray analysis. The results showed that microRNA-105 (miR-105) was significantly downregulated in chondrocytes stimulated with FGF2. Runt-related transcription factor 2 (Runx2), a key transcription factor involved in OA, has been identified as a novel potential target of miR-105. FGF2 suppressed miR-105 expression through the recruitment of the subunit of the nuclear factor kappa B transcription complex p65 to the miR-105 promoter. The knockdown of Runx2 mimicked the effect of miR-105 and abolished the ability of miR-105 to regulate the expression of a disintegrin-like and metalloproteinase with thrombospondin 4 (ADAMTS4), ADAMTS5, ADAMTS7 and ADAMTS12, both of which are responsible for the degradation of collagen 2A1 (COL2A1) and aggrecan (ACAN). miR-105 is also required for FGF2/p65-induced Runx2 activation and ADAMTS expression. Moreover, miR-105 expression was downregulated in OA patients and inversely correlated with the expression of Runx2, ADAMTS7 and ADAMTS12, which were upregulated in OA patients. These data highlight that the FGF2/p65/miR-105/Runx2/ADAMTS axis might play an important role in OA pathogenesis and that miR-105 might be a potential diagnostic target and useful strategy for OA treatment.Runx2 was identified as a novel direct target of miR-105. FGF2 inhibits miR-105 transcription through recruitment of p65 to miR-105 promoter. p65/miR-105 is essential for FGF2-mediated Runx2 and ADAMTS upregulation. miR-105 is downregulated in OA and inversely correlated with Runx2 expression.

To construct the prokaryotic expression vectors of extracellular domain (β-ED) and intracellular protein kinase domain (β-PKD) of insulin-like growth factor 1 receptor beta (IGF1R-β) subunit, purify the fusion proteins GST-IGF1R β-ED and GST-IGF1R β-PKD, and detect their activities.Human GST-IGF1R β-ED and GST-IGF1R β-PKD coding regions were amplified from human mammary cDNA library by PCR and cloned into the prokaryotic expression vector pGEX-KG. The fusion proteins GST-IGF1R β-ED and GST-IGF1R β-PKD were expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The expression of the fusion proteins were detected by Western blotting. The interactions of the proteins with mediator of epidermal growth factor receptor-2 (ERBB2)-driven cell motility (MEMO) protein were identified by GST pull-down assay.GST-IGF1R β-ED and GST-IGF1R β-PKD recombinant plasmids were successfully cloned. Double enzyme digestion and sequencing confirmed that the inserted fragments were identical to the target ones. The fusion proteins were successfully induced in Rossate and Western blotting showed the expression as expected. GST pull-down assay revealed that GST-IGF1R β-PKD could interact with MEMO in vitro.GST-IGF1R β-ED and GST-IGF1R β-PKD were successfully cloned and purified. In addition, GST-IGF1R β-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.

Estrogen exerts its physiological and pathological functions through two estrogen receptors (ERs), ERα and ERβ, which act as transcription factors. Coregulators, including coactivators and corepressors, have been shown to be crucial for regulation of ER transcriptional activity. Although many coregulators have been identified to regulate activities of ERs, novel coregulators are still needed to be investigated. Here, we show that human methyltransferase-like 17 (METTL17), whose function is unknown, physically interacts with ERα and ERβ, and functionally acts as a coactivator for ERs. METTL17 interacts with ER in vitro and in yeast and mammalian cells. Activation function-1 (AF1) and AF2 domains of ERs are responsible for the interaction between METTL17 and ERs. Knockdown of METTL17 reduces transcriptional activities of ERα and ERβ in breast cancer cells, whereas METTL17 overexpression increases ERα and ERβ transcriptional activities. Inhibition of METTL17 expression decreases mRNA and protein levels of ER target genes, including PR, cathepsin D, and pS2. Moreover, METTL17 knockdown reduces breast cancer cell growth. These results indicate that METTL17 is a novel coactivator of ERs and may play a role in breast tumorigenesis.