ZNF146 regulates cell cycle progression via TFDP1 and DEPDC1B in ovarian cancer cells.

In brief: Aberration in cell cycle progression is one of the essential mechanisms underlying tumorigenesis, making regulators of cell cycle reasonable anti-cancer therapeutic targets. Here, we dissected the regulatory mechanism involving the novel axis ZNF146/TFDP1/DEPDC1B in the cell cycle in ovarian cancer. Abstract: Ovarian cancer (OC) is the third most common kind of gynecological tumor, in addition to being the most lethal. Transcription factor Dp-1 (TFDP1) functions as a binding partner for E2F transcription factors, and its target genes include those involved in DNA synthesis, cell cycle, and apoptosis. However, the regulatory role of TFDP1 in OC remains incompletely understood. This study aimed to investigate the role and mechanism of TFDP1 in OC. TFDP1 was highly expressed in the ovarian epithelial tissues of OC patients, and the expression of TFDP1 in OC cells was higher than that in normal ovarian epithelial cells. Silencing of TFDP1 inhibited the biological activity of OC cells and hindered cell cycle entry. Zinc finger protein 146 (ZNF146) knockdown induced cell cycle arrest at the G0/G1 phase and tumor growth by blocking TFDP1 transcription, which was overturned by ectopic expression of TFDP1. TFDP1 stimulated DEP domain-containing protein 1B (DEPDC1B) expression through transcriptional activation. DEPDC1B increased the proportion of OC cells in the G2/M phase and potentiated tumor malignant progression in nude mice inhibited by sh-ZNF146. Taken together, these findings demonstrate that ZNF146 participates in TFDP1/DEPDC1B activation and plays a vital role in the cell cycle in OC.

The TFDP1 gene coding for DP1, the heterodimeric partner of the transcription factor E2F, is a target of deregulated E2F.

The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1∼E2F8), expression of activator E2Fs (E2F1∼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known. We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5' and 3' deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs. a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.

Effect of Upregulation of Transcription Factor TFDP1 Binding Promoter Activity Due to RBP4 g.36491960G>C Mutation on the Proliferation of Goat Granulosa Cells

Retinol-binding protein 4 (RBP4), a member of the lipocalin family, is a specific carrier of retinol (vitamin A) in the blood. Numerous studies have shown that RBP4 plays an important role in mammalian embryonic development and that mutations in RBP4 can be used for the marker-assisted selection of animal reproductive traits. However, there are few studies on the regulation of reproduction and high-prolificacy traits by RBP4 in goats. In this study, the 5' flanking sequence of RBP4 was amplified, and a G>C polymorphism in the promoter region -211 bp (g.36491960) was detected. An association analysis revealed that the respective first, second and third kidding number and mean kidding number of nanny goats with CC and GC genotypes (2.167 ± 0.085, 2.341 ± 0.104, 2.529 ± 0.107 and 2.189 ± 0.070 for CC and 2.052 ± 0.047, 2.206 ± 0.057, 2.341 ± 0.056 and 2.160 ± 0.039 for GC) were significantly higher (p < 0.05) than those with the GG genotype (1.893 ± 0.051, 2.027 ± 0.064, 2.107 ± 0.061 and 1.74 ± 0.05). The luciferase assay showed that luciferase activity was increased in C allele individuals compared with that in G allele individuals. A competitive electrophoretic mobility shift assay (EMSA) showed that individuals with the CC genotype had a stronger promoter region binding capacity than those with the GG genotype. In addition, transcription factor prediction software showed that the RBP4 g.36491960G>C mutation added a novel binding site for transcription factor DP-1 (TFDP1). RT−qPCR results showed that the expression of TFDP1 was significantly higher in the high-prolificacy group than in the low-prolificacy group, and the expression of RBP4 was higher in both the CC and GC genotypes than that in the GG genotype. TFDP1 overexpression significantly increased the expression of RBP4 mRNA (p < 0.05) and the expression of the cell proliferation factors cyclin-D1, cyclin-D2 and CDK4 (p < 0.05). The opposite trend was observed after interference with TFDP1. Both the EdU and CCK-8 results showed that TFDP1 expression could regulate the proliferation of goat ovarian granulosa cells. In summary, our results showed that RBP4 g.36491960G>C was significantly associated with fecundity traits in goats. The g.36491960G>C mutation enhanced the transcriptional activity of RBP4 and increased the expression of RBP4, thus improving the fertility of Yunshang black goats.

U2 small nuclear RNA auxiliary factor 2, transcriptionally activated by the transcription factor Dp-1/E2F transcription factor 1 complex, enhances the growth and aerobic glycolysis of leiomyosarcoma cells.

The dysregulation of U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) is associated with malignant behaviors of multiple types of tumors. In this study, we explored the association between U2AF2 dysregulation and the survival of patients with primary leiomyosarcoma, the regulatory effect of U2AF2 on cell growth/aerobic glycolysis, and the mechanisms of U2AF2 dysregulation at the transcriptional level. Gene expression and survival time of patients with primary leiomyosarcoma were extracted from TCGA-Sarcoma (SARC). Leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1 were utilized to construct in vitro and in vivo models. Results showed that the higher U2AF2 expression group had significantly shorter progression-free survival (HR: 2.049, 95%CI: 1.136-3.697, p = 0.011) and disease-specific survival (4.656, 95%CI: 2.141-10.13, p < 0.001) compared to the lower U2AF2 expression group. U2AF2 knockdown suppressed leiomyosarcoma cell growth and aerobic glycolysis (decreased glucose uptake, lactate production, and extracellular acidification rate) in vitro. Tumors derived from SK-LMS-1 cells with U2AF2 knockdown grew significantly slower, with lower GLUT1, PGK1, and PGAM1 protein expression than the control groups. TFDP1 and E2F1 could interact with each other in leiomyosarcoma cells. Both TFDP1 and E2F1 could bind to the promoter of U2AF2 and exert a synergistic activating effect on U2AF2 transcription. In conclusion, this study revealed that U2AF2 upregulation is associated with poor survival of leiomyosarcoma. Its upregulation enhances proliferation and aerobic glycolysis of leiomyosarcoma cells in vitro and in vivo. TFDP1 and E2F1 can form a complex, which binds to the U2AF2 gene promoter and synergistically activates its transcription.

Regulation of Multiple Fission and Cell-Cycle-Dependent Gene Expression by CDKA1 and the Rb-E2F Pathway in Chlamydomonas.

The green alga Chlamydomonas proliferates by "multiple fission": a long G1 with >10-fold cell growth followed by multiple rapid divisions. Cells above a critical size threshold are "committed" and will divide independent of light and further cell growth. The number of divisions carried out depends on the initial size of the committed mother cell. Here, I show that CDKA1, the ortholog of the yeast and animal mitotic inducer CDK1, regulates the critical size for commitment. The Rb/E2F/Dp1 pathway regulates division number as well as commitment size. Epistasis analysis indicated that CDKA1 and Rb/E2F/Dp1 regulate multiple fission by distinct mechanisms. Rb-E2F/Dp1 regulates G1/S gene expression in animals and land plants. Transcriptome analysis showed that mat3 or dp1 disruption altered regulation of a large group of cell-division-associated genes with respect to cell size, but not with respect to synchronization timing. In contrast, cdka1 inactivation disturbed both temporal and cell-size regulation of expression. These defects were enhanced by double inactivation of cdka1 and dp1, suggesting interaction between CDKA1 and the Rb-E2F/Dp1 pathways in regulating cell-cycle-specific gene expression and cell-cycle initiation. In the context of a theoretical model for regulation of Chlamydomonas multiple fission, these results suggest that CDKA1 may promote a switch into a division-competent state, and E2F/Dp1 may promote maintenance of this state.

miR-4711-5p regulates cancer stemness and cell cycle progression via KLF5, MDM2 and TFDP1 in colon cancer cells.

BACKGROUND: It is important to establish cancer stem cell (CSC)-targeted therapies to eradicate cancer. As it is a CSC marker, we focused on Kruppel-like factor 5 (KLF5) in this study. METHODS: We searched for candidate microRNAs (miRNAs) that inhibited KLF5 expression by in silico analyses and screened them in colon cancer cell lines. RESULTS: We identified one promising miRNA, miR-4711-5p, that downregulated KLF5 expression by direct binding. This miRNA suppressed cell proliferation, migration and invasion ability, as well as stemness, including decreased stem cell marker expression, reactive oxygen species activity and sphere formation ability. MiR-4711-5p inhibited the growth of DLD-1 xenografts in nude mice with no adverse effects. We found that miR-4711-5p provoked G1 arrest, which could be attributed to direct binding of miR-4711-5p to TFDP1 (a heterodimeric partner of the E2F family). Our findings also suggested that direct binding of miR-4711-5p to MDM2 could upregulate wild-type p53, leading to strong induction of apoptosis. Finally, we found that miR-4711-5p had a potent tumour-suppressive effect compared with a putative anti-oncomiR, miR-34a, in tumour cell cultures derived from five patients with colorectal cancer. CONCLUSIONS: Our data suggest that miR-4711-5p could be a promising target for CSC therapy.

Karyopherin α2-dependent import of E2F1 and TFDP1 maintains protumorigenic stathmin expression in liver cancer.

BACKGROUND: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. METHODS: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). RESULTS: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. CONCLUSION: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.

Functional Landscape of PCGF Proteins Reveals Both RING1A/B-Dependent-and RING1A/B-Independent-Specific Activities.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.

Expression of the TFDP1 gene in the endometrium of women with deep infiltrating endometriosis.

The field of endometriosis etiopathogenesis aims to identify the origin of disease in endometrial disorders. Changes in gene and protein expression related to cell adhesion, collagenases, and, mainly, cell cycle regulators have been identified. We set out to analyze the expression of the transcription factor DP-1 (TFDP1) gene, which encodes a protein that controls the G1/S phase passage of the cell cycle, in the endometrium of women with deep infiltrating endometriosis (DIE). Samples of endometrium from both endometriosis-affected women and healthy women were collected, cultured and maintained at the Cell Bank of the Pelvic Pain and Endometriosis Unit of the Federal University of Sao Paulo. This study analyzed five samples from the endometrium cell culture of healthy patients (i.e. no pelvic disease, as determined by means of laparoscopic tubal ligation) and six samples from women diagnosed with DIE. Samples were evaluated for TFDP1 gene expression by real-time PCR. We observed a downregulation of TFDP1 in the endometrium cells of women with DIE when compared to the control (a fold-change of -2.05, p value=.011). The TFDP1 gene is part of the cell cycle pathway, but its function is not yet clear. Additional studies are necessary to clarify the function of TFDP1 in endometriosis etiopathogenesis.

E2F1 and E2F7 differentially regulate KPNA2 to promote the development of gallbladder cancer.

Karyopherin alpha 2 (KPNA2) is a nuclear import factor that is elevated in multiple cancers. However, its molecular regulation at the transcriptional levels is poorly understood. Here we found that KPNA2 was significantly upregulated in gallbladder cancer (GBC), and the increased levels were correlated with short survival of patients. Gene knocking down of KPNA2 inhibited tumor cell proliferation and migration in vitro as well as xenografted tumor development in vivo. A typical transcription factor E2F1 associated with its DNA-binding partner DP1 bond to the promoter region of KPNA2 and induced KPNA2 expression. In contrast, an atypical transcription factor E2F7 competed against DP1 and blocked E2F1-induced KPNA2 gene activation. Mutation of the dimerization residues of E2F7 or DNA-binding domain of E2F1 abolished the suppressive effects of E2F7 on KPNA2 gene expression. In addition, KPNA2 mediated nuclear localization of E2F1 and E2F7, where they in turn controlled KPNA2 expression. Taken together, our data provided mechanistic insights into divergently transcriptional regulation of KPNA2, thus pointing to KPNA2 as a potential target for cancer therapy.

Differential requirement for dimerization partner DP between E2F-dependent activation of tumor suppressor and growth-related genes.

The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.

TFDP3 confers chemoresistance in minimal residual disease within childhood T-cell acute lymphoblastic leukemia.

Acquired drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. In this study, a novel gene therapy target for childhood T-ALL to overcome chemoresistance was discovered: TFDP3 increased in the minimal residual disease (MRD) positive childhood T-ALL patients. Then, we established a preclinical model of resistance to induction therapy to examine the functional relevance of TFDP3 to chemoresistance in MRD derived from Jurkat/E6-1. Jurkat xenografts in NOD/SCID mice were exposed to a four drug combination (VXLD) of vincristine (VCR), dexamethasone (DEX), L-asparaginase (L-asp) and daunorubicin (DNR). During the 4-week VXLD treatment, the level of TFDP3 increased 4-fold. High expression of TFDP3 was identified in the re-emerging lines (Jurkat/MRD) with increased chemoresistance, which is correlated with partially promoter demethylation of TFDP3. Downregulation of TFDP3 by RNA interference reversed chemoresistance in Jurkat/MRD accompanied by reinstated E2F1 activity that coincided with increased levels of p53, p73, and associated proapoptotic target genes. Importantly, TFDP3 silencing in vivo induced apparent benefit to overcome chemoresistance in combination with VXLD treatment. Collectively, TFDP3 confers chemoresistance in MRD within childhood T-ALL, indicating that TFDP3 is a potential gene therapy target for residual cancer.

COMMD9 promotes TFDP1/E2F1 transcriptional activity via interaction with TFDP1 in non-small cell lung cancer.

COMMD protein family is an evolutionarily conserved gene family implicated in a number of critical processes including inflammation, copper homeostasis, sodium balance, endosomal sorting and cancer. In an effort to profile the expression pattern of COMMD family in several non-small cell lung cancer (NSCLC) cell lines, we found that compared with that in human bronchial epithelial (HBE) cells, the mRNA expression levels of five COMMD genes including COMMD3, COMMD4, COMMD5, COMMD6 and COMMD8 were significantly down-regulated, whereas COMMD9 was up-regulated in NSCLC cell lines. Here we reported that the expression of COMMD9 protein was significantly increased in various NSCLC cell lines and tissue samples. SiRNA-induced knocking down of COMMD9 inhibited proliferation and migration, arrested cell cycle at G1/S transition and induced autophagy in NSCLC cells. Mechanistically, COMMD9 interacted with the TFDP1 through COMM domain, and DNA-binding domain of TFDP1 was required for this interaction. Moreover, decreased expression COMMD9 attenuated TFDP1/E2F1 activation accompanied with enhanced p53 signaling pathway. Taken together, these findings demonstrate that COMMD9 participates in TFDP1/E2F1 activation and plays a critical role in non-small cell lung cancer.

E2F1 and TFDP1 Regulate PITX1 Expression in Normal and Osteoarthritic Articular Chondrocytes.

We previously reported a loss-of-PITX1 expression in patients suffering of knee/hip osteoarthritis (OA). Search for the mechanism underlying this event led us to discover that PITX1 repression was triggered by the aberrant nuclear accumulation of Prohibitin (PHB1), an E2F1 co-repressor, in OA articular chondrocytes. In the current study, we assessed in details the involvement of E2F transcription factors in regulating PITX1 expression. We also analyzed other genes that are similarly regulated by E2F in regard to osteoarthritis. The transcriptional regulation of the PITX1 promoter by E2F1 was analyzed with the luciferase reporter assay, and chromatin immunoprecipitation assays, which confirmed direct E2F1-PITX1 interactions. The probable binding sites for E2F1 in the PITX1 promoter were identified by DNA pulldown experiments. In silico and in vitro analyses show that the PITX1 proximal promoter region contains 2 specific sequences that are bound by E2F1. Overexpression of E2F1 enhances PITX1 promoter activity and mRNA transcription. In primary control and osteoarthritis chondrocytes, real time RT-PCR was used to measure the mRNA expression levels of candidate genes under E2F1 transcriptional control. Transcription Factor Dp-1 (TFDP1) knockdown experiments confirmed that the E2F1-TFDP1 complex regulates PITX1. Knockdown of TFDP1, an E2F1 dimerization partner, inhibits the activating effect of E2F1 and reduces both PITX1 promoter activity and mRNA transcription. Real time RT-PCR results reveal reduced expression of TFDP1 and a similar downregulation of their targets PITX1, BRCA1, CDKN1A, and RAD51 in mid-stage OA chondrocytes. Collectively, our data define a previously uncharacterized role for E2F1 and TFDP1 in the transcriptional regulation of PITX1 in articular chondrocytes. Additional E2F1 targets may be affected in OA pathogenesis.

Rac3 regulates cell proliferation through cell cycle pathway and predicts prognosis in lung adenocarcinoma.

Lung cancer is still the leading cause of malignant deaths in the world. It is of great importance to find novel functional genes for the tumorigenesis of lung cancer. We demonstrated that Rac3 could promote cell proliferation and inhibit apoptosis in lung adenocarcinoma cell line A549 previously. The aim of this study was to investigate the function and mechanism of Rac3 in lung adenocarcinoma cell lines. Immunohistochemistry staining was performed in 107 lung adenocarcinoma tissues and matched non-tumor tissues. Multivariate analysis and Kaplan-Meier analysis were used to investigate the correlation between Rac3 expression and the clinical outcomes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and flow cytometry analysis were employed to determine the proliferative ability, cell cycle distribution, and apoptosis in H1299 and H1975 cell lines. Gene expression microarray and pathway analysis between the Rac3-siRNA group and the control group in A549 cells were performed to investigate the pathways and mechanism of Rac3 regulation. Rac3 was shown to be positively expressed in lung adenocarcinoma tissues, and the expression of Rac3 associates with longer survival in lung adenocarcinoma patients. Silencing of Rac3 significantly induced cell growth inhibition, colony formation decrease, cell cycle arrest, and apoptosis of lung adenocarcinoma cell lines, which accompanied by obvious downregulation of CCND1, MYC, and TFDP1 of cell cycle pathway involving in the tumorigenesis of lung adenocarcinoma based on the gene expression microarray. In conclusion, these findings suggest that Rac3 has the potential of being a therapeutic target for lung adenocarcinoma.

Dysregulation of TFDP1 and of the cell cycle pathway in high-grade glioblastoma multiforme: a bioinformatic analysis.

Despite extensive research, the prognosis of high-grade glioblastoma multiforme (GBM) has improved only slightly because of the limited response to standard treatments. Recent advances (discoveries of molecular biomarkers) provide new opportunities for the treatment of GBM. The aim of the present study was to identify diagnostic biomarkers of high-grade GBM. First, we combined 3 microarray expression datasets to screen them for genes differentially expressed in patients with high-grade GBM relative to healthy subjects. Next, the target network was constructed via the empirical Bayesian coexpression approach, and centrality analysis and a molecular complex detection (MCODE) algorithm were performed to explore hub genes and functional modules. Finally, a validation test was conducted to verify the bioinformatic results. A total of 277 differentially expressed genes were identified according to the criteria P < 0.05 and |log2(fold change)| ≥ 1.5. These genes were most significantly enriched in the cell cycle pathway. Centrality analysis uncovered 9 hub genes; among them, TFDP1 showed the highest degree of connectivity (43) and is a known participant in the cell cycle pathway; this finding pointed to the important role of TFDP1 in the progression of high-grade GBM. Experimental validation mostly supported the bioinformatic results. According to our study results, the gene TFDP1 and the cell cycle pathway are strongly associated with high-grade GBM; this result may provide new insights into the pathogenesis of GBM.

Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells.

Reactive oxygen species (ROS) regulate testicular function in health and disease. We previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli cells, and also induces antioxidant enzymes expression suggesting that defense systems are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and specially H2O2 may act as second messengers regulating signal transduction pathways and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase (LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and CREB/ATF-1 phosphorylation. Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant status in Sertoli cells and, more importantly, in the modulation of LDH expression which takes place through ROS generation and the Akt-CREB/ATF-1 pathway.

Intra-tumor heterogeneity in TP53 null High Grade Serous Ovarian Carcinoma progression.

BACKGROUND: High grade serous ovarian cancer is characterised by high initial response to chemotherapy but poor outcome in the long term due to acquired resistance. One of the main genetic features of this disease is TP53 mutation. The majority of TP53 mutated tumors harbor missense mutations in this gene, correlated with p53 accumulation. TP53 null tumors constitute a specific subgroup characterised by nonsense, frameshift or splice-site mutations associated to complete absence of p53 expression. Different studies show that this kind of tumors may have a worse prognosis than other TP53 mutated HGSC. METHODS: In this study, we sought to characterise the intra-tumor heterogeneity of a TP53 null HGSC consisting of six primary tumor samples, two intra-pelvic and four extra-pelvic recurrences using exome sequencing and comparative genome hybridisation. RESULTS: Significant heterogeneity was found among the different tumor samples, both at the mutational and copy number levels. Exome sequencing identified 102 variants, of which only 42 were common to all three samples; whereas 7 of the 18 copy number changes found by CGH analysis were presented in all samples. Sanger validation of 20 variants found by exome sequencing in additional regions of the primary tumor and the recurrence allowed us to establish a sequence of the tumor clonal evolution, identifying those populations that most likely gave rise to recurrences and genes potentially involved in this process, like GPNMB and TFDP1. Using functional annotation and network analysis, we identified those biological functions most significantly altered in this tumor. Remarkably, unexpected functions such as microtubule-based movement and lipid metabolism emerged as important for tumor development and progression, suggesting its potential interest as therapeutic targets. CONCLUSIONS: Altogether, our results shed light on the clonal evolution of the distinct tumor regions identifying the most aggressive subpopulations and at least some of the genes that may be implicated in its progression and recurrence, and highlights the importance of considering intra-tumor heterogeneity when carrying out genetic and genomic studies, especially when these are aimed to diagnostic procedures or to uncover possible therapeutic strategies.

Recurrent Amplification at 13q34 Targets at CUL4A, IRS2, and TFDP1 As an Independent Adverse Prognosticator in Intrahepatic Cholangiocarcinoma.

Amplification of genes at 13q34 has been reported to be associated with tumor proliferation and progression in diverse types of cancers. However, its role in intrahepatic cholangiocarcinoma (iCCA) has yet to be explored. We examined two iCCA cell lines and 86 cases of intrahepatic cholangiocarcinoma to analyze copy number of three target genes, including cullin 4A (CUL4A), insulin receptor substrate 2 (IRS2), and transcription factor Dp-1 (TFDP1) at 13q34 by quantitative real-time polymerase chain reaction. The cell lines and all tumor samples were used to test the relationship between copy number (CN) alterations and protein expression by western blotting and immunohistochemical assays, respectively. IRS2 was introduced, and each target gene was silenced in cell lines. The mobility potential of cells was compared in the basal condition and after manipulation using cell migration and invasion assays. CN alterations correlated with protein expression levels. The SNU1079 cell line containing deletions of the target genes demonstrated decreased protein expression levels and significantly lower numbers of migratory and invasive cells, as opposed to the RBE cell line, which does not contain CN alterations. Overexpression of IRS2 by introducing IRS2 in SUN1079 cells increased the mobility potential. In contrast, silencing each target gene showed a trend or statistical significance toward inhibition of migratory and invasive capacities in RBE cells. In tumor samples, the amplification of each of these genes was associated with poor disease-free survival. Twelve cases (13.9%) demonstrated copy numbers > 4 for all three genes tested (CUL4A, IRS2, and TFDP1), and showed a significant difference in disease-free survival by both univariate and multivariate survival analyses (hazard ratio, 2.69; 95% confidence interval, 1.23 to 5.88; P = 0.013). Our data demonstrate that amplification of genes at 13q34 plays an oncogenic role in iCCA featuring adverse disease-free survival, which may provide new directions for targeted therapy.

Kelch Repeat and BTB Domain Containing Protein 5 (Kbtbd5) Regulates Skeletal Muscle Myogenesis through the E2F1-DP1 Complex.

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.