TFDP3 regulates the apoptosis and autophagy in breast cancer cell line MDA-MB-231.

Cancer/testis antigen TFDP3 belongs to the transcription factor DP(TFDP) family. It can bind to E2F family molecules to form a heterodimeric transcription factor E2F/TFDP complex. The complex is an important regulatory activator of cell cycle, involved in the regulation of cell proliferation, differentiation, apoptosis and other important physiological activities. In addition, TFDP3 has also been found to be a tumor-associated antigen that only expresses in malignant tumor tissue and normal testicular tissue; Thus, it is closely related to tumor occurrence and development. In this study, our group investigated the expression of TFDP3 in mononuclear cell samples from a variety of tissue-derived malignant tumors, breast cancer and benign breast lesions. The results show that TFDP3 is expressed in the malignant form of various tissues. Moreover, our recent research had focused on the ability of TFDP3 to influence the drug resistance and apoptosis of tumor cells. To further clarify the mechanisms involved in tumor resistance, this study also examined the expression of TFDP3 and tumor cell autophagy regulation; Autophagy helps cells cope with metabolic stress (such as in cases of malnutrition, growth factor depletion, hypoxia or hypoxia) removes erroneously folded proteins or defective organelles to prevent the accumulation of abnormal proteins; and removes intracellular pathogens. Our results showed that TFDP3 expression can induce autophagy by up-regulating the expression of autophagic key protein LC3(MAP1LC3) and increasing the number of autophagosomes during chemotherapy of malignant tumors. Then, DNA and organelles damage caused by the chemotherapy medicine are repaired. Thus, TFDP3 contributes toward tumor cell resistance. When siRNA inhibits TFDP3 expression, it can reduce cell autophagy, improving the sensitivity of tumor cells to chemotherapy drugs.

Effects of cancer-testis antigen, TFDP3, on cell cycle regulation and its mechanism in L-02 and HepG2 cell lines in vitro.

TFDP3, also be known as HCA661, was one of the cancer-testis antigens, which only expressed in human tissues. The recent researches about TFDP3 mostly focused on its ability to control the drug resistance and apoptosis of tumor cells. However, the role of TFDP3 in the progress of the cell cycle is rarely involved. In this study, we examined the expression of TFDP3 in human liver tissues firstly. After that, we detect the expression of TFDP3 at the RNA level and protein level in L-02 cell line and HepG2 cell line, and the location of TFDP3 was defined by immunofluorescence technique. Furthermore, we synchronized the cells to G1 phase, S phase and G2 phase, and arrested cell mitosis. The localization of TFDP3 and co-localization with E2F1 molecules in different phases of hepatocyte lines. Finally, TFDP3 gene knockout was performed on L-02 and HepG2 cell lines, and detected the new cell cycles by flow cytometry. The result showed that the expression of TFDP3 molecule is negative in normal liver tissue, but positive in immortalized human hepatocyte cell line, and the expression level is lower than in hepatocellular carcinoma cell line. The expression level of TFDP3 was in the dynamic change of L-02 and HepG2 cell lines, and was related to the phase transition. TFDP3 can bind to E2F1 molecule to form E2F/TFDP3 complex; and the localizations of TFDP3 and E2F1 molecules and the co-localization were different in different phases of cell cycle in the nucleus and cytoplasm, which indicated that the E2F/TFDP3 complex involved in the process of regulating the cell cycle. By knocking down the TFDP3 expression level in L-02 and HepG2 cell lines, the cell cycle would be arrested in S phase, which confirmed that TFDP3 can be a potential target for tumor therapy.

TFDP3 Regulates Epithelial-Mesenchymal Transition in Breast Cancer.

Breast cancer remains a lethal disease to women due to lymph node metastasis, the tumor microenvironment, secondary resistance and other unknown factors. Several important transcription factors involved in this disease, such as PTEN, p53 and beta-catenin, have been identified and researched in-depth as candidates for targeted therapy in breast cancer. TFDP3 is a new, promising candidate for transcriptional regulation in breast cancer, although it was first identified in hepatocellular carcinoma. Here, we demonstrate that TFDP3 is expressed in a variety of malignancies, normal testis tissue and breast cancer cell lines and thus provide evidence that TFDP3 is a cancer-testis antigen. We illustrate that overexpression or silencing TFDP3 interferes with epithelial-mesenchymal transition but does not influence cell proliferation, indicating that the TFDP3 protein acts as a transcription factor during epithelial-mesenchymal transition. These data highlight that TFDP3 is expressed in breast cancer, that it is a member of the cancer-testis antigen family and that it functions as a regulator in epithelial-mesenchymal transition.

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.

Overexpression of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) supports growth of hepatocellular carcinoma.

UNLABELLED: The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. CONCLUSION: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression.

E2F-1 regulation by an unusual DNA damage-responsive DP partner subunit.

E2F activity is negatively regulated by retinoblastoma protein (pRb) through binding to the E2F-1 subunit. Within the E2F heterodimer, DP proteins are E2F partner subunits that allow proper cell cycle progression. In contrast to the other DP proteins, the newest member of the family, DP-4, downregulates E2F activity. In this study we report an unexpected role for DP-4 in regulating E2F-1 activity during the DNA damage response. Specifically, DP-4 is induced in DNA-damaged cells, upon which it binds to E2F-1 as a non-DNA-binding E2F-1/DP-4 complex. Consequently, depleting DP-4 in cells re-instates E2F-1 activity that coincides with increased levels of chromatin-bound E2F-1, E2F-1 target gene expression and associated apoptosis. Mutational analysis of DP-4 highlighted a C-terminal region, outside the DNA-binding domain, required for the negative control of E2F-1 activity. Our results define a new pathway, which acts independently of pRb and through a biochemically distinct mechanism, involved in negative regulation of E2F-1 activity.

TFDP3 inhibits E2F1-induced, p53-mediated apoptosis.

By dimerizing with E2F proteins, TFDP has profound influence on cellular E2F activities. While TFDP1 and 2 enhance the DNA binding and the transcriptional activity of E2F, the newly identified member of the DP family, TFDP3 primarily functions as a negative regulator. To further characterize the inhibitory property of TFDP3, the present study specifically examined the modulatory role of TFDP3 on E2F1-induced cell death. HEK-293 cells underwent apoptosis following ectopic expression of E2F1. This effect was virtually abolished by co-transfection with TFDP3. In the meantime, the accumulation of p53 proteins and the increased expression of the pro-apoptotic molecules, including Bax, Puma, Noxa, and Bid were found to be suppressed. These data suggest a new mechanism for the regulation of E2F1-induced apoptosis and provide further evidence for the general involvement of TFDP3 in the regulation of E2F functions.

Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F.

The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFDP3 is apparently distinct from TFDP1 in function. Although TFDP3 retained the capacity to bind to E2F proteins, the resulting heterodimers failed to interact with the E2F consensus sequence. In contrast to the stimulatory effect of TFDP1, TFDP3 inhibited E2F-mediated transcriptional activation. Consistent with this observation, we found that ectopic expression of TFDP3 impaired cell cycle progression from G(1) to S phase instead of facilitating such a transition as TFDP1 does. Sequence substitution analysis indicated that the DNA binding domain of TFDP3 was primarily responsible for the lack of DNA binding ability of E2F-TFDP3 heterodimers and the inhibition of E2F-mediated transcriptional activation. Fine mapping further revealed four amino acids in this region, which were critical for the functional conversion from activation by TFDP1 to suppression by TFDP3. In conclusion, these studies identify a new DP protein and a novel mechanism whereby E2F function is regulated.

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene.

In the present study, we show that E2Fs (E2 promoter-binding factors) regulate the expression of ASK-1 (apoptosis signal-regulating kinase 1), which encodes a mitogen-activated protein kinase kinase kinase, also known as MAP3K5. Its mRNA expression is cell-cycle-regulated in human T98G cells released from serum starvation. Moreover, overexpression and RNA interference experiments support the requirement of endogenous E2F/DP (E2F dimerization partner) activity for ASK-1 expression. Characterization of the human ASK-1 promoter demonstrates that the -95/+11 region is critical for E2F-mediated up-regulation. Chromatin immunoprecipitation assays show that E2F1-E2F4 are bound in vivo to the ASK-1 promoter in cycling cells, probably through a non-consensus E2F-binding site located 12 bp upstream of the transcription start site. Mutation of this site completely abolishes the ASK-1 promoter response to E2Fs as well as the E2F1 binding in electrophoretic mobility-shift experiments. Our results indicate that E2Fs modulate the expression of ASK-1 and suggest that some of the cellular functions of ASK-1 may be under the control of E2F transcription factors. Moreover, the up-regulation of ASK-1 may also favour the p53-independent E2F1 apoptotic activity.