Hongyan Qi

Aberrantly expressed Fra-1 by IL-6/STAT3 transactivation promotes colorectal cancer aggressiveness through epithelial–mesenchymal transition

Summary This study describes a novel mechanism of the inflammatory cytokine IL-6 induced Fra-1 upregulation through activating STAT3 by phosphorylation and acetylation, and demonstrates that this signaling pathway plays a critical role in promoting epithelial–mesenchymal transition and aggressiveness of colorectal cancer.

Interferon Regulatory Factor 1 Transactivates Expression of Human DNA Polymerase η in Response to Carcinogen N-Methyl-N′-nitro-N-nitrosoguanidine

Background: Polη implements translesion DNA synthesis but has low fidelity in replication. Results: Acetylation-stabilized IRF1 transactivates the POLH gene in response to the chemical carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Conclusion: Abnormal up-regulation of Polη through IRF1 transactivation is responsible for mutation frequency increases in cells exposed to MNNG. Significance: IRF1-induced Polη activity is a new mechanism leading to mutation accumulation and carcinogenesis in cells exposed to an environmental chemical carcinogen. DNA polymerase η (Polη) implements translesion DNA synthesis but has low fidelity in replication. We have previously shown that Polη plays an important role in the genesis of nontargeted mutations at undamaged DNA sites in cells exposed to the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Here, we report that MNNG-induced Polη expression in an interferon regulatory factor 1 (IRF1)-dependent manner in human cells. Mutagenesis analysis showed that four critical residues (Arg-82, Cys-83, Asn-86, and Ser-87) located in the IRF family conserved DNA binding domain-helix α3 were involved in DNA binding and POLH transactivation by IRF1. Furthermore, Polη up-regulation induced by IRF1 was responsible for the increase of mutation frequency in a SupF shuttle plasmid replicated in the MNNG-exposed cells. Interestingly, IRF1 was acetylated by the histone acetyltransferase CBP in these cells. Lys → Arg substitution revealed that Lys-78 of helix α3 was the major acetylation site, and the IRF1-K78R mutation partially inhibited DNA binding and its transcriptional activity. Thus, we propose that IRF1 activation is responsible for MNNG-induced Polη up-regulation, which contributes to mutagenesis and ultimately carcinogenesis in cells.

JMJD5 interacts with p53 and negatively regulates p53 function in control of cell cycle and proliferation.

JMJD5 is a Jumonji C domain-containing demethylase/hydroxylase shown to be essential in embryological development, osteoclastic maturation, circadian rhythm regulation and cancer metabolism. However, its role and underlying mechanisms in oncogenesis remain unclear. Here, we demonstrate that JMJD5 forms complex with the tumor suppressor p53 by interacting with p53 DNA-binding domain (DBD), and negatively regulates its activity. Downregulation of JMJD5 resulted in increased expression of multiple p53 downstream genes, such as the cell cycle inhibitor CDKN1A and DNA repair effector P53R2, only in p53-proficient lung cancer cells. Upon DNA damage, the JMJD5-p53 association decreased, and thereby, promoted p53 recruitment to the target genes and stimulated its transcriptional activity. Furthermore, JMJD5 facilitated the cell cycle progression in a p53-dependent manner under both normal and DNA damage conditions. Depletion of JMJD5 inhibited cell proliferation and enhanced adriamycin-induced cell growth suppression in the presence of p53. Collectively, our results reveal that JMJD5 is a novel binding partner of p53 and it functions as a positive modulator of cell cycle and cell proliferation mainly through the repression of p53 pathway. Our study extends the mechanistic understanding of JMJD5 function in cancer development and implicates JMJD5 as a potential therapeutic target for cancer.

Identification and functional implication of nuclear localization signals in the N-terminal domain of JMJD5

JMJD5 has recently been reported to participate in circadian rhythm regulation, embryological development, osteoclastogenesis and tumorigenesis. Although JMJD5 has been found mainly localized in the nucleus of cells, how it enters the nucleus remains unclear. Here we report that JMJD5 contains a functional bipartite nuclear localization signal (NLS) and a chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES). Importin α/β and transportin-1 were further identified as JMJD5-associated transport proteins, and different binding regions were determined for the two nuclear import receptors. Additionally, we demonstrate that both the active NLS and the JmjC domain of JMJD5 are necessary for cyclin A1 transcription. Chromatin immunoprecipitation (ChIP) analysis confirmed the alterations of di-methylated lysine 36 of histone H3 (H3K36me2) in the coding region of cyclin A1. These results reveal that the N-terminal domain is essential for the nuclear localization of JMJD5 and its normal enzymatic function towards substrates in the nucleus.

Characterization of human DNA polymerase κ promoter in response to benzo[a]pyrene diol epoxide.

DNA polymerase κ (Pol κ), a member of Y-family DNA polymerases, can synthesize DNA with moderate fidelity on undamaged DNAs and replicate accurately in vitro thymine glycol, 8-oxo-G and aromatic adducts such as benzo[a]pyrene diol epoxide (BPDE). However, few studies have been done on the transcriptional regulation of Pol κ. In this study, we predicted and cloned the promoter region of the human POLK gene. Through the analysis of deletion constructs of the POLK promoter, we demonstrated that the region -336/-141 contained repressing elements and the region -141/+226 contained positive regulatory elements for transcription of human Pol κ. Furthermore, quantitative RT-PCR showed that human POLK mRNA expression was dysregulated in FL cells treated by BPDE. The transcriptional activities of the POLK promoter regions -336/+437 and +20/+437 were significantly reduced by BPDE treatment, indicating that transcription factors in this two regions, such as HSF1, may regulate the transcription of human POLK gene in response to BPDE.

Phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α) alleviates benzo[a]pyrene-7,8-diol-9,10-epoxide induced cell cycle arrest and apoptosis in human cells.

Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage and inducing extensive cell responses regulating cell cycle, cell survival and apoptosis. However, the mechanism of cellular responses to BPDE exposure is not fully understood. In this study, we demonstrated the involvement of the phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α) in the cellular response to BPDE exposure and addressed the role of eIF2α phosphorylation in the regulation of the cellular stress. Phosphorylation of eIF2α was induced in a normal human FL amnion epithelial cell line, and the expression of ATF4, a conserved downstream transcriptional factor of eIF2α phosphorylation, was up-regulated after BPDE exposure; however, the four known primary kinases for eIF2α phosphorylation (GCN2, HRI, PKR, and PERK) were not found activated. While BPDE induced severe cell cycle arrest and apoptosis and decreased cell viability in FL cells, salubrinal, a selective inhibitor of eIF2α dephosphorylation, maintained the eIF2α phosphorylation and attenuated cell cycle arrest and apoptosis and promoted cell survival. The findings reveal that when BPDE causes cellular damages, it induces eIF2α phosphorylation as well, which produces a pro-survival and anti-apoptotic effect to alleviate the cellular damages. Thus, the present study proposes a new cellular defensive mechanism during the environmental mutagen and carcinogen attack.

Non-enzymatic action of RRM1 protein upregulates PTEN leading to inhibition of colorectal cancer metastasis.

Ribonucleotide reductase large subunit M1 (RRM1) forms a holoenzyme with small subunits to provide deoxyribonucleotides for DNA synthesis and cell proliferation. Here, we reported a non-RR role of the catalytic subunit protein RRM1 and related pathway in inhibiting colorectal cancer (CRC) metastasis. Ectopic overexpression of the wild-type RRM1, and importantly, its Y738F mutant that lacks RR enzymatic activity, prevented the migration and invasion of CRC cells by promoting phosphatase and tensin homolog on chromosome 10 (PTEN) transactivation. Furthermore, overexpression of the wild-type and RR-inactive mutant RRM1 similarly reduced the phosphorylation of Akt and increased the E-cadherin expression in CRC cells, which were blocked by PTEN knockdown attenuation. Examination of clinical CRC specimens demonstrated that both RRM1 protein expression and RR activity were elevated in most cancer tissues compared to the paired normal tissues. However, while RR activity did not change significantly in different cancer stages, the RRM1 protein level was significantly increased at stages T1–3 but decreased at stage T4, in parallel with the PTEN expression level and negatively correlated with invasion and liver metastasis. Thus, we propose that RRM1 protein can inhibit CRC invasion and metastasis at the advanced stage by regulating PTEN transactivation and its downstream pathways in addition to forming an RR holoenzyme for supporting cancer proliferation. Understanding of the seemingly contrary dual roles of RRM1 protein may further help to explain the complex mechanisms by which this key enzyme and its components are involved in cancer development.

Physical interaction between human ribonucleotide reductase large subunit and thioredoxin increases colorectal cancer malignancy.

Ribonucleotide reductase (RR) is the rate-limiting enzyme in DNA synthesis, catalyzing the reduction of ribonucleotides to deoxyribonucleotides. During each enzymatic turnover, reduction of the active site disulfide in the catalytic large subunit is performed by a pair of shuttle cysteine residues in its C-terminal tail. Thioredoxin (Trx) and glutaredoxin (Grx) are ubiquitous redox proteins, catalyzing thiol-disulfide exchange reactions. Here, immunohistochemical examination of clinical colorectal cancer (CRC) specimens revealed that human thioredoxin1 (hTrx1), but not human glutaredoxin1 (hGrx1), was up-regulated along with human RR large subunit (RRM1) in cancer tissues, and the expression levels of both proteins were correlated with cancer malignancy stage. Ectopically expressed hTrx1 significantly increased RR activity, DNA synthesis, and cell proliferation and migration. Importantly, inhibition of both hTrx1 and RRM1 produced a synergistic anticancer effect in CRC cells and xenograft mice. Furthermore, hTrx1 rather than hGrx1 was the efficient reductase for RRM1 regeneration. We also observed a direct protein-protein interaction between RRM1 and hTrx1 in CRC cells. Interestingly, besides the known two conserved cysteines, a third cysteine (Cys779) in the RRM1 C terminus was essential for RRM1 regeneration and binding to hTrx1, whereas both Cys32 and Cys35 in hTrx1 played a counterpart role. Our findings suggest that the up-regulated RRM1 and hTrx1 in CRC directly interact with each other and promote RR activity, resulting in enhanced DNA synthesis and cancer malignancy. We propose that the RRM1-hTrx1 interaction might be a novel potential therapeutic target for cancer treatment.

Potent antitumor activity of the Ad5/11 chimeric oncolytic adenovirus combined with interleukin-24 for acute myeloid leukemia via induction of apoptosis.

The Ad5/11 chimeric oncolytic adenovirus represents a promising new platform for anticancer therapy. Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells and is the most common malignant myeloid disorder in adults. Myeloid and other hematopoietic cell lineages are involved in the process of clonal proliferation and differentiation. In the present study, we aimed to ascertain whether chimeric oncolytic adenovirus-mediated transfer of the human interleukin-24 (IL-24) gene induces enhanced antitumor potency. Our results showed that the Ad5/11 chimeric oncolytic adenovirus carrying hIL-24 (AdCN205‑11-IL-24) produced high levels of hIL-24 in AML cancer cells, as compared with the Ad5 oncolytic adenovirus expressing hIL-24 (AdCN205-IL-24). AdCN205-11-IL-24 specifically induced a cytotoxic effect on AML cancer cells, but had little or no effect on a normal cell line. AdCN205-11-IL-24 induced higher antitumor activity in AML cancer cells by inducing apoptosis in vitro. This study suggests that transfer of IL-24 by an Ad5/11 chimeric oncolytic adenovirus may be a potent antitumor approach for AML cancer therapy.

Gemcitabine and carboplatin demonstrate synergistic cytotoxicity in cervical cancer cells by inhibiting DNA synthesis and increasing cell apoptosis

Background The present study aims to investigate the subunit expression and enzyme activity of ribonucleotide reductase in cervical cancer patients, and detect the combined effect of the ribonucleotide reductase inhibitor gemcitabine and the chemotherapeutic agent carboplatin on cervical cancer cell lines. Methods Using quantitative reverse transcription polymerase chain reaction, Western blotting, and cytidine 5′-diphosphate reduction assays, we tested the expression and activity of ribonucleotide reductase in cervical cancer patients. The antitumor activity of gemcitabine and/or carboplatin treatments to SiHa and CaSki human cervical cancer cell lines were assessed by Cell Counting Kit-8 viability assay, EdU incorporation assay, immunofluorescence assay, flow cytometry assay, and Western blotting methods. Additionally, synergistic efficacy was quantitatively analyzed using a combination index based on the Chou-Talalay method. Results The mRNA levels of three ribonucleotide reductase subunits were all upregulated in the cervical cancer tissues compared with normal tissues (P<0.0001). Consistently, the protein expression and enzyme activity of ribonucleotide reductase were also increased in the cervical cancer tissues. Interestingly, gemcitabine inhibited DNA synthesis and carboplatin induced DNA damage. Further, the combined drug regime had a significant synergistic effect on inhibiting cervical cancer cell viability (log10[combination index] <0) via enhanced DNA damage and cell apoptosis. Conclusion The expression and activity of ribonucleotide reductase was increased in cervical cancer. Our study demonstrated the synergistic cytotoxicity of gemcitabine and carboplatin, through inhibiting DNA synthesis and increasing cell apoptosis in cervical cancer cell lines. This evidence might provide a rational clue of their combined application to improve cervical cancer treatment.