Role Of WDHD1 And Its Regulation In HPV-mediated Oncogenesis And Other Tumor Cells

Human papillomaviruses (HPVs) are small DNA viruses that replicate in squamous epithelia. Specific types of HPV (high-risk HPVs) are the causative agents for cervical and several other cancers. The transforming properties of high-risk HPVs such as HPV type 16 (HPV-16) primarily depend on E7 as well as E6 oncogenes. HPV E6 and E7 proteins promote the degradation of p53 and pRb, respectively. E7 from the high-risk HPV types can abrogate cell cycle checkpoints and induces genomic instability. Although several transcription profiling studies for E7 have been conducted using DNA microarray, the HPV E7 activities downstream from, or independent of, pRb responsible for deregulation of cell cycle and induction of genomic instability are not fully understood.Cell cycle progression is regulated by cyclins, cyclin-dependent kinases (Cdks) and their regulatory proteins at several checkpoints. Once the checkpoint becomes abnormal, genomic instability may occur. Genomic instability is a hallmark of cancer progression. Polyploidy is a type of genomic instability where cells have more than two sets of chromosomes and has been recognized as a causal factor for tumorigenesis. Significantly, polyploidy can be detected in the early stage of cervical carcinogenesis. Polyploidy can be formed via re-replication, a process of successive rounds of host DNA replication without entering mitosis. Re-replication may lead to not only polyploidy but also gene amplification, DNA fragmentation, DNA breaks, and cellular DNA damage response. We recently demonstrated that HPV-16 E7 induces re-replication and the cellular DNA replication initiation factor Cdt1 plays a role in this process.DNA replication is regulated by sequential and interactive mechanisms to ensure that the genome is accurately replicated only once per cell cycle. The process of replication initiation is divided into two steps: prereplicative complex (pre-RC) assembly and activation, the latter leads to generation of replication forks. Pre-RC starts with the association of origin recognition complex (ORC), which then promotes the recruitment of two proteins, Cdc6 and Cdt1, onto origins. This is followed by recruitment of minichromosome maintenance 2-7 (MCM 2-7) onto chromatin as a result of concerted action of Cdc6 and Cdt1. Prior to S phase, origins are licensed by the binding of components of the replicative DNA helicase MCMs in eukaryotes. Afterwards, licensing proteins are downregulated or inhibited, so that no more origins can be licensed and re-replication of DNA is prevented. Cells employ a licensing checkpoint to monitor that sufficient origins are licensed, inhibiting S-phase entry until this is established. Part one. Role of WDHD1 and its regulation in HPV-mediated oncogenesis.To identify genes differentially expressed between cells with and without E7, we used NIKS cells. NIKS cells exhibit many characteristics of early-passage human keratinocytes, the natural host cells for HPV, including stratification, differentiation, and ability to sustain the HPV life cycle. For gene expression profiling analysis, the RNA-seq approach was used. From RNA-seq data, we detected a total of 20,537 transcripts that include 17,090 mRNAs. After normalization, two hundred and thirty seven genes were identified that were differentially expressed between E7 expressing and vector control NIKS cells. Among these, one hundred fifty genes were upregulated and 87 genes were downregulated in NIKS cells expressing E7. To verify the RNA-seq results, we performed real-time PCR assay for nearly a dozen genes selected on the basis of their potential E7-related biological functions.Next we performed bioinformatics analysis on genes differentially expressed in E7 expressing cells. First we used the Gene Ontology (GO) system, a gene function classification system that provides a comprehensive description of gene properties. Next we used the KEGG pathway analysis that could reveal a significant enrichment of multiple pathways that are known to enhance tumor progression. DNA replication, DNA repair and nucleotide excision repair appeared to be upregulated in E7 expressing cells when compared to control cells while the induction of programmed cell death functional group are downregulated in E7 expressing cells. Given the fact that cell cycle and DNA replication pathways and their related genes are significantly disregulated in E7 expressing cells, we selected WDHD1, which is known to be involved in both DNA replication and potentially G1 checkpoint regulation, for further analysis. The levels of WDHD1 mRNA and protein were upregulated in E7 expressing cells comparable to control cells while there were no significant changes in cells expressing HPV-6 E7, which does not degrade pRb. Meawhile we found the protein half-life of WDHD1 in E7 expressing cells was greatly increased as compared with the vector control cells (3.4 hours vs.1.6 hours).As an HMG box containing protein and a DNA replication initiation factor, WDHD1 may play a role in cell cycle control in E7 expressing cells. To test this possibility, we employed the RNAi approach by using two independent siRNAs. Consistent with what we have recently observed, upon DNA damaging agent bleomycin treatment, less cells arrested at the G1 phase in the non-silencing siRNA control transfected E7 expressing cells compared with the vector control cells, indicating abrogation of the G1 checkpoint in E7 expressing cells. Notably, knockdown of the WDHD1 with siRNAs led to an increase of G1 peak in E7-expressiong cells. To demonstrate the role of WDHD1 in promoting S-phase entry of cells more directly, we transfected siRNAs targeting WDHD1 into E7 expressing cells and measured BrdU incorporation upon bleomycin treatment. Significantly, knockdown of WDHD1 by siRNAs led to a significant reduction of BrdU incorporation. These results demonstrate an important role of WDHD1 in the G1 cell cycle control and S-phase entry of E7-expressing cells.We have recently demonstrated that cells expressing HPV-16 E7 undergo re-replication upon DNA damage and that the DNA replication initiating factor Cdt1 plays an important role in this process. Since WDHD1 has been implicated as a DNA replication initiating factor, we examined its potential role in E7-induced re-replication. We first synchronized cells with thymidine and then released them into S phase and G2 phase, as time passed,60% of cells entered G2 phase and 8% became polyploid by 6 hours, suggesting re-replication had occurred. We then used siRNAs to assess the role of WDHD1 in E7-induced re-replication upon DNA damage. Notably, the percentage of polyploidy formed was significantly reduced in E7 expressing cells after WDHD1 knockdown. As an initial step towards understanding the mechanism by which WDHD1 knockdown causes reduction in re-replication, we examined the loading of MCM3 onto chromatin by ChIP assay. It is known that DNA replication initiating factors recruit MCMs to the origin of replication. Consistent with this notion, more MCM3 (-2.5-fold) bound to chromatin in E7 expressing cells than in vector control cells. WDHD1 knockdown by siRNA significantly reduced MCM3 loading to the chromatin. Next we found MCM3 mRNA (left panel) and the steady-state levels of MCM3 went down after WDHD1 knockdown. In addition, MCM3 knockdown significantly reduced polyploidy formation. These results indicate that WDHD1 facilitates replication and re-replication in E7 expressing cells by modulating MCM3 in E7 expressing cells. These results should help to gain insights into the cellular pathways targeted during tumor development caused by HPV.Part two. The regulation mechanism of WDHD1 in E7 expressing cells and other tumor cells.Signal transducers and activators of transcription (STATs) were involved in the pathogenesis of various human diseases, including cancers, autoimmune and inflammatory disorders. The STATs share several conserved structural and functional domains and the most interesting and conserved domain is SRC homology 2 domain (SH2). The SH2 domain is required for STAT dimerization and the associated ability to bind DNA. A core member of the STATs protein family- STAT3 that has been extensively studied for its function as a transcriptional regulator is a DNA-binding transcription factor. There are 70 identified direct target genes for STAT3, shown either by chromatin immunoprecipitation (ChIP) or electrophoretic mobility shift assay (EMSA).There are three STAT3 binding sites in the promoter region of WDHD1, STAT3 and WDHD1 were found to be both highly expressed in E7 expressing cell in our RNA-seq data. We therefore predict that WDHD1 is a new target gene of STAT3. WDHD1 plays a role in DNA replication. Meanwhile in our recent study we found downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and re-replication, so we hypothesized that STAT3 plays a role in cell cycle by regulating WDHD1. All these data below are validated in E7 expression cells, colon cancer cells, and breast cancer cells. Due to page limitation, only the results using colon cancer and breast cancer cells will be presented.The role STAT3 in the G1 to S phase transition has been well-established. However, there has been no report regarding its role in DNA replication nor rereplication. Significantly, in MCF-7 cells transfected with siRNAs targeting STAT3, cells showed a concomitant reduction in DNA replication. Overexpression of STAT3 increased DNA replication. Furthermore, treatment with STAT3 activator IL-6 also increased cellular DNA replication. To explore whether cell re-replication can be influenced by STAT3, we used the colon cancer cell HCT-116 that can be induced to undergo re-replication by MLN4924. Notably, the percentage of polyploidy formed was significantly reduced in HCT116 cells after STAT3 knockdown (from 40.2% to 11.4% for si-STAT3-1 and 13.7% for si-STAT3-2). These results indicate that STAT3 plays an important role in DNA replication and re-replication in carcinoma cells.To investigate whether WDHD1 mRNA expression can be regulated by STAT3, we used real-time PCR. We first examined the WDHD1 mRNA level after treated with IL-6 or another activator EGF. Next we examined WDHD1 mRNA level in cells that have been treated with STAT3 siRNA, followed by IL-6 stimulation. Interestingly, the reduction of WDHD1 mRNA expression after STAT3 knockdown and can be rescued by IL-6 stimulation. Thus, we demonstrated the WDHD1 RNA level can be regulated by STAT3. Significantly, downregulation of STAT3 reduced the expression of WDHD1 and overexpression of STAT3 (Flag-STAT3 transfection and IL-6 treatment) increased the expression of WDHD1.STAT family proteins generally recognize a consensus DNA binding motif of TTCC (C or G) GGAA (or generically TTN5AA). Three putative STAT3 binding sites (SB), named SB1 to SB3, were found in the WDHD1 promoter. To examine possible STAT3 binding to the WDHD1 promoter, ChIP assays were performed. A significant increase in STAT3 bound to the wdhdi promoter was found in both MCF-7 and HeLa cells at most of the binding sites.Is the function of STAT3 in DNA replication or re-replication mediated by WDHD1? To explore this possibility, we overexpressed WDHD1 in STAT3 knock-down cells. Significantly, overexpression of WDHD1 rescued DNA replication reduction in MCF-7cells after STAT3 siRNA transfection. Furthermore, the DNA re-replication reduction following STAT3 knock down was rescued by overexpression of WDHD1.Above all, we described a novel biological function of STAT3 and found the WDHD1 gene as a STAT3 new target and demonstrated that STAT3 plays a role in both DNA replication and re-replication mediated by WDHD1.