Genome-Wide Transcriptome Analysis of Human Papillomavirus 16-Infected Primary Keratinocytes Reveals Subtle Perturbations Mostly due to E7 Protein Expression.

It is established that the host cell transcriptomes of natural lesions, organotypic rafts, and human papillomavirus (HPV)-immortalized keratinocytes are altered in the presence of HPV genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at 7 days postinfection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107, and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to the E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus had perturbed transcriptional regulation of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.IMPORTANCE To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.

Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells.

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.

Combinatorial PX-866 and Raloxifene Decrease Rb Phosphorylation, Cyclin E2 Transcription, and Proliferation of MCF-7 Breast Cancer Cells.

As a potential means to reduce proliferation of breast cancer cells, a multiple-pathway approach with no effect on control cells was explored. The human interactome being constructed by the Center for Cancer Systems Biology will prove indispensable to understanding composite effects of multiple pathways, but its discovered protein-protein interactions require characterization. Accordingly, we explored the effects of regulators of one protein on downstream targets of the other protein. MCF-7 estrogen receptor-positive (ER+) breast cancer cells were treated with raloxifene to upregulate the TGF-ß pathway and PX-866 to down-regulate the PI3K/Akt pathway. This resulted in highly significant downstream reduction of cell cycle proliferation in breast cancer cells with no significant proliferation reduction following similar treatment of noncancerous MCF10A breast epithelial cells. Reduced phosphorylation of p107 and substantial reduction of Rb phosphorylation were observed in response. The effects of reduced Rb and p107 phosphorylation were reflected in significant decline in E2F-1 transcriptional activity, which is dependent on pocket protein phosphorylation status. The reduced proliferation was related to decreased expression of cyclins, including E2F-1-regulated Cyclin E2, which was also in response to raloxifene and PX-866. All combinations of raloxifene and PX-866 produced significant or highly significant results for reduced MCF-7 cell proliferation, reduced Cyclin E2 transcription, and reduced Rb phosphorylation. These studies demonstrated that uncontrolled proliferation of ER+ breast cancer cells can be significantly reduced by combinational targeting of two relevant pathways. J. Cell. Biochem. 117: 1688-1696, 2016. © 2015 Wiley Periodicals, Inc.

DREAM interrupted: severing LIN-35-MuvB association in Caenorhabditis elegans impairs DREAM function but not its chromatin localization.

The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in mammalian systems, p107 and p130 are the ancestral pocket proteins. The Rb-like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an E2F-DP transcription factor heterodimer, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex. DREAM complex assembly on chromatin culminates in repression of target genes mediated by the MuvB subcomplex. Here, we examined how the Rb-like pocket protein contributes to DREAM formation and function by disrupting the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. A triple alanine substitution of LIN-52's LxCxE motif severed LIN-35-MuvB association and caused classical DREAM mutant phenotypes, including synthetic multiple vulvae, high-temperature arrest, and ectopic expression of germline genes in the soma. However, RNA-sequencing revealed limited upregulation of DREAM target genes when LIN-35-MuvB association was severed, as compared with gene upregulation following LIN-35 loss. Based on chromatin immunoprecipitation, disrupting LIN-35-MuvB association did not affect the chromatin localization of E2F-DP, LIN-35, or MuvB components. In a previous study, we showed that in worms lacking LIN-35, E2F-DP, and MuvB chromatin occupancy was reduced genome-wide. With LIN-35 present but unable to associate with MuvB, our study suggests that the E2F-DP-LIN-35 interaction promotes E2F-DP's chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket protein's association with MuvB supports DREAM function but is not required for DREAM's chromatin occupancy.

Retinoblastoma Protein Paralogs and Tumor Suppression.

The retinoblastoma susceptibility gene (RB1) is the first tumor suppressor gene discovered and a prototype for understanding regulatory networks that function in opposition to oncogenic stimuli. More than 3 decades of research has firmly established a widespread and prominent role for RB1 in human cancer. Yet, this gene encodes but one of three structurally and functionally related proteins that comprise the pocket protein family. A central question in the field is whether the additional genes in this family, RBL1 and RBL2, are important tumor suppressor genes. If so, how does their tumor suppressor activity overlap or differ from RB1. Here we revisit these questions by reviewing relevant data from human cancer genome sequencing studies that have been rapidly accumulating in recent years as well as pertinent functional studies in genetically engineered mice. We conclude that RBL1 and RBL2 do have important tumor suppressor activity in some contexts, but RB1 remains the dominant tumor suppressor in the family. Given their similarities, we speculate on why RB1 tumor suppressor activity is unique.

The Role and Clinical Implications of the Retinoblastoma (RB)-E2F Pathway in Gastric Cancer.

Gastric cancer is the most common malignant tumor in the digestive tract, with very high morbidity and mortality in developing countries. The pathogenesis of gastric cancer is a complex biological process mediated by abnormal regulation of proto-oncogenes and tumor suppressor genes. Although there have been some in-depth studies on gastric cancer at the molecular level, the specific mechanism has not been fully elucidated. RB family proteins (including RB, p130, and p107) are involved in cell cycle regulation, a process that largely depends on members of the E2F gene family that encode transcriptional activators and repressors. In gastric cancer, inactivation of the RB-E2F pathway serves as a core transcriptional mechanism that drives cell cycle progression, and is regulated by cyclins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors, p53, Helicobacter pylori and some other upstream molecules. The E2F proteins are encoded by eight genes (i.e. E2F1 to E2F8), each of which may play a specific role in gastric cancer. Interestingly, a single E2F such as E2F1 can activate or repress transcription, and enhance or inhibit cell proliferation, depending on the cell environment. Thus, the function of the E2F transcription factor family is very complex and needs further exploration. Importantly, the presence of H. pylori in stomach mucosa may affect the RB and p53 tumor suppressor systems, thereby promoting the occurrence of gastric cancer. This review aims to summarize recent research progress on important roles of the complex RB-E2F signaling network in the development and effective treatment of gastric cancer.