NFX1-123 is highly expressed in cervical cancer and increases growth and telomerase activity in HPV 16E6 expressing cells

A significant contributor to women’s cancer mortality worldwide is cervical cancer, which is caused by high-risk human papillomavirus (HR HPV). The two viral oncoproteins of HR HPV, E6 and E7, partner with host cell proteins to target oncogenic proteins and pathways. Previously, we have shown HR HPV type 16 E6 (16E6) interacts with the host protein NFX1-123 to target telomerase and cellular immortalization, requiring NFX1-123 to fully upregulate telomerase activity. We now report that NFX1-123 is highly expressed in primary cervical cancers. In vitro, cells expressing 16E6 and overexpressing NFX1-123 have extended active growth, decreased senescence marker staining, and more rapid cell cycling compared to 16E6 expressing cells with endogenous amounts of NFX1-123. These findings were associated with increased telomerase activity and augmented expression of its catalytic subunit, hTERT. In complement, HPV 16 positive cervical cancer cell lines with knocked down NFX1-123 had slowed growth and reduced hTERT over time. In cells that express HR HPV E6, greater expression of NFX1-123 can modify active cellular growth and augment hTERT expression and telomerase activity over time, potentially supporting the initiation and progression of HPV-associated cancers

HPV type 16 E6 and NFX1-123 Augment JNK Signaling to Mediate Keratinocyte Differentiation and L1 Expression

The HPV life cycle is differentiation-dependent, with cellular differentiation driving initiation of the late, productive stage of the viral life cycle. Here, we identify a role for the protein NFX1-123 in regulating keratinocyte differentiation and events of the late HPV life cycle. NFX1-123 itself increased with differentiation of epithelial cells. Greater NFX1-123 augmented differentiation marker expression and JNK phosphorylation in differentiating 16E6-expressing human foreskin keratinocytes (16E6 HFKs). This was associated with altered expression of MKK4 and MKK7, upstream kinase regulators of JNK phosphorylation. Modulating levels of NFX1-123 in HPV16-positive W12E cells recapitulated the effects on differentiation markers, JNK phosphorylation, and MKK4/7 seen in 16E6 HFKs. Crucially, levels of NFX1-123 also correlated with expression of L1, the capsid protein of HPV. Altogether, these studies define a role for NFX1-123 in mediating epithelial differentiation through the JNK signaling pathway, potentially linking expression of cellular genes and HPV genes during differentiation

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Alteration of the Epithelial Environment and Cellular Pathways by HPV type 16 E6 and NFX1-123

Thesis (Ph.D.)--University of Washington, 2018High-risk human papillomaviruses (HR HPV) are the causative agent of various anogenital and oropharyngeal cancers, accounting for approximately 5% of the global burden of cancer. The highest risk factor for development of these cancers is a persistent infection with HR HPV. Understanding what promotes long-lived HPV infection is critical for predicting and preventing serious disease outcomes. Persistent infection with HR HPV requires successful propagation in stratified squamous epithelium and is therefore driven by how HR HPVs manipulate the host cell to create an environment beneficial to the viral life cycle. The study of HR HPV, their interactions with the host cell, and how these interactions engender cellular changes that may secondarily lead to oncogenesis is the focus of this dissertation. This dissertation summarizes several ways in which a HR HPV oncoprotein, HPV type 16 E6 (16E6), partners with a host protein, NFX1-123, to manipulate cellular processes and promote its life cycle. Previous work in the laboratory has shown that 16E6 utilizes NFX1-123 to upregulate differentiation markers in keratinocytes; increase levels of Notch1, the master cell fate regulator; and increase expression of telomerase, an enzyme critical to immortalization. The studies presented here further define the cellular processes altered by 16E6 and NFX1-123 to create a cellular milieu beneficial to the viral life cycle. Evasion of the immune response by HR HPVs has been well-documented, but the many molecular mechanisms and protein partners driving these immune deregulatory functions have not yet been fully defined. The data in Chapter 3 establishes 16E6 and NFX1-123 as one such protein partnership through which HR HPVs interfere with the innate immune response and escape detection within infected epithelial cells. Overexpression of NFX1-123 in 16E6 keratinocytes 1) decreased expression of pro-inflammatory cytokines and interferon-stimulated genes (ISGs) at the mRNA and protein level 2) decreased total levels of TRAF6 and TAB2, two proteins in the signaling cascades that govern induction of these antiviral genes and 3) disturbed the normal localization of these signaling proteins. Initiating the immune response within infected keratinocytes is critical for detection of HPV infection and subsequent clearance; deregulating intracellular immune signaling allows HR HPV to avoid detection and resist immune clearance, a necessity for establishing persistent infection. The HPV life cycle is tied to the differentiation programming of epithelial cells, with cellular differentiation driving initiation of the late stage of life viral cycle. Chapter 4 identifies for the first time a role for NFX1-123 in regulating keratinocyte differentiation and late events of the HPV life cycle. NFX1-123 augmented JNK signaling in 16E6 HFKs undergoing differentiation through expression of upstream kinase regulators of JNK, MKK4 and MKK7. Consistent with the connection between the HPV life cycle and differentiation, modulating levels of NFX1-123 in HPV16-positive W12E cells affected late events of the viral life cycle. Altering levels of NFX1-123 resulted in altered mRNA expression levels of L1, the major capsid protein of HPV. 16E6 and NFX1-123 have previously been shown to target the known oncogenic protein telomerase, increasing expression and activity of its catalytic subunit, hTERT. In Chapter 5, we further explore how 16E6 and NFX1-123 targeting of telomerase affects growth and proliferative potential of cells over long term cell culture. The data presented in this chapter demonstrate that NFX1-123 is highly expressed in primary cervical cancers. In vitro, cells expressing 16E6 and overexpressing NFX1-123 had extended active growth, decreased senescence marker staining, and more rapid cell cycling compared to 16E6 expressing cells with endogenous amounts of NFX1-123. These effects on cell proliferation were associated with increased telomerase activity and expression of its catalytic subunit, hTERT. HPV 16-positive cervical cancer cell lines with knocked down NFX1-123 had slowed growth and reduced hTERT. These data indicate that 16E6 and NFX1-123 not only extend proliferative potential of cells via telomerase, but also modify active cellular growth, augment hTERT expression, and increase telomerase activity over time. Altogether, the work in this dissertation describes multiple epithelial cell pathways that HR HPV type 16 E6 and NFX1-123 modify, which subsequently alter the host cell milieu to potentially promote HPV infection and support oncogenic development

Genes Regulated by HPV 16 E6 and High Expression of NFX1-123 in Cervical Cancers

Purpose High-risk human papillomaviruses (HR HPV) cause cervical cancer, and in these cancers, HPV type 16 is the most common HR type. The HR viral oncogenes E6 and E7 partner with cellular proteins to drive cancer and modulate immune pathways; previously, we demonstrated in keratinocytes that HPV 16 E6 and high expression of the endogenous host protein partner NFX1-123 led to the increased expression of multiple genes, including Notch1, secretory leukocyte peptidase inhibitor (SLPI), and retinoic acid early transcript 1G (RAET1G). The present study was conducted to determine if NFX1-123 was highly expressed in cervical cancer and if genes increased by NFX1-123 and 16E6 in keratinocytes were also increased in cervical cancers. Materials and Methods The Cancer Genome Atlas (TCGA) database and The Human Protein Atlas database were used to compare relative mRNA and protein gene expression, respectively, in the normal cervix and cervical cancers. Formalin-fixed paraffin-embedded (FFPE) normal cervix and HPV 16 positive cervical cancer samples were analyzed for relative protein expression by immunohistochemical staining. Protein expression of a subset of regulated genes was quantified by Western blot of HPV positive and negative cell lines. Results Immunohistochemical staining of HPV 16 positive cervical dysplasias and cancers revealed high NFX1-123, Ki67, and Notch1 expression. NFX1 and NFX1L1 mRNA levels were increased in cervical cancers compared to normal cervix in the TCGA database. Fourteen genes previously identified as upregulated in keratinocytes with 16E6 and overexpressed NFX1-123 also had high mRNA expression and selected genes had high protein expression in cervical cancers and cell lines. Conclusion In cervical cancer, NFX1-123 is highly expressed, and 16E6 and NFX1-123 together alter the expression of a wide set of genes. The involvement of these genes in cell proliferation, differentiation, invasion, and metastasis provides further insight into potential ways that HR HPVs promote cancer initiation and maintenance

Human papillomavirus type 16 E6 and NFX1-123 mislocalize immune signaling proteins and downregulate immune gene expression in keratinocytes

<div><p>Human papillomavirus (HPV) is the most prevalent sexually transmitted infection, affecting an estimated 11% of the world’s population. The high-risk HPV types (HR HPV) account for approximately 5% of the global burden of cancer and thus cause high morbidity and mortality. Although it is known that persistent infection with HR HPV is the greatest risk factor for developing HPV-associated cancer, and that the HPV early proteins E6 and E7 dysregulate immune detection by its host cells, the mechanisms of immune evasion by HR HPV are not well understood. Previous work in the laboratory identified the endogenous cytoplasmic host protein NFX1-123 as a binding partner of the HR HPV type 16 oncoprotein E6 (16E6). Together NFX1-123 and 16E6 affect cellular growth, differentiation, and immortalization genes and pathways. In a whole genome microarray, human foreskin keratinocytes (HFKs) stably expressing 16E6 and overexpressing NFX1-123 showed a diverse set of innate immune genes downregulated two-fold or more when compared to 16E6 cells with endogenous NFX1-123. We demonstrated that 16E6 and NFX1-123 decreased expression of pro-inflammatory cytokines and interferon-stimulated genes (ISGs) in 16E6 HFKs at the mRNA and protein level. Knock down of NFX1-123 in 16E6 HFKs resulted in a derepression of innate immune genes, pointing to the requirement of NFX1-123 for immune regulation in the context of 16E6. Studies using immunofluorescent microscopy revealed that 16E6 and NFX1-123 disturbed the normal localization of signaling proteins involved in initiating the immune response. This study identifies NFX1-123 as a critical host protein partner through which 16E6 is able to subvert the immune response and in turn permit a long-lived HR HPV infection.</p></div

Microarray analysis of genes downregulated in 16E6 HFKs with increased NFX1-123.

<p>Whole genome expression microarrays were conducted in HFKs stably expressing 16E6 and overexpressing NFX1-123 or with endogenous levels of NFX1-123. (A) Three biologically independent HFKs were transduced with 16E6 and p53 protein levels assessed. (B) 16E6 HFK lines 1, 2, and 3 were transduced with NFX1-123 overexpression construct (FN123) or vector control (control). Levels of NFX1-123 mRNA and protein expression levels were quantified and compared. All qPCRs were normalized to the housekeeping gene 36B4, and all error bars represent 95% confidence intervals from the technical replicates shown (n = 3). GAPDH = loading control for (A and C). (D) Venn diagram of genes whose average expression was decreased at least two-fold in 16E6/FN123 cells compared to 16E6/control. Box indicates 26 genes represented in (D) (E) Heat map with hierarchical clustering of the 26 genes decreased in 16E6/FN123 cells compared to 16E6/control.</p

Subcellular localization of innate immune signaling proteins TRAF6 and TAB2.

<p>Subcellular localization of innate immune signaling proteins TRAF6 and TAB2.</p

Genes and pathways decreased in 16E6/FN123 HFKs.

<p>(A) A subset of the over 200 genes that were decreased in 16E6/FN123 cells compared to 16E6/control. Fold change shown is the average fold change over all 16E6 HFK cell lines in which that gene was decreased. (B) Microarray data were analyzed using GeneSpring GX11.5.1, and the significant Gene Ontology pathways for the collection of genes decreased in two out of three 16E6 HFK cell lines were found. Only pathways with a p-value ≤ 0.001 are shown.</p

Whole cell mean fluorescent intensity of innate immune signaling proteins.

<p>Whole cell mean fluorescent intensity of innate immune signaling proteins.</p