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NFX1-123 and human papillomavirus 16E6 increase Notch expression in keratinocytes.

ABSTRACT: The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they posttranscriptionally increase hTERT expression, the catalytic subunit of telomerase. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity and the avoidance of cellular senescence. Little is known regarding what other transcripts are dependent on or augmented by the association of NFX1-123 with 16E6. Microarray analysis revealed enhanced expression of Notch1 mRNA in 16E6-expressing keratinocytes when NFX1-123 was overexpressed. A moderate increase in Notch1 mRNA was seen with overexpression of NFX1-123 alone, but with 16E6 coexpression the increase in Notch1 was enhanced. The PAM2 motif and R3H protein domains in NFX1-123, which were important for increased hTERT expression, were also important in the augmentation of Notch1 expression by 16E6. These findings identify a second gene coregulated by 16E6 and NFX1-123 and the protein motifs in NFX1-123 that are important for this effect.

SUBMITTER: Vliet-Gregg PA

PROVIDER: S-EPMC3838236 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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NFX1-123 and human papillomavirus 16E6 increase Notch expression in keratinocytes.

Vliet-Gregg Portia A PA Hamilton Jennifer R JR Katzenellenbogen Rachel A RA

Journal of virology 20131009 24

The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they posttranscriptionally increase hTERT expression, the catalytic subunit of telomerase. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity and the avoidance of cellular senescence. Little is known regarding what ...[more]

PMID: 24109236

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Project description:PurposeHigh-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 methodsThe 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.ResultsImmunohistochemical 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.ConclusionIn 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.

Project description:GSM1056261-GSM1056272: NFX1-123 has been shown to associate with a number of RNA processing proteins, such as cytoplasmic poly(A) binding proteins (PABPC), to affect mRNA stability and translational efficiency of target genes. The high-risk human papillomavirus type 16E6 (HPV 16E6) induces telomerase activity by activation of hTERT, the catalytic subunit of telomerase. NFX1-123 can bind to hTERT mRNA and increase its stability in HPV 16E6 expressing keratinocytes (16E6 NHKs). Little is known regarding what other transcripts, and downstream signaling pathways, may be dependent on NFX1-123. In order to determine additional cellular transcripts affected by HPV 16E6 and NFX1-123, we assessed global gene expression changes in cells in which NFX1-123 was overexpressed or knocked down by short hairpin RNAs (shRNA) when compared to an isogenic scramble control, in three independently derived 16E6 NHKs GSM1212499-GSM1212510: NFX1-123 has been shown to associate with a number of RNA processing proteins, such as cytoplasmic poly(A) binding proteins (PABPC), to affect mRNA stability and translational efficiency of target genes. The high-risk human papillomavirus type 16E6 (HPV 16E6) induces telomerase activity by activation of hTERT, the catalytic subunit of telomerase. NFX1-123 can bind to hTERT mRNA and increase its stability in HPV 16E6 expressing keratinocytes (16E6 NHKs). Little is known regarding what other transcripts, and downstream signaling pathways, may be dependent on NFX1-123. In order to determine additional cellular transcripts affected solely by NFX1-123, we assessed global gene expression changes in cells in which NFX1-123 was overexpressed or knocked down by short hairpin RNAs (shRNA) when compared to an isogenic scramble control, in three independently derived NHKs. GSM1056261-GSM1056272: Three independent 16E6 expressing NHK cell lines were expanded and transduced with: short hairpin RNA (sh1) that knocked down NFX1-123 by 20%, short hairpin RNA (sh3) that knocked down NFX1-123 by 80%; a non-targeting isogenic shRNA scramble control; or a NFX1-123 overexpression construct with a FLAG-tag (FNFX1-123WT) that increased its RNA expression on average 3.5-fold. In total, twelve samples were used for the microarray, derived from the three initial NHK cell lines. GSM1212499-GSM1212510: Three independent NHK cell lines were expanded and transduced with: short hairpin RNA (sh1) that knocked down NFX1-123 by 40%, short hairpin RNA (sh3) that knocked down NFX1-123 by 83%; a non-targeting isogenic shRNA scramble control; or a NFX1-123 overexpression construct with a FLAG-tag (FNFX1-123WT) that increased its RNA expression on average 14.0-fold. In total, twelve samples were used for the microarray, derived from the three initial NHK cell lines.

Dataset Information

NFX1-123 and human papillomavirus 16E6 increase Notch expression in keratinocytes.

Publications

NFX1-123 and human papillomavirus 16E6 increase Notch expression in keratinocytes.

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