Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrinα6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

HPV-inactive cell populations arise from HPV16-transformed human keratinocytes after p53 knockout.

HPV-inactive head and neck and cervical cancers contain HPV DNA but do not express HPV E6/E7. HPV-positive primary head and neck tumors usually express E6/E7, however they may produce HPV-inactive metastases. These observations led to our hypothesis that HPV-inactive cancers begin as HPV-active lesions, losing dependence on E6/E7 expression during progression. Because HPV-inactive cervical cancers often have mutated p53, we investigated whether p53 loss may play a role in the genesis of HPV-inactive cancers. p53 knockout (p53-KO) by CRISPR-Cas9 resulted in a 5-fold reduction of E7 mRNA in differentiation-resistant HPV16 immortalized human keratinocytes (HKc/DR). E7 expression was restored by 5-Aza-2 deoxycytidine in p53 KO lines, suggesting a role of DNA methylation in this process. In-situ hybridization showed that p53 KO lines consist of mixed populations of E6/E7-positive and negative cells. Hence, loss of p53 predisposes HPV16 transformed cells to losing dependence on the continuous expression of HPV oncogenes for proliferation.

Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties.

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals andpresenta model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.

The Marine Natural Product Manzamine A Inhibits Cervical Cancer by Targeting the SIX1 Protein.

Natural products remain an important source of drug leads covering unique chemical space and providing significant therapeutic value for the control of cancer and infectious diseases resistant to current drugs. Here, we determined the antiproliferative activity of a natural product manzamine A (1) from an Indo-Pacific sponge following various in vitro cellular assays targeting cervical cancer (C33A, HeLa, SiHa, and CaSki). Our data demonstrated the antiproliferative effects of 1 at relatively low and non-cytotoxic concentrations (up to 4 µM). Mechanistic investigations confirmed that 1 blocked cell cycle progression in SiHa and CaSki cells at G1/S phase and regulated cell cycle-related genes, including restoration of p21 and p53 expression. In apoptotic assays, HeLa cells showed the highest sensitivity to 1 as compared to other cell types (C33A, SiHa, and CaSki). Interestingly, 1 decreased the levels of the oncoprotein SIX1, which is associated with oncogenesis in cervical cancer. To further investigate the structure-activity relationship among manzamine A (1) class with potential antiproliferative activity, molecular networking facilitated the efficient identification, dereplication, and assignment of structures from the manzamine class and revealed the significant potential in the design of optimized molecules for the treatment of cervical cancer. These data suggest that this sponge-derived natural product class warrants further attention regarding the design and development of novel manzamine analogues, which may be efficacious for preventive and therapeutic treatment of cancer. Additionally, this study reveals the significance of protecting fragile marine ecosystems from climate change-induced loss of species diversity.

HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression.

The homeodomain transcription factor SIX1 plays a critical role in embryogenesis, is not expressed in normal adult tissue, but is expressed in many malignancies, including cervical cancer. SIX1 drives the progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward malignancy: HKc/HPV16 express high levels of SIX1 mRNA and protein; overexpression of SIX1 in HKc/HPV16 produces pre-malignant, differentiation-resistant lines (HKc/DR); SIX1 overexpression in HKc/DR induces tumorigenicity. In this paper, we explore the consequences of inhibition of SIX1 expression in premalignant HKc/DR. Only partial inhibition of SIX1 expression could be obtained in HKc/DR by RNA interference. Decreased SIX1 expression (up to 80%) in HKc/DR resulted in slower proliferation, decreased HPV16-E6/E7 mRNA levels, and increased p53 protein levels. Gene expression changes induced in HKc/DR by anti-SIX1 shRNA were indicative of mesenchymal-epithelial transition (MET) and changes in TGF-beta signaling. We conclude that HPV16-transformed cells depend on SIX1 for survival, HPV16 E6/E7 gene expression and epithelial-mesenchymal transition.

Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to Human Papillomavirus 16 DNA.

Human papillomavirus (HPV) infection of the genital tract is common; however, only about 10 to 15% of infections persist, and approximately 10 to 15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. However, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in three-dimensional anchorage-free suspension to assess their spheroid-forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and fluorescence-activated cell sorting-purified populations of basal stem-like keratinocytes, expressing low levels of epidermal growth factor receptor and high levels of integrin alpha 6 (EGFRlo/ITGα6hi), responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance than autologous mass-cultured cells. Conversely, cells committed to terminal differentiation (EGFRhi/ITGα6lo) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass-cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation-resistant phenotype upon immortalization by HPV16.IMPORTANCE This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to preneoplastic and neoplastic progression of HPV-positive lesions.

Six1 promotes colorectal cancer growth and metastasis by stimulating angiogenesis and recruiting tumor-associated macrophages.

The homeoprotein Six1 is overexpressed in many human cancers and is associated with increased tumor progression and metastasis. Recent studies have shown that Six1 is associated with poorer overall survival in advanced-stage colorectal cancer (CRC). In the current study, we explored the functional changes and molecular events associated with Six1 overexpression in a mouse model of CRC. An orthotopic model and a splenic injection metastasis model were used to investigate the role of Six1 in CRC tumor growth and metastasis using mouse colon adenocarcinoma MC38 cells overexpressing Six1. We found that overexpression of Six1 dramatically promotes CRC tumor growth and metastasis in vivo. Six1 overexpression in MC38 increased protein levels of aldehyde dehydrogenase-1 and expanded CD44+/CD166+ populations, indicating Six1 increased features of cancer stem cells. In addition, Six1 overexpression stimulated angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF). Six1-overexpressing tumor cells recruited tumor-associated macrophages (TAM) by increasing the expression of macrophage-specific colony stimulating factor, chemokine (C-C motif) ligand 2/5 and VEGF, further facilitating CRC tumor growth and metastasis. Furthermore, we determined that Six1 activated mitogen-activated protein kinase (MAPK) signaling in CRC cells. In summary, our studies strongly suggest that Six1 overexpression promotes CRC growth and metastasis and remodels tumor stroma by stimulating angiogenesis and recruiting TAM. MAPK activation may be a pivotal event in Six1-associated tumor progression, which may provide opportunities for pharmacologic intervention.

Identification and characterization of HPV-independent cervical cancers.

BACKGROUND: Human papillomavirus (HPV) initiates cervical cancer, and continuous expression of HPV oncogenes E6 and E7 is thought to be necessary to maintain malignant growth. Current therapies target proliferating cells, rather than specific pathways, and most experimental therapies specifically target E6/E7. We investigated the presence and expression of HPV in cervical cancer, to correlate HPV oncogene expression with clinical and molecular features of these tumors that may be relevant to new targeted therapies. RESULTS: While virtually all cervical cancers contained HPV DNA, and most expressed E6/E7 (HPV-active), a subset (8%) of HPV DNA-positive cervical cancers did not express HPV transcripts (HPV-inactive). HPV-inactive tumors occurred in older women (median 54 vs. 45 years, p = 0.02) and were associated with poorer survival (median 715 vs 3046 days, p = 0.0003). Gene expression profiles of HPV-active and -inactive tumors were distinct. HPV-active tumors expressed E2F target genes and increased AKT/MTOR signaling. HPV-inactive tumors had increased WNT/ß-catenin and Sonic Hedgehog signaling. Substantial genome-wide differences in DNA methylation were observed. HPV-inactive tumors had a global decrease in DNA methylation; however, many promoter-associated CpGs were hypermethylated. Many inflammatory response genes showed promoter methylation and decreased expression. The somatic mutation landscapes were significantly different. HPV-active tumors carried few somatic mutations in driver genes, whereas HPV-inactive tumors were enriched for non-synonymous somatic mutations (p-value < 0.0000001) specifically targeting TP53, ARID, WNT, and PI3K pathways. MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA) cervical cancer data were analyzed. CONCLUSIONS: Many of the gene expression changes and somatic mutations found in HPV-inactive tumors alter pathways for which targeted therapeutics are available. Treatment strategies focused on WNT, PI3K, or TP53 mutations may be effective against HPV-inactive tumors and could improve survival for these cervical cancer patients.

Human Papillomavirus Type 16 L2 DNA Methylation in Exfoliated Cervical Cells From College-Age Women.

OBJECTIVES: The Carolina Women's Care Study (CWCS) at the University of South Carolina followed 467 young women with the goal of identifying biomarkers of human papillomavirus (HPV) persistence. In this study, we analyzed the methylation of HPV16 DNA. METHODS: The aims of this study were to determine the methylation status of the HPV16 L2 gene in DNA isolated from exfoliated cervical cells collected longitudinally as part of the CWCS and to determine the prevalence of polymorphisms (single nucleotide polymorphisms [SNPs]) in folate metabolizing enzymes and DNA repair enzymes known to affect DNA methylation in blood-derived genomic DNA from CWCS participants. For methylation studies, DNA samples were bisulfite converted and amplified with the EpiTect Whole Bisulfitome kit. Polymerase chain reaction was performed for amplicons containing 5 CpG sites in L2. Pyrosequencing was carried out using EpigenDx and analyzed with PyroMark Software. Taqman genotyping assays were performed to determine selected SNP alleles in the CWCS cohort. RESULTS AND CONCLUSIONS: Methylation data were obtained for 82 samples from 27 participants. Of these, 22 participants were positive for HPV16 for 3 or more visits (≥12 months). Methylation in L2 was detectable, but methylation levels varied and were not associated with HPV16 persistence. No linearity of methylation levels over time was observed in participants for whom longitudinal data could be analyzed. Analysis of 9 selected SNPs did not reveal an association with persistence. We conclude that at early stages of infection methylation of HPV16 L2 DNA in Pap test samples is not a predictive biomarker of HPV persistence.

Human papillomavirus status and gene expression profiles of oropharyngeal and oral cancers from European American and African American patients.

BACKGROUND: Disparities in prevalence, human papillomavirus (HPV) status, and mortality rates for head and neck cancer have been described between African American and European American patients. METHODS: We studied the HPV status and gene expression profiles in 56 oropharyngeal/oral cavity tumors and 9 normal tissue samples from European American and African American patients treated in South Carolina between 2010 and 2012. RESULTS: Overall, 59% of tumors were HPV DNA-positive, but only 48% of those expressed E7 mRNA (HPV-active). The prevalence of HPV-active tumors was 10% in African American patients and 39% in European American patients. Tumors positive for HPV DNA but negative for HPV mRNA exhibited gene expression profiles distinct from those of both HPV-active and HPV-negative cancers, suggesting that HPV DNA-positive/RNA-negative tumors may constitute a unique group. CONCLUSION: This study provides a direct assessment of differential expression patterns in HPV-related oropharyngeal cancer arising from African American and European American patients, for which there is a paucity of data. © 2015 Wiley Periodicals, Inc. Head Neck 00: 000-000, 2015.

Neuroendocrine Merkel cell carcinoma is associated with mutations in key DNA repair, epigenetic and apoptosis pathways: a case-based study using targeted massively parallel sequencing.

INTRODUCTION: Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma with a poorly understood molecular etiology. We implemented a comprehensive deep sequencing approach to identify mutations in the tumor DNA from a cohort of patients treated at our institution over the past 15 years. Our results indicate mutations that may constitute therapeutic targets in MCC. METHODS: Five patients were treated for MCC within the study interval. Patients with adequate tissue (n = 4), positive neuroendocrine differentiation (chromogranin, synaptophysin, and cytokeratin 20), and histopathological confirmation of MCC were included in the study. DNA was extracted from archival tumor tissue samples and analyzed by massively parallel sequencing using a targeted, multiplex PCR approach followed by semiconductor sequencing. RESULTS: We demonstrate high-penetrance nonsense mutations in PDE4DIP (n = 4) as well as various missense mutations in the DNA damage response (PRKDC, AURKB, ERCC5, ATR, and ATRX) and epigenetic modulating enzymes (MLL3). CONCLUSION: We describe several mutations in potential disease-relevant genes and pathways. These targets should be evaluated in a larger cohort to determine their role in the molecular pathogenesis of MCC.

Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT.

Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial-mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes.

Disparity in the persistence of high-risk human papillomavirus genotypes between African American and European American women of college age.

BACKGROUND: Cervical cancer incidence and mortality rates are higher in African Americans than in European Americans (white, non-Hispanic of European ancestry). The reasons for this disparity are not known. METHODS: We recruited a population-based longitudinal cohort of 326 European American and 113 African American female college freshmen in Columbia, South Carolina, to compare clearance of high-risk human papillomavirus (HR-HPV) infection between ethnicities. HPV testing and typing from samples obtained for Papanicolaou testing occurred every 6 months. RESULTS: African American participants had an increased risk of testing positive for HR-HPV, compared with European American participants, but the frequency of incident HPV infection was the same in African American and European American women. Thus, exposure to HPV could not explain the higher rate of HPV positivity among African American women. The time required for 50% of participants to clear HR-HPV infection was 601 days for African American women (n = 63) and 316 days for European American women (n = 178; odds ratio [OR], 1.61; 95% confidence interval [CI], 1.08-2.53). African American women were more likely than European American women to have an abnormal result of a Papanicolaou test (OR, 1.58; 95% CI, 1.05-2.39). CONCLUSIONS: We propose that the longer time to clearance of HR-HPV among African American women leads to increased rates of abnormal results of Papanicolaou tests and contributes to the increased rates of cervical cancer observed in African American women.

Six1 promotes epithelial-mesenchymal transition and malignant conversion in human papillomavirus type 16-immortalized human keratinocytes.

Six1, a member of the Six family of homeodomain transcription factors, is overexpressed in various human cancers, and SIX1 overexpression is associated with tumor progression and metastasis. Six1 messenger RNA levels increase during in vitro progression of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we show that HKc/DR-overexpressing Six1 exhibited a more mesenchymal phenotype, as characterized by a fibroblastic appearance and increased invasion. We utilized Whole Human Genome Microarrays to explore the gene expression changes associated with Six1 overexpression in HKc/DR. We found that overexpression of Six1 downregulated epithelial-related genes and upregulated mesenchymal-related genes, which suggests that Six1 overexpression induces epithelial-mesenchymal transition (EMT). Pathway analysis of the microarray data showed alterations in the transforming growth factor-beta (TGF-ß) pathway, including enhanced expression of the TGF-ß receptor type II (TßRII), and activation of the mitogen-activated protein kinase (MAPK) pathway in HKc/DR-overexpressing Six1, suggesting that Smad-independent pathways of TGF-ß signaling may be involved in Six1-mediated EMT. p38 MAPK activation was required for sustained Six1-induced EMT and TßRII overexpression. Finally, we determined that Six1 overexpression in HKc/DR resulted in malignant conversion and increased the cancer stem cell (CSC)-like population. Thus, Six1 overexpression promotes EMT, CSCs properties and malignant conversion in HKc/DR through MAPK activation, which supports the possible use of p38-TßRII inhibitors for the treatment of cancers overexpressing Six1.

The translational significance of epithelial-mesenchymal transition in head and neck cancer.

Positive markers of epithelial-mesenchymal transition (EMT) in head and neck cancers complicate clinical management and are associated with reduced survival. We discuss recent translational discoveries in EMT and suggest additional actionable molecular pathways, biomarkers, and clinical agents.

TGF-ß regulation of gene expression at early and late stages of HPV16-mediated transformation of human keratinocytes.

In our in vitro model for HPV16-mediated transformation, HPV16-immortalized human keratinocytes (HKc/HPV16) give rise to differentiation resistant, premalignant cells (HKc/DR). HKc/DR, but not HKc/HPV16, are resistant to growth inhibition by transforming growth factor beta (TGF-ß), due to a partial loss of TGF-ß receptor type I. We show that TGF-ß activates a Smad-responsive reporter construct in HKc/DR to about 50% of the maximum levels of activation observed in HKc/HPV16. To investigate the functional significance of residual TGF-ß signaling in HKc/DR, we compared gene expression profiles elicited by TGF-ß treatment of HKc/HPV16 and HKc/DR on Agilent 44k human whole genome microarrays. TGF-ß altered the expression of cell cycle and MAP kinase pathway genes in HKc/HPV16, but not in HKc/DR. However, epithelial-mesenchymal transition (EMT) responses to TGF-ß were comparable in HKc/HPV16 and HKc/DR, indicating that the signaling pathways through which TGF-ß elicits growth inhibition diverge from those that induce EMT in HPV16-transformed cells.

Partial loss of Smad signaling during in vitro progression of HPV16-immortalized human keratinocytes.

BACKGROUND: Disruption of the transforming growth factor-beta (TGF-ß) signaling pathway is observed in many cancers, including cervical cancer, resulting in TGF-ß resistance. While normal human keratinocytes (HKc) and human papillomavirus type 16-immortalized HKc (HKc/HPV16) are sensitive to the growth inhibitory effects of TGF-ß, HKc/HPV16 develop resistance to TGF-ß1 as they progress in vitro to a differentiation resistant phenotype (HKc/DR). The loss of sensitivity to the antiproliferative effects of TGF-ß1 in HKc/DR is due, at least partially, to decreased expression of the TGF-ß receptor type I. In the present study, we explored in detail whether alterations in Smad protein levels, Smad phosphorylation, or nuclear localization of Smads in response to TGF-ß could contribute to the development of TGF-ß resistance during in vitro progression of HKc/HPV16, and whether TGF-ß induction of a Smad-responsive reporter gene was altered in HKc/DR. METHODS: Western blot analysis was used to assess Smad protein levels. In order to study Smad nuclear localization we performed indirect immunofluorescence. In addition, we determined Smad-mediated TGF-ß signaling using a luciferase reporter construct. RESULTS: We did not find a decrease in protein levels of Smad2, Smad3 or Smad4, or an increase in the inhibitory Smad7 that paralleled the loss of sensitivity to the growth inhibitory effects of TGF-ß1 observed in HKc/DR. However, we found diminished Smad2 phosphorylation, and delayed nuclear Smad3 localization in response to TGF-ß1 in HKc/DR, compared to normal HKc and TGF-ß sensitive HKc/HPV16. In addition, we determined that TGF-ß1 induction of a Smad responsive promoter is reduced by about 50% in HKc/DR, compared to HKc/HPV16. CONCLUSIONS: These results demonstrate that alterations in Smad protein levels are not associated with the loss of response to the antiproliferative effects of TGF-ß in HKc/DR, but that diminished and delayed Smad phosphorylation and nuclear localization, and decreased Smad signaling occur in response to TGF-ß in HKc/DR.

HPV prevalence at enrollment and baseline results from the Carolina Women's Care Study, a longitudinal study of HPV persistence in women of college age.

BACKGROUND: Cervical cancer, a rare outcome of high-risk human papillomavirus (HPV) infection, disproportionately affects African American women, who are about twice more likely than European American women to die of the disease. Most cervical HPV infections clear in about one year. However, in some women HPV persists, posing a greater risk for cervical dysplasia and cancer. The Carolina Women's Care Study (CWCS) was conducted to explore the biological, genetic, and lifestyle determinants of persistent HPV infection in college-aged European American and African American women. This paper presents the initial results of the CWCS, based upon data obtained at enrollment. METHODS: Freshman female students attending the University of South Carolina were enrolled in the CWCS and followed until graduation with biannual visits, including two Papanicolaou tests, cervical mucus collection, and a questionnaire assessing lifestyle factors. We recruited 467 women, 293 of whom completed four or more visits for a total of 2274 visits. RESULTS AND CONCLUSION: CWCS participants were 70% European American, 24% African American, 3% Latina/Hispanic, and 3% Asian. At enrollment, 32% tested positive for any HPV. HPV16 infection was the most common (18% of infections). Together, HPV16, 66, 51, 52, and 18 accounted for 58% of all HPV infections. Sixty-four percent of all HPV-positive samples contained more than one HPV type, with an average of 2.2 HPV types per HPV-positive participant. We found differences between African American and European American women in the prevalence of HPV infection (38.1% African American, 30.7% European American) and abnormal Papanicolaou test results (9.8% African-American, 5.8% European American). While these differences did not reach statistical significance at enrollment, as the longitudinal data of this cohort are analyzed, the sample size will allow us to confirm these results and compare the natural history of HPV infection in college-aged African American and European American women.

Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer.

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-ß) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-ß. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-ß treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-ß-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.