Cervical Keratinocytes Research Articles

PIWIL2/PDK1 Axis Promotes the Progression of Cervical Epithelial Lesions via Metabolic Reprogramming to Maintain Tumor-Initiating Cell Stemness.

When PIWIL2 expression is restored via heterogeneous integration of human papillomavirus, cellular reprogramming is initiated to form tumor-initiating cells (TICs), which triggers cervical squamous intraepithelial lesions (SIL). TIC stemness is critical for the prognosis of SIL. However, the mechanisms underlying TIC stemness maintenance and tumorigenicity remain unclear. Here, it is revealed that aberrant pyruvate dehydrogenase kinase 1 (PDK1) expression is closely related to aerobic glycolysis in SIL and poor survival in patients with cervical cancer. Mechanistically, that PIWIL2, which induced by stable transfection of either PIWIL2 or HPV16 oncogene E6 in human primary cervical basal epithelial cells and keratinocyte cell line HaCaT, upregulates PDK1 expression via the LIN28/let-7 axis, hence reprogramming metabolism to activate glycolysis and synchronize with TIC formation. It is further demonstrate that PDK1 is critical for TIC stemness maintenance and tumorigenicity via the PI3K/AKT/mTOR pathway both in vitro and in vivo, revealing a previously unclear mechanism for SIL progression, regression or relapse. Therefore, this findings suggest a potential rationale for prognostic predictions and selecting targeted therapy for cervical lesions.

Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.Graphical abstract

N-Acetyl-L-Cysteine Reduces Cervical Carcinogenesis by Promoting Apoptosis.

Cervical cancer is the fourth leading cause of cancer death in women, and is one of the most common malignant tumors of the reproductive system. However, more effective treatment for cervical cancer is needed. In this study, we aim to investigate whether N-acetyl-L-cysteine (NAC) could inhibit the proliferation of human papillomavirus (HPV)-positive cells, and reduce cervical carcinogenesis. The cervical cancer cell lines SiHa, HeLa, HPV-negative cell line C33A, and the immortalized human cervical keratinocyte cells S12 were used. The protein expression was determined using Western blot assay. mRNA expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was determined by Cell Counting Kit-8 assay. Cell apoptosis was evaluated using Annexin V-FITC apoptosis kits. The numbers of colonies were measured using colony-forming assay. Xenograft tumor necrosis and HPV16 E7 expression were determined using hematoxylin and eosin (H&E) staining and immunohistochemistry. Our results showed that NAC treatment at the concentration of 1.5mM significantly promoted cell apoptosis and reduced cell growth by inhibiting HPV16 E7 expression. NAC inhibited HPV16-oncoprotein-induced hypoxia-inducible factor (HIF)-1α protein expression and Akt activation invitro. Additionally, NAC suppressed tumor growth, as evidenced by the smaller tumor size in the xenograft mouse model and decreased HPV16 E7 expression in tumor tissues. Our findings demonstrate that NAC exhibits the potential to promote HPV-positive cell apoptosis, and suppress the proliferation of HPV-positive cells by inhibiting cell inhibitor of apoptosis protein 2 and HIF-1α.

An in vitro carcinogenesis model for cervical cancer harboring episomal form of HPV16.

Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity.

AIB1 is a novel target of the high-risk HPV E6 protein and a biomarker of cervical cancer progression.

The high‐risk human papillomaviruses (HPV‐16, ‐18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ‐containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high‐risk E6 but not low‐risk E6 expression led to increases in AIB1 transcript levels by real‐time RT‐PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV‐16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.

Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells.

The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property. Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells. The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells. MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis. The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells. The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

Development of an in vitro carcinogenesis model of human papillomavirus-induced cervical adenocarcinoma.

Cervical adenocarcinoma (ADC) is the second most common pathological subtype of cervical cancer after squamous cell carcinoma. It accounts for approximately 20% of cervical cancers, and the incidence has increased in the past few decades, particularly among young patients. The persistent infection of high‐risk human papillomavirus (HPV) is responsible for most cervical ADC. However, almost all available in vitro models are designed to study the carcinogenesis of cervical squamous cell carcinoma. To gain better insights into molecular background of ADC, we aimed to establish an in vitro carcinogenesis model of ADC. We previously reported the establishment of an in vitro model for cervical squamous cell carcinoma by introducing defined viral and cellular oncogenes, HPV16 E6 and E7, c‐MYC, and activated RAS to human cervical keratinocytes. In this study, the expression of potential lineage‐specifying factors and/or SMAD4 reduction was introduced in addition to the defined four oncogenes to direct carcinogenesis toward ADC. The cell properties associated with the cell lineage were analyzed in monolayer and organoid cultures and the tumors in mouse xenografts. In the cells expressing Forkhead box A2 (FOXA2), apparent changes in cell properties were observed, such as elevated expression of columnar cell markers and decreased expression of squamous cell markers. Strikingly, the histopathology of tumors expressing FOXA2 resembled cervical ADC, proposing that FOXA2 plays a vital role in dictating the histopathology of cervical cancers.

Mechanism of hydrogen on cervical cancer suppression revealed by high‑throughput RNA sequencing.

Cervical cancer is considered one of the diseases with the highest mortality among women and with limited treatment options. Hydrogen (H2) inhalation has been reported to have a variety of tumor-suppressive effects, but the exact mechanism remains unclear. In the present study, HeLa cervical cancer cells and HaCaT keratinocytes treated with H2, and a HeLa xenograft mouse model subjected to H2 inhalation were established. TUNEL, Cell Counting Kit-8 and Ki67 staining assays were used to detect cell apoptosis and proliferation. Oxidative stress was determined according to the levels of reactive oxygen species, malondialdehyde and superoxide dismutase. Tumor growth was recorded every 3 days, and the excised tumors were stained with hematoxylin and eosin. High-throughput RNA sequencing and subsequent Gene Ontology (GO) enrichment analysis were performed in HeLa-treated and un-treated HeLa cells. The expression of hypoxia-inducible factor (HIF)-1α and NF-κB p65 was verified by western blotting, immunohistochemistry and reverse transcription-quantitative PCR. The results revealed an increased apoptosis rate, and reduced cell proliferation and oxidative stress in H2-treated HeLa cells but not in HaCaT cells. Similarly, decreased tumor growth and cell proliferation, and enhanced cell apoptosis were observed in H2-treated HeLa tumors. RNA sequencing and GO analysis suggest that downregulated HIF1A (HIF-1α mRNA) and RelA (NF-κB p65) levels, and reduced NF-κB signaling were associated with the antitumor effect of H2. Finally, decreased HIF-1α and NF-κB p65 expression both at the transcriptional and translational levels were observed in H2-treated HeLa cells and in HeLa-derived tumors. In conclusion, the present study reveals a novel mechanism of H2 against cervical cancer, which may serve as a potential therapeutic target in clinical practice.

Autophagy buffers Ras-induced genotoxic stress enabling malignant transformation in keratinocytes primed by human papillomavirus

Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRasG12V activity in E6E7–HPV–keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRasG12V.

The Potentials and Pitfalls of a Human Cervical Organoid Model Including Langerhans Cells.

Three-dimensional cell culturing to capture a life-like experimental environment has become a versatile tool for basic and clinical research. Mucosal and skin tissues can be grown as “organoids” in a petri dish and serve a wide variety of research questions. Here, we report our experience with human cervical organoids which could also include an immune component, e.g., Langerhans cells. We employ commercially available human cervical keratinocytes and fibroblasts as well as a myeloid cell line matured and purified into langerin-positive Langerhans cells. These are then seeded on a layer of keratinocytes with underlying dermal equivalent. Using about 10-fold more than the reported number in healthy cervical tissue (1–3%), we obtain differentiated cervical epithelium after 14 days with ~1% being Langerhans cells. We provide a detailed protocol for interested researchers to apply the described “aseptic” organoid model for all sorts of investigations—with or without Langerhans cells.

The Transcriptional Cofactor VGLL1 Drives Transcription of Human Papillomavirus Early Genes via TEAD1.

The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here, we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD1 target sites in the HPV16 long control region (LCR) by in vitro DNA pulldown assays; 8 of these sites contributed to the transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1 both in vitro and in vivo Furthermore, introducing HPV16 and HPV18 whole genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to the upregulation of TEADs. These results suggest that multiple VGLL1/TEAD1 complexes are recruited to the LCR to support the efficient transcription of HPV early genes.IMPORTANCE Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.

High resolution analysis of the intracellular proteome of cervical cancer cell lines unveils novel regulators of cervical carcinogenesis.

Cervical cancer remains the fourth most common and most lethal type of cancer in women, despite the applied regular screening and prevention strategies, while the available treatment schemes still pose a threat to fertility. Substantial understanding of the underlying mechanisms and development of novel diagnostic, prognostic and therapeutic approaches are critical steps for improving cervical cancer management. Towards this goal, a comparative proteomic analysis was conducted between three cervical cancer cell lines (HeLa: HPV18+, SiHa: HPV16+, C33A: HPV‑) and normal cervical keratinocytes (HCK1T). The total cell extract of each cell line was analyzed by liquid chromatography coupled to tandem mass spectrometry (LC‑MS/MS). Differential expression analysis revealed 919, 826 and 1,370 deregulated proteins in the comparisons of HeLa, SiHa and C33A with HCK1T cell lines, respectively. Pathway enrichment analysis of the differentially expressed proteins highlighted common cancer characteristics such as high metabolic demands and increased cell turnover, confirming the validity of the proteomic results. Extensive literature mining of the consistently differentially expressed proteins that resulted from the three comparisons was performed leading to a shortlist of 21 proteins that are potentially involved in cervical malignancy. The criteria for this shortlisting were the association of the proteins with various types of cancer, while there is no study as yet associating their expression to cervical cancer. Moreover, the expression trend of two of the shortlisted proteins was validated using western blot analysis. The proteomic datasets generated in this study can be utilized to enrich the current knowledge on cervical cancer pathology and unveil key molecular mechanisms of carcinogenesis. In conclusion, the shortlist of consistently deregulated proteins between cervical cancer cell lines and normal cervical keratinocytes can be used for validation in clinical samples and in functional investigation experiments that could ultimately lead to the discovery of novel disease biomarkers and drug targets.

Carbon-ion irradiation overcomes HPV-integration/E2 gene-disruption induced radioresistance of cervical keratinocytes.

To date, only few data exist on mechanisms underlying the human papillomavirus (HPV)-associated irradiation response. It has been suggested, that the viral E2 gene plays an important role in that context. The aim of the current study is to compare the effect of photon- and carbon-ion (12C)-radiation therapy (RT) on cells with different HPV and E2 gene status. We hypothesized that 12C-RT might overcome the radioresistance of E2 gene-disrupted cells.We analyzed four different cell lines that differed in HPV status or E2 gene status. Cells were irradiated with either photons or 12C. Clonogenic survival, cell cycle and expression of Rb and p53 were analyzed.Radiosensitivity seemed to be dependent on E2 gene status and type of RT. 12C-RT led to lower surviving fractions, indicating higher radiosensitivity even in cells with disrupted E2 gene. The observed relative biological effectiveness (RBE) of 12C-RT for C33a/Caski and W12/S12 was 1.3/4 and 2.7/2.5, respectively. Cell cycle regulation after both photon- and 12C-RT was dependent on HPV status and on E2 gene status. Furthermore, the effect of RT on expression of p53 and Rb seemed to be dependent on E2 gene status and type of RT.We showed that 12C-RT overcomes HPV-integration induced radioresistance. The effect of RT on cell cycle regulation as well as on expression of p53 and Rb seemed to be dependent on HPV status, E2 gene status and type of RT. Differences in Rb expression and cell cycle regulation may play a role for enhanced radiosensitivity to 12C-RT of cells with disrupted E2 gene.

Biological Characteristics of Cervical Precancerous Cell Proliferation.

High-grade squamous intraepithelial lesions (HSILs) are regarded as precancerous lesions that can progress to cervical carcinoma; however, it is very difficult to effectively differentiate these precancerous cells from cancerous cells based on morphology alone. Additionally, the difference between precancerous cells and cancerous cells in regard to biological behaviour remains unclear. We previously cultured primary normal uterine cervical keratinocytes from human normal cervical tissue and cervical precancerous cells that were naturally infected with human papillomavirus from small-sized neoplastic cervical tissues. Here, we extended our study to further observe the in vitro proliferative characteristics of cervical precancerous cells at the cellular and molecular levels. In this study, we found that the growth rate of precancerous cells was significantly faster than that of normal cervical cells and slower than that of Caski cells. However, the proliferative capacity of such precancerous cells was similar to that of cancerous cells of the cervix at the molecular level. These results suggest that the surrounding environment of the cells may play an important role in the development of cervical cancer, which provides an important basis for the further study of precancerous and cancerous lesions of the cervix.

Human papillomavirus E6 regulates the trafficking of gap junction protein Cx43

High-risk human papillomavirus (HR-HPV) infects epithelial cells and is the major cause of anogenital and oropharyngeal cancers. HR-HPV oncogenic activity is through E6 control of p53, but E6 binds and degrades PDZ proteins such as the tumour suppress protein hDlg (human homologue of DrosophilaDiscs Large). The E6/hDlg complex also contains Connexin 43 (Cx43) (MacDonald, Sun et al. 2012), the major building block of gap junctions that allow intercellular molecular communication. In HPV16-positive non-tumour cervical keratinocytes (W12NT: low E6 levels) Cx43/hDlg is on the plasma membrane but in the cytoplasm in W12T tumour epithelial cells (W12T: high E6 levels) correlating with loss of gap junction cell-cell communication. E6 siRNA depletion in W12T cells restored Cx43 to the cell membrane, while overexpressing E6 in HPV-negative cervical cancer cells C33a resulted in Cx43 moving to the cytoplasm. E6 could control Cx43 trafficking through controlling hDlg or by altering cell signalling. In the absence of E6 in HEK293, HaCaT and normal immortalised keratinocytes (NIKS), Cx43 and hDlg could be co-immunoprecipitated and they co-localised on the plasma membrane. Thus, the Cx43/hDlg interaction is not carcinoma cell-specific, is not dependent on HR-HPV E6, and may have a functional role in non-cancer cells. siRNA depletion of hDlg, led to reduction in Cx43 protein levels and some relocation to the cytoplasm. This indicates that HPV E6 controls Cx43 through interaction with and degradation of hDlg. However, we cannot discount that E6 itself may have additional effects on Cx43 levels and trafficking besides via hDlg.

Genome-Wide Profiling of Cervical RNA-Binding Proteins Identifies Human Papillomavirus Regulation of RNASEH2A Expression by Viral E7 and E2F1.

RNA-binding proteins (RBPs) control mRNA processing, stability, transport, editing, and translation. We recently conducted transcriptome analyses comparing normal (i.e., healthy) cervical tissue samples with human papillomavirus (HPV)-positive cervical cancer tissue samples and identified 614 differentially expressed protein-coding transcripts which are enriched in cancer-related pathways and consist of 95 known RBPs. We verified the altered expression of 26 genes with a cohort of 72 cervical samples, including 24 normal cervical samples, 25 cervical intraepithelial neoplasia grade 2 (CIN2) and CIN3 samples, and 23 cervical cancer tissue samples. LY6K (lymphocyte antigen 6 complex locus K), FAM83A (family member with sequence similarity 83), CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A were identified as novel candidate genes associated with cervical lesion progression and carcinogenesis. HPV16 or HPV18 infection was found to alter the expression of 8 RBP genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) in human vaginal and foreskin keratinocytes. Both viral E6 and E7 decreased NOVA1 expression, but only E7 increased the expression of RNASEH2A in an E2F1-dependent manner. Proliferating cell nuclear antigen (PCNA) directs RNASEH2 activity with respect to DNA replication by removing the RNA primers to promote Okazaki fragment maturation, and two factors are closely associated with neoplasia progression. Therefore, we predict that the induction of expression of RNASEH2A via viral E7 and E2F1 may promote DNA replication and cancer cell proliferation.IMPORTANCE High-risk HPV infections lead to development of cervical cancer. This study identified the differential expression of 16 novel genes (LY6K, FAM83A, CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A) in HPV-infected cervical tissue samples and keratinocytes. Eight of these genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) encode RNA-binding proteins. Further studies indicated that both HPV16 and HPV18 infections lead to the aberrant expression of selected RBP-encoding genes. We found that viral E6 and E7 decrease NOVA1 expression but that E7 increases RNASEH2A expression via E2F1. The altered expression of these genes may be utilized as biomarkers for high-risk (HR)-HPV carcinogenesis and progression.

Isolation of Biopsy-Derived, Human Cervical Keratinocytes Propagated as Monolayer and Organoid Cultures

The successful isolation and propagation of patient-derived keratinocytes from cervical lesions constitute a more appropriate model of cervical disease than traditional cervical cancer-derived cell lines such as SiHa and CaSki. Our aim was to streamline the growth of patient-obtained, cervical keratinocytes into a reproducible process. We performed an observational case series study with 60 women referred to colposcopy for a diagnostic biopsy. Main outcome measures were how many samples could be passaged at least once (n = 11), and where enough cells could be established, to precisely define their proliferation profile over time (n = 3). Altering cell culture conditions over those reported by other groups markedly improved outcomes. We were also successful in making freeze backs which could be resuscitated to successfully propagate multi-layered, organoids from cervical keratinocytes (n = 3). For best results, biopsy-intrinsic factors such as size and tissue digestion appear to be major variables. This seems to be the first systematic report with a well characterized and defined sample size, detailed protocol, and carefully assessed cell yield and performance. This research is particularly impactful for constituting a sample repository-on-demand for appropriate disease modelling and drug screening under the umbrella of personalized health.

Suppression of miR-22, a tumor suppressor in cervical cancer, by human papillomavirus 16 E6 via a p53/miR-22/HDAC6 pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that function to down-regulate gene expression involving in various cellular processes related to carcinogenesis. Recently, miR-22 was identified as a tumor-suppressing miRNA in many human cancers. However, the regulatory mechanism and the specific function of this miRNA in cervical cancer remain unclear. In the present study, we carried out gene transfection, western blot and quantitative RT-PCR to explore the regulatory mechanism and the functional role of miR-22 in cervical cancer. We verified that miR-22 was down-regulated in cervical cancer tissues and cervical cancer cell lines relative to matched non-tumor tissues and normal human cervical keratinocyte line (HCK1T). By contrast, histone deacetylase 6 (HDAC6) was inversely correlated with miR-22 in both cervical tissues and cancer cell lines. Mechanically, HDAC6 was down-regulated by miR-22 at the post-transcriptional level, via a specific target site within the 3’UTR, identified by a luciferase reporter assay. Moreover, we also showed that the correlation between miR-22 and HDAC6 expression was regulated by an E6/p53 pathway in HCK1Ts expressing HPV16 E6. For functional study, an ectopic expression of miR-22 could inhibit cell proliferation and migration, and could induce apoptosis of cervical cancer cell lines. These findings demonstrated that miR-22 was down-regulated in cervical cancer and inversely collated with its downstream target HDAC6. MiR-22 acts as tumor suppressor by inhibiting proliferation and migration, and by inducing apoptosis of cervical cancer cell lines by targeting the 3’UTR of HDAC6. This newly identified E6/p53/miR-22/HDAC6 regulatory network might be a candidate therapeutic target for cervical cancer.

Effect of Productive Human Papillomavirus 16 Infection on Global Gene Expression in Cervical Epithelium.

Human papillomavirus (HPV) infection is the world's most common sexually transmitted infection and is responsible for most cases of cervical cancer. Previous studies of global gene expression changes induced by HPV infection have focused on the cancerous stages of infection, and therefore, not much is known about global gene expression changes at early preneoplastic stages of infection. We show for the first time the global gene expression changes during early-stage HPV16 infection in cervical tissue using 3-dimensional organotypic raft cultures, which produce high levels of progeny virions. cDNA microarray analysis showed that a total of 594 genes were upregulated and 651 genes were downregulated at least 1.5-fold with HPV16 infection. Gene ontology analysis showed that biological processes including cell cycle progression and DNA metabolism were upregulated, while skin development, immune response, and cell death were downregulated with HPV16 infection in cervical keratinocytes. Individual genes were selected for validation at the transcriptional and translational levels, including UBC, which was central to the protein association network of immune response genes, and top downregulated genes RPTN, SERPINB4, KRT23, and KLK8 In particular, KLK8 and SERPINB4 were shown to be upregulated in cancer, which contrasts with the gene regulation during the productive replication stage. Organotypic raft cultures, which allow full progression of the HPV life cycle, allowed us to identify novel gene modulations and potential therapeutic targets of early-stage HPV infection in cervical tissue. Additionally, our results suggest that early-stage productive infection and cancerous stages of infection are distinct disease states expressing different host transcriptomes.IMPORTANCE Persistent HPV infection is responsible for most cases of cervical cancer. The transition from precancerous to cancerous stages of HPV infection is marked by a significant reduction in virus production. Most global gene expression studies of HPV infection have focused on the cancerous stages. Therefore, little is known about global gene expression changes at precancerous stages. For the first time, we measured global gene expression changes at the precancerous stages of HPV16 infection in human cervical tissue producing high levels of virus. We identified a group of genes that are typically overexpressed in cancerous stages to be significantly downregulated at the precancerous stage. Moreover, we identified significantly modulated genes that have not yet been studied in the context of HPV infection. Studying the role of these genes in HPV infection will help us understand what drives the transition from precancerous to cancerous stages and may lead to the development of new therapeutic targets.

Abstract B12: HPV-positive tumor cell lines exhibit major alterations in Toll-like receptor pathways and depend on HMGB1 expression

Abstract Cervical cancer is one of the leading causes of cancer death in women worldwide. Persistent infection with high-risk human papillomavirus (HPV) types is the main risk factor for the development of cervical cancer precursor lesions and is usually characterized by innate immune system evasion. The innate immune system is the first line of defense against pathogens and homeostatic changes. Toll-like receptors (TLR) are a family of membrane proteins that actively participate in this process by recognizing molecular patterns derived from pathogens and from cellular danger signals. Alterations in TLR expression and activation may be important for the control of HPV infections and could play a role in the progression of HPV-associated lesions and cancers. In the present study, we analyzed the expression of 84 genes involved in TLR signaling pathways in cervical cancer cell lines and primary human keratinocytes. We observed that 80% of the differentially expressed genes were downregulated in cervical cancer cell lines relative to normal keratinocytes. Major alterations were detected in genes coding for several TLR signaling proteins, including adaptor molecules MyD88 and SARM1, NFκB activation complex components such as Ube2N and TRAF6, and effector proteins such as HMGB1. Specifically, HMGB1, a protein related to stimulation of inflammation after TLR activation, showed increased expression levels in the HPV-positive cell lines HeLa and SiHa relative to normal keratinocytes. HMGB1 silencing also reduced cell viability, proliferation, and colony formation capacity of cervical cancer cell lines. In addition, silenced cervical cancer cell lines exhibited increased activated caspase-3 levels and hypodiploid cell population suggestive of apoptosis triggering. HPV infection may interfere with TLR pathways at different levels, resulting in the impairment of their activation and function and potentially leading to innate immune system evasion. Our results suggest that TLR pathway proteins such as TLR4, SARM1, and HMGB1 may play other roles related to tumor development. In conclusion, HMGB1 is a key factor in the survival of HPV-positive tumor cells, which warrants further studies to evaluate its potential as a therapeutic target or tumor biomarker. Citation Format: Mirian Galliote Morale, Walason da Silva Abjaude, Aline Montenegro da Silva, Luisa Lina Villa, Enrique Boccardo. HPV-positive tumor cell lines exhibit major alterations in Toll-like receptor pathways and depend on HMGB1 expression [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B12.