Cervical Squamous Cell Carcinoma Cell Lines Research Articles

CDKN2A inhibited ferroptosis through activating JAK2/STAT3 pathway to modulate cisplatin resistance in cervical squamous cell carcinoma.

Cervical squamous cell carcinoma (CESC) is a significant threat to women's health. Resistance to cisplatin (DDP), a common treatment, hinders the therapeutic efficacy. Understanding the molecular basis of DDP resistance in CESC is imperative. Cyclin-dependent kinase inhibitor 2A (CDKN2A) expression was evaluated through quantitative real-time-PCR and western blot in clinical samples from 30 CESC patients and human cervical epithelial cells and CESC cell lines (SiHa, C33A, and Caski). It was also evaluated through bioinformatics analysis in Timer, Ualcan, and GEPIA database. Cell viability was detected by CCK-8. Apoptosis was detected by Calcein AM/PI assay. Lipid reactive oxygen species (ROS), malondialdehyde, glutathione, Fe2+, and iron level were detected by kits. Protein level of JAK2, STAT3, p-JAK2, p-STAT3, ACSL4, GPX4, SLC7A11, and FTL were detected by western blot. In CESC, elevated CDKN2A expression was observed. Cisplatin exhibited a dual effect, inhibiting cell proliferation and inducing ferroptosis in CESC. CDKN2A knockdown in a cisplatin-resistant cell line suppressed proliferation and induced ferroptosis. Moreover, CDKN2A was identified as an inhibitor of erastin-induced ferroptosis. Additionally, targeting the JAK2/STAT3 pathway enhanced ferroptosis in cisplatin-resistant cells. CDKN2A could inhibit ferroptosis in CESC through activating JAK2/STAT3 pathway to modulate cisplatin resistance.

N6-methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16-infected cervical squamous cell carcinoma.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.

The immune landscape and prognostic analysis of CXCL8 immune-related genes in cervical squamous cell carcinoma.

Cervical squamous cell carcinoma (CESC), one of the most common malignancies in women, imposes a significant burden on women's health worldwide. Despite extensive research, the molecular and pathogenic mechanisms of cervical squamous cell carcinoma and CESC remain unclear. This study aimed to explore the immune-related genes, immune microenvironment infiltration, and prognosis of CESC, providing a theoretical basis for guiding clinical treatment. Initially, by mining four gene sets and immune-related gene sets from public databases, 14 immune-related genes associated with CESC were identified. Through univariate and multivariate COX regression analyses, as well as lasso regression analysis, four CESC-independent prognostic genes were identified, and a prognostic model was constructed, dividing them into high and low-risk groups. The correlation between these genes and immune cells and immune functions were explored through ssGSEA enrichment analysis, revealing a close association between the high-risk group and processes such as angiogenesis and epithelial-mesenchymal transition. Furthermore, using public databases and qRT-PCR experiments, significant differences in CXCL8 expression between normal cervical cells and cervical cancer cells were discovered. Subsequently, a CXCL8 knockdown plasmid was constructed, and the efficiency of CXCL8 knockdown was validated in two CESC cell lines, MEG-01 and HCE-1. Through CCK-8, scratch, and Transwell assays, it was confirmed that CXCL8 knockdown could inhibit the proliferation, invasion, and migration abilities of CESC cells. Targeting CXCL8 holds promise for personalized therapy for CESC, providing a strong theoretical basis for achieving clinical translation.

USP39 interacts with SIRT7 to promote cervical squamous cell carcinoma by modulating autophagy and oxidative stress via FOXM1

BackgroundSirtuin 7 (SIRT7) is an oncogene that promotes tumor progression in various malignancies, however, its role and regulatory mechanism in cervical squamous cell carcinoma (CSCC) is unknown. Herein, we attempted to investigate the functional role and molecular mechanism of SIRT7 underlying CSCC progression.MethodsSIRT7 expression was evaluated in CSCC cells using various assays. We then used a series of function gain-and-loss experiments to determine the role of SIRT7 in CSCC progression. Furthermore, mechanism experiments were conducted to assess the interaction between SIRT7/USP39/FOXM1 in CSCC cells. Additionally, rescue assays were conducted to explore the regulatory function of USP39/FOXM1 in CSCC cellular processes.ResultsSIRT7 was highly expressed in CSCC patient tissues and cell lines. SIRT7 deficiency showed significant repression on the proliferation, and autophagy of CSCC cells in vitro and tumorigenesis in vivo. Similarly, apoptosis and ROS production in CSCC cells were accelerated after the SIRT7 knockdown. Moreover, SIRT7 and USP39 were found colocalized in the cell nucleus. Interestingly, SIRT7 was revealed to deacetylate USP39 to promote its protein stability in CSCC cells. USP39 protein was also verified to be upregulated in CSCC tissues and cells. USP39 silencing showed suppressive effects on CSCC cell growth. Mechanistically, USP39 was revealed to upregulate SIRT7 by promoting the transcriptional activity of FOXM1. Rescue assays also indicated that SIRT7 promoted autophagy and inhibited ROS production in CSCC cells by regulating USP39/FOXM1.ConclusionThe SIRT7/USP39/FOXM1 positive feedback network regulates autophagy and oxidative stress in CSCC, thus providing a new direction for CSCC-targeted therapy.Graphical

E2F Transcription Factor 1 Activates FKBP Prolyl Isomerase 4 to Promote Angiogenesis in Cervical Squamous Cell Carcinoma Via the PI3K/AKT Signaling Pathway.

Angiogenesis, namely the formation of blood vessels, is crucial for tumor growth, metastasis and development. E2F transcription factor 1 (E2F1) has been linked to tumorigenesis in several human cancers. This work examines the role of E2F1 and its downstream targets in angiogenesis in cervical squamous cell carcinoma (CSCC). E2F1 was predicted as a candidate oncogene in CSCC using a GSE63514 dataset. Increased E2F1 expression was detected in CSCC tumor samples and cell lines by RT-qPCR, immunohistochemistry, and western blot assays. E2F1 downregulation reduced the angiogenesis activity of HUVECs and the invasiveness of CSCC cells. In vivo, E2F1 knockdown also reduced the xenograft tumor growth and promoted tumor necrosis in mice. FKBP prolyl isomerase 4 (FKBP4) was identified as a target of E2F1. E2F1 bound to FKBP4 promoter for transcriptional activation. Further upregulation of FKBP4 blocked the tumor-suppressive role of E2F1 silencing. FKBP4 was enriched in the PI3K/AKT signaling. In cells and xenograft tumors, the E2F1/FKBP4 axis promoted PI3K and AKT phosphorylation. Activation of the PI3K/AKT signaling restored the angiogenesis activity in cells blocked by E2F1 silencing. In summary, this work demonstrates that E2F1 promotes FKBP4 transcription to activate the PI3K/AKT pathway, which augments the angiogenesis and invasiveness of CSCC.

Metallothionein-like 5 expression is correlated with poor prognosis and promotes proliferation of cervical squamous cell carcinoma

ABSTRACT Cervical cancer represents one of the most important female genital cancers. Cervical squamous cell carcinoma (CESC) accounts for about 90% of all cervical malignancies and the prognosis are unsatisfied. Here we aimed to investigate the clinical relevance of metallothionein-like 5 (MTL5), a novel metallothionein-like protein, in CESC. RT-qPCR and immunohistochemistry staining showed that MTL5 was upregulated in CESC tissues than nontumorous cervix tissues, which is consistent with the data from TCGA database. Kaplan–Meier survival analysis revealed that higher MTL5 can help predict worse prognosis. In addition, Cox hazard regression analysis verified an independent predictive role of MTL5 in CESC. To further investigate the involvement of MTL5 in CESC, we conducted knockdown experiments in two CESC cell lines. As a result, silencing MTL5g significantly inhibited proliferation of CESC cells. Finally, we validated that silencing MTL5 can suppress CESC tumor growth in vivo using the mice subcutaneous xenografts model. Taken together, higher MTL5 indicates worse survival of CESC after surgical resection. Targeting MTL5 represents a potential therapy of CESC by inhibiting tumor growth, which deserves further investigations.

Long non-coding RNA LINC00514 promotes the proliferation and invasion through the miR-708-5p/HOXB3 axis in cervical squamous cell carcinoma.

Long non-coding RNA (lncRNA) LINC00514 is a cancer-related lncRNA that has been proven to be implicated in the progression of several cancers. However, the biological function of LINC00514 in cervical squamous cell carcinoma (CSCC) remains unclear. Thus, we aimed to identify the LINC00514 expression profile in CSCC and determine its exact mechanism. Our results showed that the expression of LINC00514 was up-regulated in human CSCC tissues and cell lines. Knockdown of LINC00514 significantly inhibited the proliferation and invasion of CSCC cells, as well as suppressed tumorigenesis of CSCC in vivo. In addition, LINC00514 was found to work as a miRNA sponge for miR-708-5p and suppressed the expression of miR-708-5p in CSCC cells. Homeobox B3 (HOXB3) was found to be a target gene of miR-708-5p. Rescue assays demonstrated that miR-708-5p inhibitor attenuated the effects of LINC00514 knockdown on cell proliferation and invasion in CSCC cells. In addition, overexpression of HOXB3 reversed the inhibitory effects of miR-708-5p mimics on cell proliferation and invasion. Taken together, our findings for the first time elucidated that lncRNA LINC00514 promotes the proliferation and invasion through the miR-708-5p/HOXB3 axis in CSCC. Thus, LINC00514/miR-708-5p/HOXB3 axis might be a promising therapeutic target for the treatment of CSCC.

CCNB1 Expedites the Progression of Cervical Squamous Cell Carcinoma via the Regulation by FOXM1.

BackgroundCervical squamous cell carcinoma (CSCC) is responsible for 80–85% of cervical cancer. Cyclin B1 (CCNB1) represents a hub gene during the development of cervical cancer. However, the oncogenic role of CCNB1 in CSCC remains unclear. Our study aims to explore the mechanism underlying CCNB1 regulation on cell cycle progression in CSCC cells.MethodsFirst, we analyzed differentially expressed genes from CSCC dataset GSE63678 and conducted gene function enrichment analysis. Subsequently, CCNB1 expression was knocked down in CSCC cell lines to assess cell proliferation, apoptosis, and cell cycle distribution. After the validation of the binding relationship between forkhead box protein M1 (FOXM1) and the promoter of CCNB1, the effect of FOXM1 on CCNB1 expression and on CSCC cell growth and apoptosis was verified. We further analyzed the histone ChIP-Seq data of CCNB1 in CSCC cells and measured the acetylation levels of the CCNB1 promoter histones.ResultsCCNB1 was overexpressed in CSCC tissues and cells, and CCNB1 silencing inhibited the growth of CSCC cells, and promoted cell cycle arrest and apoptosis. FOXM1 potentiated CCNB1 transcription by binding to its promoter and recruiting CBP/P300, a histone acetyltransferase. Further increasing FOXM1 expression or increasing P300 activity in CSCC cells with CCNB1 knockdown elevated CCNB1 expression and proliferation and cell cycle progression of CSCC cells. Knockdown of CCNB1 activated the p53 pathway in cells.ConclusionFOXM1 inhibited the activation of the p53 pathway by recruiting CBP/P300, which promoted the transcription of CCNB1, resulting in the growth and cell cycle progression of CSCC cells.

MiR-106a Regulates Cell Proliferation and Autophagy by Targeting LKB1 in HPV-16-Associated Cervical Cancer.

miR-106a is aberrantly regulated in various tumors and plays an important role in carcinogenesis. However, the biological role and molecular mechanism by which miR-106a contributes to cervical squamous cell carcinoma (CSCC) remains elusive. In this study, we verified that miR-106a was elevated in both human papilloma virus (HPV) 16-positive CSCC tissues and cell lines. ROC curve analysis showed that miR-106a could well distinguish HPV-16-positive CSCC tissues from normal cervical squamous epithelium tissues. High expression of miR-106a was associated with malignant clinicopathologic parameters in CSCC tissues. Exogenous expression of miR-106a greatly promoted cervical cancer cell proliferation while attenuated autophagy. Furthermore, a novel target of miR-106a, liver kinase B1 (LKB1), a proven tumor suppressor in cervical cancer was verified. Here we confirmed LKB1 was negatively correlated with malignant clinicopathologic parameters in CSCC tissues. Overexpression of LKB1 neutralized the effect of miR-106a on proliferation and autophagy in cervical cancer cell lines. In addition, the role of miR-106a in cell proliferation and autophagy was via LKB1 and its downstream pathway AMP-activated protein kinase-mammalian target of rapamycin. Of note, miR-106a was upregulated by HPV-16 E7 protein. The function of HPV-16 E7 to cell proliferation was suppressed when knockdown miR-106a in HPV-16 E7-expressing cells. IMPLICATIONS: Our study highlights the tumorigenic role and regulatory mechanism of miR-106a in CSCC. miR-106a may be a potential therapeutic target in HPV-associated cervical cancer.

LncRNA MAGI2-AS3 Affects Cell Invasion and Migration of Cervical Squamous Cell Carcinoma (CSCC) via Sponging miRNA-233/EPB41L3 Axis.

IntroductionThe incidence of cervical squamous cell carcinoma (CSCC) has expanded in recent years. However, the function of long non-coding RNA (lncRNA) MAGI2-AS3 in the occurrence and progression of CSCC remains unclear. Therefore, the role of lncRNA MAGI2-AS3 in cervical squamous cell carcinoma (CSCC) was investigated in our study.MethodsWe used qRT-PCR analysis to identify the level of MAGI2-AS3 mRNA expression in CSCC clinical samples and cell lines. We investigated cell migration and invasion of CSCC cells transfected with MAGI2-AS3, miR-233 mimic, or EPB41L3 with transwell assays. Bioinformatics analysis and a luciferase reporter assay were employed to predict the interaction between MAGI2-AS3 and miR-233.ResultsWe found that MAGI2-AS3 and EPB41L3 were both downregulated in CSCC and the expression of this two was positively correlated. Bioinformatics analysis showed that MAGI2-AS3 might bind to miR-233, which could directly target EPB41L3. In CSCC cells, overexpression of MAGI2-AS3 led to upregulated, while overexpression of miRNA-233 led to downregulated expression of EPB41L3. However, MAGI2-AS3 and miR-233 did not affect the expression of each other. In addition, overexpression of MAGI2-AS3 and EPB41L3 led to inhibited cancer cell invasion and migration, while overexpression of miR-233 played an opposite role and attenuated the effects of overexpressing MAGI2-AS3.ConclusionMAGI2-AS3 may sponge miR-233 to upregulate EPB41L3, thereby inhibiting CSCC cell invasion and migration.

Long Noncoding RNA loc285194 Expression in Human Papillomavirus-Positive and -Negative Cervical Squamous Cell Carcinoma, C33A, and SiHa Cells and Transforming Growth Factor-β1.

BackgroundThis study aimed to investigate the expression of long noncoding RNA (lncRNA) loc285194 in cervical squamous cell carcinoma (CSCC) biopsies that were positive and negative for human papillomavirus (HPV) and in human CSCC cell lines SiHa and C33A and to investigate the overexpression of lncRNA loc285194.Material/MethodsCervical biopsy tissue and plasma samples from 66 patients with histologically confirmed CSCC, that were HPV16-positive (N=22), HPV18-positive (N=27), and HPV-negative (N=17), and healthy controls (N=20) and human CSCC cell lines SiHa (HPV16-positive) and C33A (HPV-negative) were studied. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the expression of lncRNA loc285194 in cervical biopsies and plasma. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to measure levels of transforming growth factor-β1 (TGF-β1). A lncRNA loc285194 expression vector was constructed and transfected into SiHa and C33A cells that underwent a transwell assay for cell migration.ResultsExpression of lncRNA loc285194 was downregulated in HPV-positive and HPV-negative tissue samples and plasma from patients with CSCC and distinguished between patients and healthy controls. Plasma levels of loc285194 and TGF-β1 were significantly correlated with the presence of CSCC. In SiHa and C33A cells, TGF-β1 expression was downregulated, and cell migration was inhibited following lncRNA loc285194 overexpression. Although lncRNA loc285194 expression was not affected by TGF-β1 treatment, its effects on cell migration were reduced by TGF-β1.ConclusionsThe expression of lncRNA loc285194 inhibited the migration of CSCC cells in vitro through the inactivation of TGF-β1.

LncRNA ANCR downregulates hypoxia‑inducible factor1α and inhibits the growth of HPV‑negative cervical squamous cell carcinoma under hypoxic conditions.

Long non‑coding RNA (lncRNA) anti‑differentiation non‑coding RNA (ANCR) has been reported to participate in numerous types of malignancies. The present study aimed to investigate the function of lncRNA ANCR in cervical squamous cell carcinoma (CSCC). The expression of ANCR in the cervical tissues (tumor tissues in patients with CSCC) and serum of patients with CSCC in addition to healthy female controls was detected using reverse transcription‑quantitative polymerase chain reaction. Diagnostic values of ANCR expression in cervical tissue and serum for CSCC were determined using receiver operating characteristic curve analysis. LncRNA ANCR and hypoxia‑inducible factor 1α (HIF‑1α) expression vectors were constructed and transfected into CSCC cell lines, and cell proliferation under normal O2 and hypoxic conditions (8% O2) was detected using a Cell Counting kit‑8 assay. Expression of HIF‑1α was determined using western blot analysis. It was observed that ANCR was downregulated in human papillomavirus (HPV)‑negative patients with CSCC compared with in normal female cases and HPV‑positive patients with CSCC in cervical tissues and in the serum, and the downregulation of ANCR effectively distinguished HPV‑negative patients with CSCC from healthy controls. ANCR overexpression inhibited the proliferation of HPV‑negative CSCC cells under hypoxic conditions, whilst HIF‑1α overexpression reversed this effect. ANCR overexpression inhibited HIF‑1α expression in HPV‑negative CSCC cells, while HIF‑1α overexpression exhibited no significant effect on ANCR expression. It was therefore concluded that ANCR may inhibit the growth of HPV‑negative cervical squamous cell carcinoma under hypoxic conditions by downregulating HIF‑1α.

LncRNA HAND2-AS1 inhibits cancer cell proliferation, migration and invasion by downregulating ROCK1 in HPV-positive and negative cervical squamous cell carcinoma.

Long non-coding (lnc)RNA HAND2 antisense RNA 1 (HAND2-AS1) exhibited tumor suppression activity in different types of cancer. However, its role in cervical squamous cell carcinoma (CSCC), which is frequently diagnosed among females, has not been elucidated. The current study revealed that lncRNA HAND2-AS1 was downregulated, while Rho-associated protein kinase 1 (ROCK1) was upregulated in serum of human papillomavirus (HPV)-positive and -negative patients with CSCC compared with healthy controls. Correlation analysis revealed that the expression levels of lncRNA HAND2-AS1 were negatively correlated with the expression levels of ROCK1 in HPV-positive and -negative patients with CSCC but not in healthy controls. Downregulation of lncRNA HAND2-AS1 distinguished patients with CSCC from healthy controls. Additionally, lncRNA HAND2-AS1 overexpression led to reduced expression levels of ROCK1 in HPV-positive and -negative human CSCC cell lines but not in normal cervical cell lines. ROCK1 overexpression did not significantly affect the expression of lncRNA HAND2-AS1 in all the cell lines investigated. lncRNA HAND2-AS1 overexpression inhibited, while ROCK1 overexpression promoted the proliferation, migration and invasion of HPV-positive and -negative human CSCC cell lines but not normal cervical cell lines. ROCK1 overexpression attenuated the effects of lncRNA HAND2-AS1 overexpression on cancer cell proliferation, migration and invasion. lncRNA HAND2-AS1 may inhibit cancer cell proliferation, migration and invasion by downregulating ROCK1 in HPV-positive and -negative CSCC.

Cancer-derived exosomal miR-221-3p promotes angiogenesis by targeting THBS2 in cervical squamous cell carcinoma.

Recently, cancer-derived exosomes were shown to have pro-metastasis function in cancer, but the mechanism remains unclear. Angiogenesis is essential for tumor progression and is a great promising therapeutic target for advanced cervical cancer. Here, we investigated the role of cervical cancer cell-secreted exosomal miR-221-3p in tumor angiogenesis. miR-221-3p was found to be closely correlated with microvascular density in cervical squamous cell carcinoma (CSCC) by evaluating the microvascular density with immunohistochemistry and miR-221-3p expression with in situ hybridization in clinical specimens. Using the groups of CSCC cell lines (SiHa and C33A) with miR-221-3p overexpression and silencing, the CSCC exosomes were characterized by electron microscopy, western blotting, and fluorescence microscopy. The enrichment of miR-221-3p in CSCC exosomes and its transfer into human umbilical vein endothelial cells (HUVECs) were confirmed by qRT-PCR. CSCC exosomal miR-221-3p promoted angiogenesis in vitro in Matrigel tube formation assay, spheroid sprouting assay, migration assay, and wound healing assay. Then, exosome intratumoral injection indicated that CSCC exosomal miR-221-3p promoted tumor growth in vivo. Thrombospondin-2 (THBS2) was bioinformatically predicted to be a direct target of miR-221-3p, and this was verified by using the in vitro and in vivo experiments described above. Additionally, overexpression of THBS2 in HUVECs rescued the angiogenic function of miR-221-3p. Our results suggest that CSCC exosomes transport miR-221-3p from cancer cells to vessel endothelial cells and promote angiogenesis by downregulating THBS2. Therefore, CSCC-derived exosomal miR-221-3p could be a possible novel diagnostic biomarker and therapeutic target for CSCC progression.

High Expression of Ubiquitin-Specific Protease 8 (USP8) Is Associated with Poor Prognosis in Patients with Cervical Squamous Cell Carcinoma.

BackgroundCervical cancer is one of the most common female malignancies in the world. The ubiquitin-specific protease 8 (USP8) functions by removing ubiquitin from protein substrates, and its potential role in cancer development was recently uncovered in lung cancer. The aim of this study was to investigate the expression and function of USP8 in cervical squamous cell carcinoma (CSCC).Material/MethodsImmunohistochemical staining and quantitative PCR were performed to explore the expression of USP8 in both CSCC tissues and adjacent normal cervical tissues. Univariate and multivariate analyses were conducted to evaluate the clinical significance of USP8 in CSCC. Proliferation, migration, and invasion abilities of 2 CSCC cell lines were assessed after overexpression or silencing USP8, respectively.ResultsBoth the RNA and protein levels of USP8 were upregulated in CSCC tissues compared to normal cervical tissues. High expression of USP8 was correlated with advanced tumor stage and high recurrence risk. Moreover, USP8 was identified as a novel independent prognostic factor for CSCC patients. Cellular studies showed that USP8 can enhance the proliferation, migration, and invasion abilities of CSCC cells, thereby promoting tumor progression.ConclusionsHigh expression of USP8 is frequent in CSCC tissues, which promotes tumor proliferation and invasion, and is correlated with a poor overall survival. Targeting USP8 may be a novel direction for drug development for CSCC therapy.

Genomic characterization of human papillomavirus-positive and -negative human squamous cell cancer cell lines.

Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.

Functional Role of NRF2 in Cervical Carcinogenesis.

Nuclear factor erythroid-2-related factor 2 (NFE2L2) is a transcription factor associated with resistance to chemotherapy and increased tumor growth. NRF2 is repressed by the inhibitor Keap1. The Keap1-NRF2 pathway is dysfunctional in multiple tumor types. Among Uighur women, the incidence of cervical squamous cell carcinoma (CSCC) and cervical intraepithelial neoplasia (CIN) was associated with elevated nuclear expression of NRF2 and decreased cytoplasmic expression of Keap1. Up-regulation of nuclear NRF2 was significantly associated with reduced cytoplasmic Keap1 expression. NRF2 positivity and Keap1 negativity were frequently associated with more advanced tumors (i.e., higher histological grade, lymph node involvement, and higher tumor stages) (p<0.05 for all). Methylated CpG islands in the Keap1 gene promoter in cervical cancer tissue were identified using MassARRAY. Moreover, promoter hypermethylation of this gene was significantly associated with decreased protein expression and increased nuclear NRF2 expression in cervical cancer tissues. Overexpression and knockdown of NRF2 in CSCC cell lines showed that NRF2 promotes proliferation, inhibits apoptosis, and enhances migration and invasion. These studies support the concept that epigenetic changes regulate expression of Keap1 in cervical cancer tissues. The association of NRF2 expression with aggressive tumor behavior suggests that NRF2 may be a marker of poor prognosis in patients with cervical cancer.

Targeted blockade of interleukin-8 abrogates its promotion of cervical cancer growth and metastasis

Surgery and radiotherapy have been used for decades to treat cervical cancer; however, high recurrence and lymph node metastasis rates are observed after these procedures. New therapeutic agents are needed to improve survival rates of patients by reducing tumor growth and metastasis. We previously demonstrated that interleukin-8 (IL-8) was associated with lymph node metastasis of early cervical squamous cell carcinoma (SCC). The current study assessed the role of IL-8 in growth and metastasis of cervical SCC and evaluated the effects of targeting IL-8 with small hairpin RNA (shRNA) and a human anti-IL-8 antibody. The human cervical SCC cell lines CaSki (high IL-8 producers), SiHa, HeLa, and SiHa transfected with the IL-8 gene were used for the studies. IL-8 stimulated proliferation, migration, and invasion but prevented apoptosis of SCC cells in vitro. Suppressing IL-8 expression with shRNA reduced cell growth and invasion of SCC cells in vitro. In a xenograft model, SCC cells were inoculated subcutaneously into athymic mice to evaluate the effect of IL-8 and its antibody on tumor growth and metastasis and animal survival. IL-8 enhanced tumor growth and metastasis in vivo concomitant with reduced animal survival. IL-8 antibody treatment of tumor-bearing animals resulted in smaller tumor volume, decreased lymph node metastasis, and longer animal survival. Blockade of IL-8 with an antibody demonstrated significant anti-tumor effects in a xenograft model and may thus provide a potential alternative approach for the treatment of cervical cancer.

Mast cell chymase is present in uterine cervical carcinoma and it detaches viable and growing cervical squamous carcinoma cells from substratum in vitro

Increased numbers of mast cells is a typical feature of a variety of human cancers. The major mediators in the secretory granules of the MC(TC) type of mast cells, serine proteinases tryptase and chymase, may be involved in squamous cell carcinoma (SCC) lesions by inducing matrix remodeling and epithelial cell detachment. The objective of this study was to analyze immunohistochemically whether MC(TC) mast cells as well as protease inhibitors, squamous cell carcinoma antigens (SCCAs), are present in the uterine cervical SCC. In addition, the effect of tryptase and chymase on uterine cervical SCC cell lines was studied in vitro. Here we report that tryptase- and chymase-positive mast cells are present in significant numbers in the peritumoral stroma of SCC lesions. Also, weak SCCA-2 immunoreactivity is observed in the SCC lesions, but only SCCA-1 in uterine cervical specimens with nonspecific inflammation. In cell cultures, especially chymase, but not tryptase, was shown to induce effective detachment of viable, growing and non-apoptotic SiHa SCC cells from substratum. Chymase also detached viable ME-180 SCC cells from substratum as well as degraded fibronectin. In contrast, normal keratinocytes underwent apoptotic cell death after similar prolonged chymase treatment. No inhibition of chymase was detected by SiHa cell sonicates nor did these cells express marked SCCA immunopositivity. MC(TC) mast cells containing tryptase and chymase are present in the peritumoral stroma of uterine cervical SCC and the malignant cells are only weakly immunoreactive for the chymase inhibitor SCCA-2. It is chymase that appears to be capable of inducing effective detachment of viable and growing SCC cells and therefore, it may release SCC cells from a tumor leading to spreading of malignant cells.

Molecular Cytogenetic Analysis of Cervical Squamous Cell Carcinoma Cells Demonstrates Discordant Levels of Numerical and Structural Chromosomal Instability and Identifies ‘Selected’ Chromosome Rearrangements

The processes underlying the karyotype complexity and heterogeneity characteristic of cervical squamous cell carcinoma (SCC) cells are not well understood, nor is it known which chromosome abnormalities provide a selective advantage. We used molecular cytogenetics to quantify for the first time the prevalence of all chromosomal abnormalities detectable in 8 cervical SCC cell lines. Of 225 rearrangements identified, 118 occurred frequently (≧70% metaphases) in one or more lines, indicating selection. Most of these selected rearrangements (107; 48% of the total) were included in published lists of abnormalities detected in independent cell stocks (although their significance was not previously recognised), indicating that they arose early in the establishment of the lines and were therefore most likely to have been present in the original tumours. The 8 cell lines showed 4.7- and 11.0-fold variation in levels of numerical and structural chromosomal instability (N-CIN and S-CIN) respectively, as determined by quantifying non-clonal abnormalities. We observed discordance between N-CIN and S-CIN (p = 0.148), with modal chromosome number showing a strong association with N-CIN (p = 0.007) but not with S-CIN (p = 0.602). These data imply that different mechanisms underlie N-CIN and S-CIN in cervical SCC. Despite ongoing S-CIN, newly arisen in vitro rearrangements were infrequently selected, supporting the view that selection is an important determinant of somatic evolution in malignancy. There was a positive association between numbers of non-clonal and clonal rearrangements (p = 0.002 for non-clonal vs. selected rearrangements), suggesting that factors affecting S-CIN determine the total numbers of rearrangements present in established cervical SCC cells.