TGF-beta signalling pathway factors in HPV-induced cervical lesions

Human papillomaviruses (HPV) are considered the etiological agents of cervical cancer, especially high-risk genotypes. TGF-beta (transforming growth factor-beta) is well known for its anti-proliferative effects but the neoplastic cells often lose their sensitivity to TGF-beta. A characteristic alteration associated with malignant progression is the loss of responsiveness to TGF-beta1-induced cell growth inhibition. The aim of the present study was to establish the possible role of some members of TGF-beta signalling pathway during cervical cancer development and the possible relationship with HPV infection. In order to establish TGF-beta gene expression levels in cervical oncogenesis, TGF-beta1, TGF-beta1 receptors and Smad2 were investigated in precancerous and cervical cancer samples (Quantitative Real-Time PCR). The study revealed that 84.5% of patients were positive for HPV DNA. The most prevalent HPV genotypes were high-risk HPV 16 and 18 in single or co-infections. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia to cervical carcinoma. Furthermore, we observed that cervical lesions without HPV infection expressed significantly less TGF-beta1. TGF-betaRI and Smad2 gene expression levels were found to be decreased in SCC and AC samples in contrast with CIN1 and CIN2/3 samples. Our results showed that in human cervical cancer the disruption of TGF-beta/Smad signalling pathway might contribute to the malignant progression of cervical dysplasia. These data emphasize the importance of canonical TGF-beta pathway integrity in carcinogenesis.     

Downregulation of ERp57 expression is associated with poor prognosis in early-stage cervical cancer

Abstract

Objective: We investigated the clinical significance of ERp57 in the progression of cervical cancer.

Methods: mRNA and protein expression of ERp57 in cervical neoplasias were examined.

Results: ERp57 mRNA expression was significantly decreased in cervical cancers. Immunohistochemistry revealed that ERp57 expression in 123 cervical cancers was down-regulated compared to cervical intraepithelial neoplasias or normal tissues (p?<?0.001). Low ERp57 expression was significantly associated with worse overall survival (HR?=?12.19, p?=?0.018).

Conclusions: Low ERp57 expression independently predicts a poor outcome for patients with cervical cancer, supporting the notion that ERp57 may be a promising novel cancer target.     

Combined analysis of HPV DNA and p16INK4a expression to predict prognosis in ASCUS and LSIL pap smears

Human papillomavirus (HPV) is known to play an important etiological role in the genesis of cervical cancer, but only a very small proportion of infected women develop invasive cervical cancer. The purpose of cervical cancer prevention is early diagnosis of its precursors. The molecular detection of HPV DNA as a diagnostic test to cervical carcinogenesis gave a low positive predictive value as compared to the use of biomarkers. p16INK4a has been proposed as putative surrogate biomarkers that would allow identification of dysplastic cervical epithelia. Serial consecutive cervical smears were test for high-risk HPV stained with immunocytochemistry for p16INK4a and followed-up for 36 months. The aim of the study was to evaluate the immunohistochemical expression of pl6INK4a as a marker of progression risk in low-grade dysplastic lesions of the cervix uteri. In the present series, significant pl6 overexpression was observed in the group that progressed from low to high-grade squamous intraepithelial lesion when compared with the group that did not progress. In conclusion, overexpression of p16INK4a acts as potential biomarkers for cervical cancer progression from premalignant lesions.     

TSHR蛋白在宫颈癌的表达及其与HPV-16感染的关系

目的研究促甲状腺激素受体（TSHR）在子宫颈癌组织的表达及其与乳头瘤病毒（HPV－16）的关系。方法应用免疫组织化学链霉菌抗生物素过氧化物酶（SP）法检测79例子宫颈癌和30例子宫颈炎组织HPV－16与TSHR蛋白表达。79例癌症患者中病理分级〈Ⅱ级33例,≥Ⅱ级46例;病理分期〈Ⅱ期56例,≥Ⅱ期23例;无淋巴结转移66例,有淋巴结转移13例;肿瘤大小〈3cm44例,肿瘤大小≥3cm35例。结果HPV－16在子宫颈癌表达率55．70％明显高于宫颈炎5％（P〈0．05）,TSHR在子宫颈癌表达率68．35％明显高于宫颈炎26．67％（P〈0．05）。HPV－16表达与肿瘤的大小、肿瘤分级、分期、淋巴结转移不相关。TSHR表达与肿瘤的大小呈正相关,P〈0．05,与肿瘤分级、分期及淋巴结转移不相关。HPV－16与TSHR在宫颈癌表达呈正相关。结论HPV感染对宫颈癌病变起到强烈的预警作用。TSHR不仅在甲状腺滤泡上皮细胞表达,在子宫颈癌细胞也表达,TSHR过表达能促进宫颈细胞的异常增殖,其异常功能可能是恶性肿瘤特定的临床表型。HPV与TSHR在子宫颈癌变过程中起协同作用。     

CEACAM1 in cervical cancer and precursor lesions: association with human papillomavirus infection.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an adhesion molecule expressed in a wide variety of tissues including epithelial cells, leukocytes, and tumors that may establish both homotypic and heterotypic interactions. The aim of this work was to study the protein expression pattern of CEACAM1 in cervical cancer and precursor lesions in the context of human papillomavirus (HPV) infection. We used immunohistochemistry to analyze CEACAM1 expression in formalin-fixed, paraffin-embedded cervical tissues from 15 healthy women, 15 patients with low-grade squamous intraepithelial lesions (SIL), 15 patients with high-grade SIL, and 15 patients with squamous carcinomas. HPV types were identified by PCR. CEACAM1 was either undetectable (13/15) or low (2/15) in normal cervical tissues. By contrast, CEACAM1 expression was increased in high-grade SIL (10 samples staining intermediate/high and 4 samples staining low) as compared with low-grade SIL with undetectable (n=3) or low (n=12) expression. CEACAM1 expression was undetectable or low in cervical carcinoma. Our results suggest that CEACAM1 may be an interesting progression marker in SIL and cervical cancer, in particular due to reported immunoregulatory properties.     

HPV16 oncoproteins induce MMPs/RECK-TIMP-2 imbalance in primary keratinocytes: possible implications in cervical carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.     

1235例妇科门诊患者高危型HPV感染状况及亚型分布调查研究

目的了解本地区妇科门诊患者宫颈高危型HPV感染状况及亚型分布,为今后的宫颈癌前病变、宫颈癌防治提供临床依据。方法采用基因芯片技术对1 235例妇科门诊患者进行HPV筛查,筛查出的阳性患者应用流式荧光杂交法进行高危型HPV亚型检测,分析比较宫颈炎、宫颈鳞癌及宫颈腺癌患者高危型HPV感染情况及亚型分布差异。结果六安市金安区妇幼保健院妇科门诊患者HPV感染率高达60%,其中高危型HPV感染率为43. 2%,主要以HPV16、HPV18为主;低危型HPV感染率为30.0%,主要以HPV11为主;单一感染阳性率为34. 1%,而混合型感染高达65. 9%,且两者均以HPV16型和HPV18型为主。宫颈炎患者HPV16型、HPV18型及HPV16 ＋ HPV18型的检出率明显低于宫颈鳞癌和宫颈腺癌患者,其中宫颈腺癌患者HPV16 ＋ HPV18型混合感染率最高。结论妇科门诊患者HPV感染率较高,宫颈癌患者HPV16及18型检出率显著高于宫颈炎患者,加强HPV高危基因型的监测有助于预警宫颈癌尤其是宫颈腺癌的发生。     

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.     

ERp57 is a multifunctional thiol-disulfide oxidoreductase

The thiol-disulfide oxidoreductase ERp57 is a soluble protein of the endoplasmic reticulum and the closest known homologue of protein disulfide isomerase. The protein interacts with the two lectin chaperones calnexin and calreticulin and thereby promotes the oxidative folding of newly synthesized glycoproteins. Here we have characterized several fundamental structural and functional properties of ERp57 in vitro, such as the domain organization, shape, redox potential, and the ability to catalyze different thiol-disulfide exchange reactions. Like protein disulfide isomerase, we find ERp57 to be comprised of four structural domains. The protein has an elongated shape of 3.4 +/- 0.1 nm in diameter and 16.8 +/- 0.5 nm in length. The two redox-active a and a' domains were determined to have redox potentials of -0.167 and -0.156 V, respectively. Furthermore, ERp57 was shown to efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins. The implications of these findings for the function of the protein in vivo are discussed.     

Correlation of immunochemical detection of HPV L1 capsid protein in pap smears with regression of high-risk HPV positive mild/moderate dysplasia

OBJECTIVE: To immunostain Pap smears of high-risk (hr) HPV DNA-positive early squamous lesions for detecting HPV L1 protein. STUDY DESIGN: Routinely stained archival slides from 84 mild and moderate hrHPV DNA-positive dysplasias were immunostained using a panreactive HPV L1 antibody. Follow-up smears were taken from women with remission for a mean period of 22.8 months (range, 6-46). Conization was done in patients with persistence or progression (3 and 48 patients, respectively) after a mean time of 12 months (range, 9-48). RESULTS: Twenty-nine of 84 smears (34.5%) had positively stained squamous epithelial cell nuclei. In 9 of 29 (31%) women progressive disease occurred (2 cervical intraepithelial neoplasia [CIN] 2 and 7 CIN 3 lesions on conization) 20 (69%) had remission. Of the 55 L1-negative cases, 13 (23.6%) had remission, 42 (76.4%) progressed (3 CIN 2, 38 CIN 3, 1 microinvasive carcinoma). The difference in follow-up between L1 positive and negative cases was statistically significant (chi2 test, p< or =0.001). CONCLUSION: Low and moderate dysplastic squamous lesions without immunochemically detectable HPV L1 protein are significantly more likely to progress than are L1-positive cases. Immunochemical L1 capsid detection in routine Pap smears thus offers prognostic information about early dysplastic lesions.     

宫颈鳞癌演进过程P16INK4a及Hh-Gli信号通路相关蛋白表达及其相关性研究

探讨P16^INK4a及Sonic hedgehog(Hh-Gli)信号通路蛋白在宫颈癌及癌前病变(CIN)中的表达相关性及其意义．采用Western-blot方法检测HPV16阳性及HPV18阳性宫颈癌细胞系P16^INK4a及Hh-Gli信号通路蛋白Smo、Ptch及Gli表达．免疫组化检测组织芯片P16^INK4a、Shh、Smo、Ptch及Gli表达,包括20例正常宫颈、18例癌旁组织、54例CIN及28例宫颈鳞癌组织．分析P16^INK4a与Hh-Gli信号通路蛋白间表达相关性及与临床病理因素的关系．结果显示P16^INK4a、Smo、Ptch及Gli蛋白在HPV16及HPV18阳性宫颈癌细胞系中表达无显著差异(P>0.05)．P16^INK4a、Shh、Smo、Ptch及Gli蛋白在宫颈癌中表达强度显著高于癌旁及正常组织(P<0.05),在CINⅠ与正常组织间差异不显著(P>0.05)．P16^INK4a、Shh、Smo及Gli蛋白,在CINⅠ、CINⅡ与CINⅢ之间均有显著性差异(P<0.05)．相关分析显示,CINⅡ-CINⅢ中P16^INK4a与Shh和Smo蛋白表达正相关,浸润癌中P16^INK4a与Shh、Smo和Gli蛋白正相关．结论认为,P16^INK4a及Hh-Gli信号通路异常激活与宫颈癌发生及演进密切相关,且二者间具有相关性．Hh-Gli信号通路的激活可能是Shh配体增高调控Smo高表达而上调Gli蛋白所致．     

安康地区人乳头瘤病毒感染特征及其与宫颈癌的关系研究

目的:调查安康地区女性人乳头瘤病毒(HPV)感染的基因型别及年龄分布特征,分析其与宫颈癌的关系,为宫颈癌防治及HPV疫苗研发提供可靠的依据。方法:收集2010年6月-2012年8月间在本院及安康市部分县级医院妇产科就诊的2736名女性的液基细胞学和组织学标本,分为8个年龄组:16-24岁119例、25-29岁230例、30-34岁343例、35-39岁472例、40-44岁574例、45-49岁512例、50-54岁206例、55-86岁280例,进行病理学分类及HPV分型检测,分析不同年龄组及不同类型宫颈组织中的HPV感染率。结果:2736例女性中发生HPV感染720例(26.32%),共检出21种型别,感染率最高的基因型别是HPV16(25.05%),其他常见型别依次为HPV58、HPV52、HPV6、HPV11。单一感染占76.25%,多重感染占23.75%。HPV感染率在16-24岁、35-39岁和55-86岁三个年龄段出现高峰;而高危型HPV的感染率在35-39岁和55-86岁两个年龄段分别出现高峰。HPV的检出率随着宫颈病变的严重程度而增加,其中正常或炎症人群的HPV感染率显著低于宫颈病变及宫颈鳞状细胞癌患者(均P0.05),且意义未明的不典型鳞状细胞(ASCUS)、CIN1-3及宫颈鳞状细胞癌患者的HPV感染率对比结果存在显著差异(P0.05)。CIN1组、CIN2-CIN3组及宫颈鳞状细胞癌组单一感染率逐渐增加(P0.05),且其二重、三重感染率比较差异均有统计学意义(P0.05)。结论:安康地区HPV16型别感染较广,临床需加强对HPV16型单一感染宫颈病变患者的癌症预防工作。     

Human Papillomavirus Genotype Distribution in Czech Women and Men with Diseases Etiologically Linked to HPV

Background

The HPV prevalence and genotype distribution are important for the estimation of the impact of HPV-based cervical cancer screening and HPV vaccination on the incidence of diseases etiologically linked to HPVs. The HPV genotype distribution varies across different geographical regions. Therefore, we investigated the type-specific HPV prevalence in Czech women and men with anogenital diseases.

Methods

We analyzed 157 squamous cell carcinoma samples, 695 precancerous lesion samples and 64 cervical, vulvar and anal condylomata acuminate samples. HPV detection and typing were performed by PCR with GP5+/6+ primers, reverse line blot assay and sequencing.

Results

Thirty different HPV genotypes were detected in our study, HPV 16 being the most prevalent type both in precancerous lesions (45%) and squamous cell carcinomas (59%). In benign lesions, HPV 6 (72%) was the most common type. Altogether, 61% of carcinoma samples and 43% of precancerous lesion samples contained HPV 16 and/or 18. The presence of HPV types related to the vaccinal ones (HPV 31, 45, 33, 52, 58) were detected in 16% of carcinoma samples and 18% of precancerous lesion samples. HPV 16 and/or 18 were present in 76% of cervical cancer samples, 33% of CIN1, 43% CIN2 and 71% of CIN3 samples. HPV types 6 and/or 11 were detected in 84% samples of condylomata acuminate samples.

Conclusions

The prevalence of vaccinal and related HPV types in patients with HPV-associated diseases in the Czech Republic is very high. We may assume that the implementation of routine vaccination against HPV would greatly reduce the burden of HPV-associated diseases in the Czech Republic.     

Human papillomavirus genotyping by the DNA chip in the cervical neoplasia

Human papillomavirus (HPV) is implicated as an etiologic agent in neoplasitc lesions of the cervix. In this study, we used an HPV DNA chip to detect the type-specific sequence of HPV from cervical swabs in women with biopsy- proven neoplastic lesions of the cervix. Four hundred seventy-one patients were involved and classified into four groups based on the cytopathologic diagnosis: group I (normal, n = 290), group II (low-grade squamous intraepithelial lesions (SIL), n = 68), group III (high-grade SIL, n = 51), and group IV (invasive cervical cancer, n = 55). HPV detection rates were 17.6% (51 of 290), 73.5% (50 of 68), 92.2% (47 of 51), and 95.2% (59 of 62) in patients of group I to group IV, respectively. HPV-16 was the most frequent type (21.8%) in all specimens tested, and significantly increased the prevalence by advancing the grade of the cervical lesions (P < 0.01). The next frequent virus types were HPV-18 and HPV-58. The prevalence of multiple HPV infections was 37.3, 43.7, 27.7, and 28.8%, and no significant difference was detected between each group (P > 0.05). This suggests that the HPV DNA chip is a sensitive diagnostic tool for the detection of HPV in cervical specimens, and that it would provide more useful information on viral genotype and multiple HPV infections. Taken together, molecular biological data on HPV might be beneficial for the prevention and management of cervical neoplastic lesions.