Elsevier

Gynecologic Oncology

Volume 104, Issue 2, February 2007, Pages 352-361
Gynecologic Oncology

Coexpression of Notch1 and NF-κB signaling pathway components in human cervical cancer progression

https://doi.org/10.1016/j.ygyno.2006.08.054Get rights and content

Abstract

Objectives.

Features of deregulated Notch1 signaling and NF-κB activation have independently been reported in cervical cancers. Here, we have extended these observations and examined both these pathways simultaneously in human cervical cancer tissue. Further, we have investigated the potential cross-talk between these pathways in a human cervical cancer derived cell line CaSki, which mirrors features of Notch activation as in the majority of human cervical cancers.

Methods.

Cervical tissue samples were analyzed for the expression of Notch1, Jagged 1, Hes1, pAKT, NF-κB p50, NF-κB p65, IκB-α, Bcl-2, CyclinD1, Cdk9, c-Fos, and p53 by immunohistochemistry. A total of 352 samples were analyzed which included 69 normal cervical tissue, 132 preinvasive lesions and 151 squamous cell carcinomas of the uterine cervix. Dual immunofluorescent analysis was performed to evaluate the coexpression of Notch1 and NF-κB. Transcriptional reporter assays and xenografts were undertaken with CaSki cells.

Results.

Features of Notch1 activation as measured by intracellular Notch1, high levels of Jagged1, Hes1 and Cdk9 were paralleled by nuclear translocation of both NF-κB p50 and p65 with target gene expression (IκB-α, Bcl-2, and CyclinD1) in human cervical cancer sections. Reporter assays in CaSki cells are consistent with Notch being an upstream regulator of NF-κB. Further, the xenografts recreate key aspects of human cancer tissue.

Conclusions.

Results from this study suggest that there is a co-activation of Notch1 and NF-κB signaling pathways at the cellular level in the majority of human cervical cancers, with Notch as an upstream regulator.

Introduction

Cancer of the cervix is one of the most frequent malignancies and is the second most prevalent cancer in women worldwide. There is conclusive evidence of the linkage between high-risk human papillomaviruses and cervical cancer [1]. Studies addressing the additional cellular events that sustain cervical carcinogenesis are presently underway. Following the observations of Zagouras et al. [2], demonstrating the presence of intracellular forms of Notch1 in human cervical cancers, we have undertaken a series of studies on this subject. Our work has shown that there are features of disregulated Notch signaling in a major proportion of human cancers [3], [4]. The human Notch protein constitutes a family of transmembrane receptors that plays a pivotal role in cell fate determination, proliferation and apoptosis [5]. The Notch receptor interacts with ligands, such as Delta-like or Jagged, which results in two successive proteolytic cleavages that liberate the cytoplasmic portion of Notch (Notch-IC) [5]. The cleaved intracellular Notch C-terminal fragment translocates to the nucleus and recruits CSL (CBF1, Suppressor of Hairless, Lag1) proteins that transcriptionally regulate target genes like Hes1 (Hairy Enhancer of Split). Our cumulative observations have suggested that features of Notch signaling essentially coincide with the progression of high-grade precursor lesions to invasive cervical cancers. In brief, this involves the generation of intracellular forms of Notch1 accompanied by the marked expression of Jagged 1, down regulation of Manic Fringe, a negative regulator of Jagged 1-Notch signaling, and upregulation of Hes1. [3].

Further, Notch signaling cooperates with papillomavirus oncogenes E6 and E7 in both in vitro and in vivo transformation assays [6], [7], [8]. Of particular interest is the enhanced in vitro and in vivo transformation that activated Notch1 alleles show with an oncogenic form of E6 (83 aa) that accumulates in the progression of human cervical cancers [9]. We have identified PI3K-AKT and Myc as potential mediators of Notch signaling in the context of transformation functions [8], [10], [11]. In contrast, activated Notch signaling has been shown to negatively regulate cell growth in human cervical cancer derived cell lines [12]. Lathion et al. [6] in an extensive study have shown that there is a dose-dependent effect of Notch signaling and indeed this pathway does cooperate with papillomavirus oncogenes E6 and E7 in human primary keratinocytes in xenografts. Consistent with a functional role for Notch signaling in human cervical cancers, Weijzen's group [13] and us [3] have independently shown that inhibiting Notch signaling leads to growth inhibition of the cell line CaSki.

The other cell signaling pathway that has been shown to be disregulated in human cervical cancers is NF-κB [14], [15]. NF-κB complexes are formed of homo or heterodimers, of which NF-κBp50 and p65 are the major active form. They are present in the cytoplasm bound with their inhibitory proteins IκBs (IκB-α, IκB-β, IκB-ε), of which the function of IκB-α is well characterized. Phosphorylation of IκB-α at serines 32 and 36 by the IκB kinase (IKK) complex results in its ubiquitination and subsequent proteosome mediated degradation leading to translocation of NF-κB to the nucleus where it can bring about transcription of target genes [16], [17], [18]. Increased NF-κB signaling could block apoptosis, increase proliferation or induce EMT [19] through induction of various target genes. Some of the well-documented downstream target genes of NF-κB are c-Myc, VEGF, IL-6, IL-8, Bcl-2, Bcl-XL, CyclinD1 and IκB-α[20]. Two independent studies showed constitutive nuclear localization and DNA binding ability of NF-κB, either p50/p65 heterodimer [15] or p50/p50 homodimer [14] in invasive cervical cancer.

Notch and NF-κB have been shown to cross-talk in diverse cellular situations [21], [22], [23], [24], [25]. In this study on human cervical cancers, we have extended the analysis of both the Notch and NF-κB pathways. Further, using the human cervical cancer CaSki, which best resembles a major proportion of human cervical cancers in the context of Notch activation, we examine the functional linkage between Notch and NF-κB.

Section snippets

Patient samples

Human cervical tissue samples for the study were obtained from patients from the Gynaecological Department at Kidwai Memorial Institute of Oncology, Bangalore and the Gynecologic Oncology and Surgical Oncology Clinics of the Regional Cancer Centre, Thiruvananthapuram. The study was approved by the Research Advisory Committees, Institutional Review Board and Ethical Committees of Kidwai Memorial Institute of Oncology, National Centre For Biological Sciences (NCBS), Bangalore and Regional Cancer

Results

Immunohistochemical analyses for Notch pathway genes and NF-κB pathway genes were done on a total of 352 samples, which include 69 normal cervical tissues, 132 preinvasive lesions and 151 squamous cell carcinomas of the uterine cervix. The representative pictures for each of the molecule analyzed are shown in Fig. 1. The upper inset is the representation of staining on normal cervical epithelium for the respective molecule. An immunostaining analysis was undertaken of normal, preinvasive and

Discussion

In this study, we have extended previous observations on the role of Notch and NF-κB pathway in human cervical cancer progression. An important component of the approach undertaken in this paper is the parallel analysis from two independent centers. There is near identical convergence of the data generated by immunohistochemistry from these two independent analyses. Our previous report had revealed features of disregulated Notch signaling in a major proportion of human cervical cancers [3]. To

Acknowledgments

We sincerely thank Sreekala Nair, for her contribution to the study. Bharathi Ramdass and the project were supported by a research grant from the Department of Biotechnology (BT/PR3351/Med/14/445/2002). Confocal imaging of PET sections was carried out in the Wellcome Trust-aided imaging facility at NCBS, supervised by Dr. H. Krishnamurthy. We are grateful to Dr. Satyajit Mayor and Sudha Kumari for providing reagents for immunofluorescence. We are thankful to D. Subramanyam and S. Srivastava for

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