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Received: October 22^nd 2014

Accepted: May 15^th 2015

Wnt signalling pathway and cervical cancer

Moisés Ramos-Solano,^a,b Monserrat Álvarez-Zavala,^a,b Beatriz García-Castro,^b Luis Felipe Jave-Suárez,^a Adriana Aguilar-Lemarroy^a

^aCentro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social

^bDoctorado en Ciencias Biomédicas, Universidad de Guadalajara

Guadalajara, Jalisco, México

Communication with: Adriana Aguilar-Lemarroy

Teléfono (33) 3617 0060, extensión 31926

Email: adry.aguilar.lemarroy@gmail.com

Cervical cancer (CC) is a pathology that arises in the cervical epithelium, whose major cause of risk is human papillomavirus (HPV) infection. Due to the fact that HPV infection per se is not enough to generate a carcinogenic process, it has been proposed that alterations in the Wnt signaling pathway are involved in cervical carcinogenesis. The Wnt family consists of 13 receptors and 19 ligands, and it is highly conserved phylogenetically due to its contribution in different biological processes, such as embryogenesis and tissue regeneration. Additionally, this signaling pathway modulates various cellular functions, for instance: cell proliferation, differentiation, migration and cell polarity. This paper describes the Wnt signaling pathways and alterations that have been found in members of this family in different cancer types and, especially, in CC.

Keywords: Wnt receptors, Cervical intraepithelial neoplasia, Papilloma.

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Overview of the Wnt signaling pathway

Wnt ligands comprise a large family of proteins that activate a diverse group of signaling pathways by which various processes associated with the development and physiology are regulated, such as embryogenesis, polarity, migration, and cellular differentiation.¹ However, a malfunction of this pathway has important implications in carcinogenesis and the development of degenerative diseases; also, this pathway has been implicated in other processes such as inflammation and regeneration of injuries.^2-4 The term Wnt derives from the contraction of the names Wingless and Integrase-1. The first was coined to name the gene whose absence generates a wingless phenotype in Drosophila melanogaster, and the second names the gene that, as reported, is integrated into the virus of the murine mammary tumor in mice; over the years it was determined that the two genes were homologous, so they were renamed Wnt (from wingless-related integration site).^5,6 19 Wnt genes have been identified in mammals, which code for cysteine-rich proteins, susceptible to post-translational modifications such as glycosylation and palmitoylation, giving them hydrophobic characteristics, so that when secreted they provide autocrine and paracrine signals that mediate communication between cells.⁵    

The family of Wnt ligands includes phylogenetically conserved members; this makes them keep important similarities that allow the indiscriminate interaction of these ligands with different receptors. The best-characterized receptors for Wnt ligands are those of the frizzled family (FZD); however, other receptors/co-receptors have been involved in the activation of this signaling pathway, including LDL-related protein (LRP), tyrosine-protein kinase transmembrane receptor (ROR), receptor-like tyrosine kinase (RYK) and protein tyrosine kinase 7 (PTK7).^3,7-9

Canonical pathway: Wnt/beta-catenin

There are several Wnt signaling pathways (Figure 1). Among them, the canonical pathway (so named for being the first described and best-characterized for this family of ligands) is considered relevant in the development of cancer. In this pathway, the main role is played by the transcriptional co-factor beta-catenin. Usually, in the absence of a signal, beta-catenin is associated with two proteins: E-cadherin in the adherens junctions, and a complex called degradosome, in the cytoplasm. The degradosome is formed by the proteins APC, axin, and GSK3-beta; its function is to phosphorylate beta-catenin in the serine/threonine residues of its amino-terminus in order to generate recognition sites for beta-TRCP and to induce ubiquitylation subsequent to degradation in the proteasome. Thus the beta-catenin levels are controlled, allowing only its action at adherens junctions. The absence of beta-catenin in the nucleus causes the TCF transcription factor to bind to a transcriptional repressor complex that inhibits expression of the target genes of the canonical Wnt pathway.¹⁰     

Activation of the canonical pathway is initiated in the cell membrane upon interaction of a Wnt ligand and its FZD/LRP receptor; this interaction in turn allows the association of the DVL protein to the FZD receptor and the sequestration of the APC-Axin-GSK3-beta complex. The incorporation of this complex into DVL-FZD promotes GSK3-beta kinase activity on the LRP5/6 co-receptor in the serine of the PPPSP domains and its surroundings by casein kinase Iγ (CkIγ). These modifications recruit the axin protein to the phosphorylated residues of the LRP5/6 co-receptor, which causes the dissociation of the degradation complex and promotes the release of beta-catenin, which is stored successively in the cytoplasm, to then migrate to the nucleus.² Already in the nucleus, beta-catenin activates the transcription of multiple genes in collaboration with transcriptional co-factors, such as TCF/LEF, p300/CBP, Brg1, parafibromin/Hyrax, and PYGO, the latter through union with BCL9/legless. Many genes activating beta-catenin are involved in the regulation of key processes such as proliferation, cell adhesion, and apoptosis.^2,11 

Non-canonical Wnt pathways

Currently various non-canonical Wnt pathways have been described; all are characterized by not requiring beta-catenin stabilization to complete its signaling cascade. Activation of different non-canonical pathways is determined by the different receptors that the WNT ligands can bind to.^9,12,13 It has been described in general terms that the interaction of Wnt ligands with FZD, RYK, ROR, and MUSK (musculoskeletal receiver) receptors activate non-canonical pathways.^12,14,15 

The non-canonical pathways include the pathways of planar cell polarity (PCP), Wnt-RAP1, Wnt-ROR2, Wnt-PKA, Wnt-GSK3MT, Wnt-zPKC, Wnt-RYK, Wnt-mTOR, and the Wnt/calcium pathway.¹⁴ The most-studied to date are Wnt-PCP and Wnt/calcium, in which effector molecules are involved such as G proteins (RHOA, RHOU, RAC, and CDC42), c-Jun N-terminal kinase, Nemo-like kinase, and nuclear factor of activated T-cells (NFAT).¹⁶

Wnt-calcium

According to various published reports, the FZD, RYK, and ROR receptors are attached to heterodimeric G proteins, which can be activated when the Wnt ligand bonds to its receptor; these G proteins in turn activate phospholipase C (PLC), leading to a modest increase in the concentration of some intracellular signaling molecules such as phosphatidylinositol 1,4,5-trisphosphate (IP3), 1,2 diacylglycerol (DAG), and Ca2Þ.^14,16,17

The signaling pathway triggered by IP3 produces a rapid increase in intracytosolic free calcium because IP3 can diffuse through the cytosol, until reaching the smooth endoplasmic reticulum (SER), where it binds to its receptor, stimulating the release of calcium from deposits found in the SER. Free calcium in the cytoplasm can promote cell polarization, or bond to the calmodulin-dependent protein kinase II (CaMKII), forming a complex that activates a serine/threonine phosphatase enzyme called calcineurin, which is essential in the activation of nuclear factor associated with T-cells (NFAT). In turn, CaMKII can activate TAK1-NLK kinases which are responsible for inhibiting the Wnt canonical pathway. Alternatively, the Wnt-FZD-G protein complex stimulates p38 kinases and activates phosphodiesterase 6 (PDE6), which hydrolyses the cyclic GMP (cGMP), resulting in inactivation of the G protein kinases, increasing cytoplasmic calcium and subsequent activation of the calmodulin/calcineurin system.^16,17 NFAT factor activation can increase the expression of several genes in neurons, cardiac muscle and skeletal cells, as well as proinflammatory genes in lymphocytes.¹⁴

Meanwhile DAG is a hydrophobic molecule that remains in the plasma membrane after it has formed as a product of membrane phospholipid degradation. The combination of DAG and intracytosolic calcium promotes conformational change of PKC (protein kinase C) in the membrane, activating it and making its catalytic region free to join the substrate. PKC activates IKB kinases, which phosphorylate serine amino acids of the inhibitors of nuclear factor kB (NF-kB), allowing their release and transfer to the nucleus, where it contributes to the activation of gene transcription of cytokines, chemokines, and proliferation.^14,16,17

This pathway has been instrumental in the development of dorsoventral polarity and convergent extension of movement, as well as in the formation of several organs.¹⁴ Alterations in this pathway have also been correlated with neoplastic processes because it has the ability to inhibit beta-catenin nuclear function. Because it antagonizes the canonical pathway, the Wnt/calcium pathway has been considered as a tumor-suppressing pathway.¹⁴ Several studies have reported that activation of the Wnt/calcium pathway decreases the proliferation of tumor cells, such as neuroblastoma, squamous cell carcinoma of the esophagus, acute myeloid lymphoma, acute lymphoblastic lymphoma, breast cancer, and colon carcinoma. Therefore, it has been suggested that activation of this pathway may be important for tumor regression and decrease in tumor growth.¹⁴ 

Planar cell polarity pathway

The planar cell polarity pathway refers to the polarization of cells in an epithelial layer, which occurs for instance during the orientation of cilia or hairs. Planar polarity is transmitted locally from cell to cell and is dependent on the FZD receptors and dishevelled (Dsh) protein. This pathway can also regulate cell polarity in a non-epithelial context, such as in convergent extension in gastrulation and in control of the orientation of cell groups in the eye.²

The events governing the signaling of this pathway begin upon the binding of Wnt-FZD, enabling Dsh recruitment and its subsequent phosphorylation by casein kinase II (CK2), which makes this protein an adapter for a signaling complex analogous to Grb-2, thereby promoting the activation of Ras and Rac, where both are responsible for the activation of the MAPK-kinases pathway that culminates in the activation of c-Jun N-terminal kinase (JNK). The latter phosphorylates c-Jun, promoting its translocation to the nucleus, where it forms part of the AP-1 transcription factor by binding to Fos.¹⁸ The GTPases of the Rho family are also involved in this signaling pathway, as when this protein is activated they promote cytoskeletal reorganization. Activation of this pathway regulates polarized cell movements and the planar polarity of epithelial cells.¹⁸

Wnt and cancer

The association of members of the Wnt family with carcinogenic process began as soon as this family was described, as Nusse and Varmus’s 1982 report noted that Wnt1 (then called int1) activation coincided with the induction of breast tumors through the mammary tumor virus.¹⁹ This association grew when it was discovered that the tumor suppressor gene APC (Adenomatous Polyposis Coli) was associated with negative regulation of beta-catenin function and that a mutation in this gene was associated with a loss of function and a familial heritable condition called familial adenomatous polyposis, which leads to the early generation of polyps in the intestine, which degenerate into colorectal cancer.²⁰ It was also found that this effect was generated by beta-catenin stabilization in the cytoplasm and its subsequent translocation to the nucleus, where it functions as a co-transcription factor.    

Different target genes have been described for the canonical Wnt pathway involved in the progression of a tumor process. One of the major upregulated genes is those of the Myc family (c-myc and n-myc), which encode a series of transcription factors that regulate the expression of various genes involved in cellular proliferation.²¹ Another upregulated gene is CCDN1 (Cyclin D1), which encodes a kinase that generates the cell cycle progression from a phase G1 to a phase S.^22,23 The upregulation of these genes makes this signaling pathway active in different types of cancer diseases.   

Beta-catenin stabilization and therefore the activation of the canonical Wnt pathway can be generated by various changes in the components of the signaling pathway. One of these alterations, as already mentioned, is the mutation in component of the beta-catenin degradation complex as APC, which disables the degradation of this molecule. Another way that this stabilization happens is by mutations in the same gene of beta-catenin. These mutations affect the recognition sites of the degradation complex, but do not affect the binding sites of transcription factors such as TCF/LEF.²⁴   

It has been shown that there are epigenetic modifications that contribute to carcinogenesis, as demonstrated in a 1995 report by Laird, in which suppression of methyltransferase inhibits polyp formation in a mouse model.²⁵ Later it was shown that these modifications act more frequently at the level of antagonists of Wnt ligands,^26,27 although involvement has been reported of epigenetic modifications in the silencing of genes encoding ligands associated with an inhibition of tumorigenesis, such as Wnt7a (Table I).²⁸

Several studies have addressed the depth of expression Wnt ligands and receptors in different cell models, such as the report by Sercan et al., which determined the expression profile of these molecules in hematopoietic cells. This group found a differential expression of these components between cells from healthy donors compared to lines of established leukemia, suggesting that ligands expressed in leukemic lines may be involved in maintaining a proliferative state.²⁹ Recently it has also been determined that the loss of expression of the Wnt7a and Wnt4 ligands is a characteristic of cells derived from leukemia (Garcia-Castro, manuscript in revision).³⁰ Furthermore, it has also been reported that the expression of certain ligands (Wnt7a, Wnt3A, and Wnt1) generates a transformation in epithelial cells of the breast.^31,32 These findings together allow one to hypothesize that the use of different ligands in the treatment of various cancer diseases could be useful in eliminating these diseases.

Wnt and cervical cancer

Specifically, the relationship between members of the Wnt family and cervical carcinogenesis is less studied than other cancers. One of the most important reports in this area is from Uren et al., which describes that activation of the canonical Wnt pathway in keratinocytes transfected with the HPV oncoproteins E6 and E7 creates the adoption of a malignant phenotype. Note that HPV infection per se is not sufficient for the transformation, so they propose that one of the second major changes in cervical carcinogenesis is the modification of components of the Wnt pathway.³³ These results are supported by the report from Bulut et al., which determined that activation of the Wnt pathway in a mouse model in the presence of HPV oncoproteins E6 and E7 accelerates the progression of cervical carcinogenesis.³⁴ In addition, it is thought that there is a direct interaction of oncoprotein E6 with different molecules that decrease the activity of proteins involved in the complex of beta-catenin degradation and thus the stabilization of this molecule and its role as transcription co-factor.³⁵ Another working group reported that E6 proteins of non-European HPV-18 variants modulate the Wnt signaling pathway.³⁶ Together these reports further reinforce the possibility that a change in the components of the Wnt pathway is required for the progression of cervical cancer.    

One report that more deeply studies the role of Wnt ligands is from Carmon and Loose, which determines that Wnt7a induces activation of different signaling pathways, depending on the FZD receptor that it bonds with; the canonical pathway is activated by FZD5, and the PCP pathway by FZD10 in endometrial cancer, and that this effect is regulated by the soluble receptor (sFRP4), demonstrating that Wnt ligands have the ability to generate a completely different effect with the change of a single component.³⁷ 

It has been reported that one of the target genes of the canonical Wnt pathway is the COX2 (cyclooxygenase 2) gene, a gene described as proinflammatory and one that probably, together with the production of proinflammatory cytokines by macrophages in the tumor microenvironment, contributes to a steady (chronic) state of inflammation that would improve tumor progression.^38,39 This has been described as a hallmark of the progression of a tumor.⁴⁰ Our working group has shown that cell lines derived from cervical cancer poorly express Wnt7a and Wnt4, and that a restoration of the expression of these ligands causes a significant inhibition of cell proliferation (Ramos-Solano et al.; in revision).   

Because the presence of some viral type of HPV has been reported in 99.7% of cervical cancer samples,⁴¹ and the involvement of HPV infection has been demonstrated in the modulation of Wnt pathway components, alterations in this pathway could be considered a hallmark of cervical carcinogenesis, as well as targets or therapeutic agents in the treatment of this pathology.

Deeper studies of the mechanisms of cervical cancer regulated by components of the Wnt family could give promising results for the possible use of members of this family, either as markers of prognosis or treatment.

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