The Potential of Wnt Signaling Pathway in Cancer: A ...

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Cancer Transl Med 2016;2(2):55–60 doi: 10.4103/2395-3977.181437

Cancer Translational Medicine

Review

The Potential of Wnt Signaling Pathway in Cancer: A Focus on Breast Cancer Mahnaz M. Kazi, Trupti I. Trivedi, Toral P. Kobawala, Nandita R. Ghosh Department of Cancer Biology, Division of Molecular Endocrinology, Gujarat Cancer & Research Institute, Asarwa, Ahmedabad, Gujarat, India Address for correspondence: Dr. Nandita R. Ghosh, Department of Cancer Biology, Division of Molecular Endocrinology, Gujarat Cancer & Research Institute, NCH Compound, Asarwa, Ahmedabad 380016, Gujarat, India. E‑mail: [email protected] Received January 12, 2016; Accepted March 07, 2016

Cancer development and progression as well as tumor recurrence are largely due to the presence of cancer stem cells (CSCs) that are maintained through various pathways. Wnt/β‑catenin signaling is the fundamental pathway, which when deregulated leads to tumor development by sustaining CSC population. Along with the upregulation of its various components, Wnt pathway is highly active in cancer cells resulting in increased expression of the target genes. In breast cancer condition, convincing results are available wherein the Wnt pathway activation in breast cancer cells increases the cell motility while its blockade suppresses their aggressive behavior in vitro. Further, numerous reports on breast cancer patients have documented the importance of activation of Wnt pathway and its components to an extent that the regulation can be exploited therapeutically with promising results. In addition, recent research has laid emphasis on the significance of Wnt pathway in the triple‑negative breast cancer, a molecular subtype of breast cancer, which lacks targeted therapy till date. Hence, understanding of Wnt signaling and its targeting to treat such patients can be an assuring approach. Key words: Cancer stem cells, triple negative breast cancer, ‑catenin

INTRODUCTION Cancer stem cells (CSCs) have increasingly grabbed the focus in cancer research. They are identified in multiple solid tumors such as colon cancer, melanoma, breast cancer, pancreatic cancer, liver cancer, brain cancer, and lung cancer.1‑7 The major setbacks of cancer management – drug resistance, disease relapse, and metastasis – are attributed to CSCs, and hence, it is now required to redirect the cancer therapies to target these CSCs.8,9 CSCs are regulated by several pathways that govern the processes of development, self‑renewal, and cell fate,10 among which Wnt/β‑catenin pathway is the fundamental one and by far the best characterized. This Wnt/β‑catenin pathway can be dysregulated in many types of cancers and hence could provide an excellent therapeutic target.11‑13 Therefore, this review will recapitulate the contribution of Wnt/β‑catenin pathway in cancer progression with a focus on breast cancer.

WNT SIGNALING PATHWAY Wnt/β‑catenin pathway is initiated by evolutionarily conserved growth factors of the wingless and integration site growth factor (Wnt) family. Wnts are encoded by 19 different Wnt genes and share a high degree of sequence homology.14 They bind to cell surface

receptors to activate the Wnt pathway and thus trigger signaling cascades that are important in many physiological settings.15 Wnt signaling pathway actively functions in embryonic development and helps in homeostasis in mature tissues by regulating diverse processes including cell proliferation, survival, migration and polarity, specification of cell fate, and self‑renewal property.16,17 Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes. These proteins are hydrophobic and are found in association with cell membranes and the extracellular matrix. They become palmitoylated in the endoplasmic reticulum of the Wnt‑producing cells in the presence of acyltransferase porcupine. This palmitate modification is thought to assist in ligand reception on This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: [email protected]

How to cite this article: Kazi MM, Trivedi TI, Kobawala TP, Ghosh NR. The Potential of Wnt Signaling Pathway in Cancer: A Focus on Breast Cancer. Cancer Transl Med 2016;2(2):55-60.

© 2016 Cancer Translational Medicine | Published by Wolters Kluwer ‑ Medknow

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Cancer Transl Med 2016;2(2)

Wnt‑responding cells. Once modified, the proteins are transported and secreted using secretory vesicles controlled by the multi‑pass transmembrane protein Wntless/Evi (evenness interrupted), which is present in the Golgi and/or on the plasma membrane. This facilitates the release of Wnt protein from the cells to get associated with the seven‑pass transmembrane receptor Frizzled (Fzd). Fzd is present on the surface of responding cells and possesses a large extracellular domain containing a conserved motif which comprises 10 cysteine residues called the cysteine‑rich domain. There are various co‑receptors – low‑density lipoprotein receptor‑related proteins 5 or 6 (LRP5/6) or ROR2 that aid in the binding of Wnt proteins to the receptor.18 The co‑receptor engaged then determines the downstream effect of the successful ligand binding, initiating either the noncanonical or the canonical pathways.19

NONCANONICAL WNT PATHWAYS The noncanonical pathway is further divided into planar cell polarity (PCP) pathway and Wnt/Ca+2 pathway. The Wnt4, Wnt5a, and Wnt11 are the proposed ligands for these noncanonical pathways.20‑22 In PCP pathway, Wnt ligand binds to the Fzd receptor and the signal is transduced to Dishevelled (Dvl), leading to its activation. Consequently, Dvl-associated activator of morphogenesis 1 (Daam1) binds to PDZ domain of Dvl and activates Rho GTPase which then leads to the activation of the Rho‑associated coiled coil‑containing protein kinase and myosin.23 The second branch of signaling takes place through the DEP domain of Dvl which activates the Rac GTPase independent of Daam1. The activated Rac in turn stimulates JNK activity that plays a role in cell polarization and directional migration in co‑ordination with Rho.24 Another domain of the noncanonical Wnt signaling pathway termed as Wnt/Ca2+ pathway is dependent on G‑proteins. It stimulates intracellular Ca2+ release from endoplasmic reticulum by activating inositoltrisphosphate through phospholipase C. The calcium release and intracellular accumulation activate several Ca2+ sensitive proteins, including protein kinase C (PKC), calcineurin, and calcium/calmodulin‑dependent kinase II.25 The noncanonical pathways mainly regulate the cell motility, tissue polarity, cell migration, and also inhibit canonical Wnt pathway.11 There are still other emerging pathways which are initiated by Wnt ligands and are found to overlap with components of the PCP and Wnt/Ca2+ branches, but they have distinct outcomes.26

CANONICAL WNT PATHWAY Canonical Wnt pathway, also referred to as β‑catenin‑dependent Wnt pathway, is the best characterized of the three Wnt signaling pathways. Normally, in the absence of Wnt signaling, the cytoplasmic β‑catenin is maintained at a low level through ubiquitin‑proteasome‑mediated degradation. It is regulated by a multiprotein destruction complex comprising Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase‑3 β (GSK‑3 β). As shown in Figure 1, the signaling pathway is initiated upon engagement of the Wnt ligand with Fzd receptor protein in combination with either LRP5 or LRP6, forming a ternary complex on the extracellular membrane. LRP5/6 is a transmembrane receptor with a large extracellular domain critical for Wnt binding and a short intracellular tail. LRP5/6 also acts as the receptor for the 56

secreted agonists of the Wnt pathway, R‑spondin family of proteins. R‑spondin uniquely synergizes with the Wnt proteins and leads to enhancement of the signal responses.27,28 The upstream ligand binding then results in the activation of the kinases which induces phosphorylation of serine residues in the intracellular cytoplasmic tail of LRP5/6, resulting in the initiation of the Wnt‑mediated signaling cascade.8 Consequently, phosphoprotein Dvl is recruited to the formed complex at the plasma membrane that leads to translocation of Axin and GSK‑3 β from the cytoplasm to the receptor complex. As a result, the destruction complex dissociates and disrupts, due to which the cytoplasmic concentration of β‑catenin increases. Hence, the accumulated β‑catenin translocates into the nucleus where it forms a complex with members of the T‑cell transcription factor/lymphoid enhancer‑binding factor (LEF) family of transcription factors. Here, β‑catenin acts as a transcription activator by displacing Grouchos and recruits the co‑activators cAMP response element‑binding protein binding protein (CBP) or its homolog p300 and also other components of the basal transcription machinery (such as CtBP, Foxo, TNIK, Bcl9, and Pygopus).29 The binding of CBP and p300 activates the Wnt pathway. CBP‑mediated Wnt signaling is shown to be associated with colonic cell proliferation, and p300‑mediated Wnt activity promotes differentiation.30,31 This results in the expression of the downstream target genes, c‑jun, fra‑1, c‑myc, cyclin D1, etc., that are normally involved in developmental stages and adult tissue homeostasis [Figure 1]. Canonical Wnt pathway supports the formation and maintenance of CSCs and thereby cancer formation. Hence, the aberrant activation of this pathway and the target genes maintains the pluripotency of the stem cells rather than differentiating them and results in the neoplastic proliferation. Overexpression or mutation in any of the pathway components leads to malignant growth.

CANONICAL WNT PATHWAY IN CANCER A relation of Wnt pathway with cancers was identified for the 1st time in 1980s when the proto‑oncogenic properties of Wnts were discovered in mouse models of breast cancer. Then after, the role of Wnt pathway has been shown in a number of malignancies. In colorectal cancer, aberrant activation of Wnt pathway is well established with an incidence of about 93%. Mutations in APC gene and loss of its function or activating mutations of CTNNB1 (β‑catenin gene) are most frequent and found to be the principal cause for Wnt pathway upregulation, shown in about 80% of the cases.32 There are still a number of other Wnt pathway components, ranging from the ligand receptor to the target genes, which are involved in colorectal tumor formation. The expression of Wnt ligand, receptor Fzd, and co‑receptor LRP6 in colonic mucosa leads to β‑catenin accumulation, thereby promoting intestinal tumor growth and invasion.33,34 Modulation of Wnt pathway, epigenetically, is also widespread in colorectal cancer wherein negative regulators and antagonists of the pathway are silenced. These antagonists include Fzd‑related proteins (FRPs), Dickkopf (DKK), Wnt‑inhibiting factor, and Sox that are frequently hypermethylated and suppressed. In addition, Axin2 which is originally involved in the degradation of β‑catenin is suppressed due to hypermethylation. This suppression of the pathway antagonists leads to β‑catenin accumulation and pathway activation resulting in progressive colorectal tumorigenesis.35‑39 Therefore, the importance of aberrant Wnt signaling in colorectal


Wnt signaling in breast cancer

Figure 1. Schematic representation of canonical Wnt signaling pathway

cancer is definitively clear, but the pathway mutations are not limited to it and play a role in many other types of malignancies too. In cervical cancer, Wnt pathway is activated by Sox 14, leading to proliferation and invasion of cervical cells and cancer progression.40 Furthermore, in ovarian cancer, there is an abundant presence of Wnt7a which ultimately activates the Wnt pathway directly promoting cell functions associated with tumor growth.41 Besides, overexpression of β‑catenin is evident in advanced epithelial ovarian cancer.42 Similarly, β‑catenin expression is an adverse underlying factor in carcinogenesis and progression of esophageal cancer that is related to poor survival.43,44 In addition, in hepatocarcinogenesis, Wnt pathway is suggested to play a role where there is an overexpression of Wnt3, the receptor Fzd7, and the co‑receptor LRP6, contributing to the hyperactivation of the pathway.45,46 Likewise, in B‑cell chronic lymphocytic leukemia, several isoforms of Wnt ligand, the transcription factor LEF‑1, and its downstream target cyclin D1 were overexpressed, and the pathway is shown to contribute to the defect in apoptosis.47‑49

CANONICAL WNT PATHWAY IN BREAST CANCER Wnt/β‑catenin pathway plays an important role in breast cancer, which still needs extensive research. The pathway is frequently deregulated in breast cancer cells, like other malignancies, which maintains the stem‑cell phenotype. Various studies have demonstrated the role of Wnt and its downstream effectors in primary human breast tumors as well as breast cancer cell lines.50,51 Wnt signaling pathway and the related components are increasingly expressed in breast CSC‑enriched populations in comparison with normal breast stem cell‑enriched populations.52,53 Wnt pathway activation in breast cell lines, owing to the treatment with Wnt ligands, significantly increased the cell motility,52,54 in contrast blocking the pathway by knocking down the Wnt ligand, Dvl or β‑catenin, considerably suppressed aggressive behavior of the breast cancer cells.52,53,55 Second, ectopic expression of the pathway antagonists such as sFRP and DKK blocks the Wnt signaling pathway, thereby decreasing the cells’ ability to form mammosphere and grows as xenografts in mammary glands.50,52,54 Moreover, a study on transgenic mouse model of ErbB2‑driven tumor progression has shown a significant upregulation of direct β‑catenin transcriptional targets in the mammary tumors. Blocking the endogenous β‑catenin by RNA interference in

transgenic mouse‑derived clonal mammary tumor cells exhibited a pronounced defect in invasion.56 Furthermore, Wnt/β‑catenin pathway along with the downstream molecules has been found to play a crucial role in primary breast cancer patients. However, the mechanism for aberrant activation of the pathway in breast cancer tissue has not yet been clearly understood. Of all the molecules involved, β‑catenin is the most studied and a substantiate marker in breast cancer patients and is considered a hallmark for Wnt pathway activation. It is suggested to be a marker of advanced breast cancer, representing a new diagnostic or therapeutic target. The prominent expression of β‑catenin in the nucleus and/or cytoplasm stimulates cell proliferation and significantly correlates with the poor survival of breast cancer patients.57 Moreover, the subcellular localization of β‑catenin considerably affects the signal transduction pathways in cells leading to breast tumor formation. Loss of β‑catenin expression at the membrane resulting in its cytoplasmic accumulation is associated with worse prognosis in invasive ductal carcinoma.58 However, there are still other components of the pathway that are involved in its regulation. Axin2 polymorphism and gene expression of APC, casein kinase Iα, GSK‑3 β, and protein phosphatase 2A, i.e., the constituents of destruction complex are found to be associated with breast cancer susceptibility.59 In addition, the Wnt receptor ‑ Fzd7 pair of ligand receptor is upregulated in a subset of breast cancer patients and when targeted, results in a significant reduction in tumor growth. This reveals the essentiality of Fzd7 for activating Wnt signaling pathway and leading to tumorigenesis and breast cancer.60,61 Subsequently, the aberrant expression of the co‑receptors LRP5 and LRP6 is strongly implicated in mammary gland tumorigenesis in breast cancer patients. Silencing these co‑receptors reduces Wnt signaling, cell proliferation, and in vivo tumor growth.62,63 Alterations in Wnt antagonists through their gene promoters’ hypermethylation could also be a possibility for the aberrations in the pathway. These alterations are found in the pathway antagonists (APC, FRP1, FRP2, CDH1, and DKK3) along with increased nuclear accumulation of β‑catenin in breast cancer patients. Therefore, the molecules are suggested to play an important role in the development of breast cancer and have significant clinical importance as prognostic markers.64,65 These data demonstrate the importance of Wnt signaling pathway in breast cancer patients and their outcome, which exhibits the promising therapeutic approach, targeting various components of Wnt pathway. 57


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Breast cancer being the most common cancer among women, there are enormous studies carried out for breast cancer treatment, but the outcome seems to be unsatisfactory, demanding further extended investigations, which could reveal the specific targets of breast cancer. The present scenario for treating breast cancer depends on the presence of estrogen receptor (ER), progesterone receptor (PR), and expression of Her2‑neu protein that divides breast cancer patients into luminal (ER+), Her2‑positive (Her2+), and triple‑negative subtype (ER−, PR−, and Her2−). The patients belonging to luminal sub‑type are the most frequent ones and are benefitted by the hormonal therapy agents such as tamoxifen due to expression of ER on the cell surface. The patients who lack ER but overexpress Her2 receptor can be cured by targeting the Her2 protein with monoclonal antibodies (trastuzumab).66 However, there is still a third sub‑type of breast cancer, i.e., triple‑negative breast cancer (TNBC) where patients do not exhibit any of the three cell surface receptors, due to which none of the hormonal or targeted therapy can be employed on such patients. The only option left for treating TNBC is to utilize general chemotherapies. Hence, TNBC is the deadliest one that shows worst prognosis in breast cancer due to the potential aggressive nature of the disease and limited number of therapeutic options available.67,68 Therefore, it is essential to explore new pathways that are expressed in TNBC specifically so as to acquire targeted therapy for the same. Although Wnt signaling pathway is frequently upregulated in breast cancer, presently, it is being explored more exclusively in TNBC due to call for a targeted therapy.

WNT PATHWAY IN TRIPLE‑NEGATIVE BREAST CANCER Wnt signaling pathway has found to be particularly activated in TNBC patients with the upregulation of its components.69 Recent studies have shown that β‑catenin expression and thereby Wnt signaling activation is more frequently observed in basal‑like invasive breast cancers, a sub‑type that significantly overlaps with TNBC.58,70 In addition, β‑catenin is found to be the predictor of a poor outcome in TNBC patients.70 Dey et al.71 have studied the tumor specimens from two breast cancer cohorts and together with meta‑analysis of other breast cancer microarray studies confirmed the Wnt pathway activation in TNBC sub‑types. Wnt pathway and its activated components are shown to be associated with high grade, poor prognosis, and metastatic disease. Wnt pathway plays an important role in the regulation of metastasis‑associated phenotypes in breast tumor cells, and its activation significantly increases the risk of brain and lung metastases, especially in TNBC patients.71 Furthermore, Geyer et al.72 have studied the sub‑cellular localization of β‑catenin in invasive breast carcinoma patients and found that the aberrant β‑catenin nuclear expression and thereby Wnt pathway activation are preferentially activated in TNBC sub‑type.72 In addition, researchers have explored for the presence of any mutations in β‑catenin encoding gene – CTNNB1 in TNBC patients and found that the gene was not mutated in any of the studied cases, but the β‑catenin protein was overexpressed, leading to Wnt pathway activation. This suggests that the overexpression is not driven by the gene mutations, and the constitutive activation of the pathway may involve a number of other components too.71,73 Hence, activated Wnt pathway is implied as a hallmark of TNBC disease and presents an attractive therapeutic target for the same. Further detailed studies on the mechanism of pathway function may be elucidated that might be helpful in the development of 58

Table 1: Wnt pathway inhibitors in clinical trials Compound name

Target

References Manufacturer

LGK 974

Porcupine

74

Novartis

OMP‑18R5

Fzd7

61

OncoMed

OMP‑54F28 Fzd8 (Fzd8‑Fc fusion)

75

OncoMed

OTSA101

Fzd10

76

Centre Leon Berard

PRI‑724

β‑catenin

77

Prism Pharma

CWP232291 β‑catenin

8

JW Pharmaceutical

anti‑TNBC drugs targeting Wnt signaling pathway. However, several biological agents specifically targeting Wnt signaling pathway for therapeutic blockade have succeeded to enter the early phase of clinical trials, and many are still in the discovery phase or preclinical phase. Table 1 shows the Wnt pathway inhibitors that are in the clinical phase I for various malignancies. Although Wnt pathway and its components have provided promising results as therapeutic targets in cancer treatment, it still needs further investigation. Fevr et al.78 have shown the rapid loss of intestinal epithelial cells in adult mice upon deletion of β‑catenin resulting in a complete block of intestinal homeostasis. However, successful therapy can be accomplished when the cancer cells are efficiently eradicated without interfering with the precarious role of the Wnt pathway in normal tissues’ homeostasis and repair. Hence, it is important to target the CSCs alone via Wnt pathway without hampering its regular function.

CONCLUSION Wnt signaling pathway and its components are activated in various cancers where they regulate the CSCs that lead to development and progression of tumor. The role of the pathway components is definitive in colorectal cancer and has now emerged for the breast cancer too. Wnt signaling could aptly be targeted in TNBC which so far does not have any targeted therapy. Hence, a proficient cure for TNBC by exploitation of Wnt signaling pathway is immensely anticipated, which could assist for winning the victory against this cancer. Financial support and sponsorship

This study was supported by the Gujarat Cancer Society and the Gujarat Cancer and Research Institute. Conflicts of interest

There are no conflicts of interest.

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