Roles of the WWOX in pathogenesis and endocrine therapy of breast cancer

Abstract

Breast cancer is one of the most common malignancies, often with complicated etiology and poor clinical outcome. In recent years, a critical role has emerged for the WW domain-containing oxidoreductase (WWOX) in breast cancer. WWOX is a tumor suppressor; it is deleted or attenuated in 29–63.2% of breast cancer tissues and is associated with a poor prognosis of breast cancer patients. WWOX heterozygous knockout mice show a higher incidence of mammary tumors and impaired branching morphogenesis. At the molecular level, WWOX interacts with AP2γ, ErbB4, SMAD3, and WBP2 suppressing their transcription activities in breast cancer cell lines. This review provides comprehensive insights into the current knowledge of WWOX activities in the pathogenesis and endocrine therapy of breast cancer.

Introduction

WW domain-containing oxidoreductase (WWOX) is cloned and mapped to the chromosome region 16q23. The Wwox gene spans a genomic locus of more than 1 Mb but encodes an open reading frame of 1.2 kb. The Wwox gene overlaps the second chromosomal fragile site FRA16D, which exhibits an increased incidence of gaps or breaks when exposed to extrinsic or intrinsic factors such as UV radiation, ionizing radiation, and hypoxia.^1–4

The full-length WWOX is a 414-amino acid protein possessing two typical N-terminal WW domains and a short-chain dehydrogenase reductase (SDR) domain. The WW domain is needed for the classical WW–PPxY interaction. WWOX interacts with the proteins that possess PPxY motifs, including ErbB4, AP-2γ, Run2, p73, and C-Jun. WWOX sequesters these proteins in the cytoplasm and suppresses their transcriptional functions.^5–9 Proteins with the SDR domain are involved in oxidation and reduction of various substrates, such as lipid hormones, sugars, alcohols, and retinoids.¹⁰ The SDR domain-containing protein WWOX is found to be highly expressed in hormone-dependent tissues, such as the mammary gland, prostate, and ovary. Thus, WWOX may be involved in steroid metabolism.^11,12

The subcellular localization and function of WWOX are regulated by its phosphorylation. Tyr33 phosphorylation of the WWOX protein can be activated by steroid hormone 17β-estradiol (E₂) independent of the estrogen receptor (ER).¹³ Tyr33 phosphorylation of WWOX enhances the recognition of the PPxY motif and promotes the WW–PPxY interaction.^5,6 WWOX polyubiquitination and degradation are associated with its phosphorylation at Tyr287 by CDC42-associated tyrosine kinase 1 (ACK1).¹⁴

WWOX is an important tumor suppressor and loss or deregulation of WWOX contributes to development of various tumors. Numerous studies have been conducted on the roles of WWOX in bone homeostasis and osteosarcoma and their findings have been reviewed previously.^9,15,16 In this minireview, we focus on the roles of WWOX in the pathogenesis and endocrine therapy of breast cancer.

WWOX is involved in mammary gland development and breast tumorigenesis

WWOX has been found to be highly expressed in hormone-dependent tissues, such as the mammary gland, prostate, and ovary.¹² Aldaz et al. conditionally knocked out the Wwox gene in the mouse mammary epithelium utilizing two transgenic lines expressing Cre recombinase, BK5-Cre, and MMTV-Cre. They found that Wwox knockout (KO) mice showed a significant impairment in mammary gland branching morphogenesis, with rudimentary mammary epithelium and little or no evidence of ductal structures.¹⁷ These results indicate that WWOX may be involved in mammary gland development.

The Wwox KO mouse is a useful tool to study the roles of WWOX in vivo. The single and double allelic targeted ablations result in two different phenotypes of Wwox KO mice. It has been found that Wwox^−/− mice suffer metabolic disorders and eventually postnatal lethality, while Wwox^+/− mice show higher incidence of mammary and lung tumors than wild-type mice. When Wwox^+/− mice are given a single dose of ethyl-nitrosourea (a powerful chemical mutagen), 80% of Wwox^+/− mice develop mammary, lymphoblastic, or lung tumors.^12,18 Molecular analysis of global gene expression in Wwox ablated mammary epithelium has revealed that Wwox deletion results in the up-regulation of cancer-related genes, such as the Wnt signaling pathway genes, the tissue remodeling related genes, and the cell migration- and adhesion-related genes.¹⁷ These results suggest that WWOX may be involved in breast tumorigenesis.

Taken together, WWOX is necessary for mammary gland development and loss of WWOX contributes to breast tumorigenesis.

WWOX expression is associated with breast cancer progression and prognosis

Loss or reduction of WWOX expression is associated with breast cancer. Immunohistochemical staining shows strong WWOX expression in the normal human breast tissue.¹⁹ WWOX expression is reduced in 55–63.2% of breast cancer tissues and lost in 29% of breast cancer tissues.^20–22 However, breast tumor tissues have increased expressions of short WWOX variants, when compared to normal breast tissues. The FORIII transcript, which lacks the majority of the WWOX SDR domain, is expressed in 50% of the breast tumors and 90% of the breast cancer cell lines.²³ These results suggest that the WWOX gene, especially the SDR domain of WWOX, may be involved in breast cancer progression. The above studies show that the SDR domain may play a crucial role in the function of WWOX proteins as a breast tumor suppressor.

Further analysis shows that the WWOX expression level is highly positively related with hormone receptor status, but negatively correlated with clinical stages of breast cancer. ER or progestogen receptor (PR) positive cancer has a higher level of WWOX expression than ER or PR negative cancer.²⁴ Stage III breast cancer has higher WWOX expression than stage III breast cancer.²² Ovarian cancer, another hormone-dependent tumor, is associated with the expression of WWOX in the same manner as breast cancer. Normal ovarian tissue samples show consistently strong WWOX expressions while 37% ovarian carcinomas show reduced or undetectable WWOX protein expression levels. Reduced WWOX expressions have been found to be significantly associated with clinical stage IV, negative PR status, and negative ER in ovarian cancer tissues.^25,26 The studies on breast and ovarian cancers indicate that reduced or lost WWOX expression may be involved in the progression of hormone-dependent tumors.

As shown in Table 1, reduced WWOX expression has been found to be associated with basal-like, triple-negative, and invasive breast cancer subtypes.^27–29 Basal-like and triple-negative subtypes are associated with high local recurrence rates, distant metastases, lack of effective targeted therapies, and poor disease-free survival in young women. Distant metastases occur in 28% of triple-negative breast cancers within five and 10 years and the most characteristic sites of metastases include the brain and lungs. Ninety percent of metaplastic breast carcinomas, as well as the majority of medullary carcinomas, consistently show a basal-like phenotype.^30–32 These results indicate that reduced WWOX expression is associated with a poor prognosis of breast cancer patients.

Table 1.

Dysregulation of WWOX expression in breast cancer tissues

Tissues	Technique	Frequency	Ref
Breast cancer	Immunohistochemistry	29% (negative)	22
Breast cancer	Immunohistochemistry	63.2% (reduced)	28
Breast cancer	RT-PCR	55% (reduced)	33
Ductal carcinoma in situ	Immunohistochemistry	68% (reduced)	20
Invasive breast cancer	Immunohistochemistry	77.1% (negative or reduced)	29
Triple negative breast cancer	Immunohistochemistry	90% (negative or reduced)	29

WWOX is associated with cancer metastasis and invasion. WWOX protein expression is lost or reduced in nearly 100% of metastatic breast cancer tissues.²⁸ Reduced protein level of WWOX increases migration of cancer cells through the basal membrane. Integrin α3 is a transmembrane receptor that mediates the attachment between cells and extracellular matrix (ECM) and plays a vital role in migration and tumor invasion. Loss of WWOX expression induces the membranous integrin α3 protein expression and modulates the interaction between cancer cells and ECM, resulting in migration of cancer cells through the basal membrane.³⁴

Taken together, WWOX is lost or reduced in breast cancer tissues, and the expression of WWOX is highly positively related with hormone receptor status, but negatively correlated with clinical stages of breast cancer. Loss or reduction of WWOX expression has significant correlation with breast cancer progression and prognosis.

Molecular function of WWOX in breast cancer

Decreased WWOX expression in breast cancer suggests that loss of WWOX may contribute to the pathogenesis of breast cancer. WWOX has been shown to partner with several proteins involved in the pathogenesis of breast cancer, including activator protein 2γ (AP2γ), erythroblastic leukemia viral oncogene homolog 4 (ErbB4), and mothers against DPP homolog 3 (SMAD3). Figure 1 shows the regulatory effects of WWOX on proteins involved in the pathogenesis of breast cancer. AP2γ is an essential regulator of breast cancer-related genes in vitro and associates with poor prognosis of breast cancer.^35,36 Transiently overexpressed WWOX physically interacts with the PPPY motif of AP2γ via the first WW domain and inhibits the oncogenic activity of AP2γ by sequestering AP2γ in the cytoplasm.⁶ ErbB4 plays an important role in cellular proliferation and differentiation and is involved in the pathogenesis and progression of breast cancer.³⁷ Recent results have suggested that ectopic WWOX is associated with the expression of ErbB4 and inhibits the transactivation function of ErbB4.⁵ SMAD3 is a transcription factor directly phosphorylated and activated by TGF-β type 1 receptor kinase. After phosphorylation, SMAD3 is translocated into the nucleus, where it induces the transcription of cell cycle inhibitors such as CDKN2B (p15) and CDKN1A (p21).³⁸ Transiently overexpressed WWOX can inhibit TGF-β signaling by interacting with SMAD3 and sequestering it in the cytoplasm of breast cancer cells.³⁹ Thus, WWOX can suppress the pathogenesis of breast cancer through down-regulation of some transcription factors. However, TGF-β1 induces re-location of endogenous WOX1 to the nuclei by binding to cell surface hyaluronidase Hyal-2 in murine L929 fibroblasts. The Hyal-2·WOX1 complexes relocate in the nuclei and further recruits Smad4, enhancing the Smad promoter activity.⁴⁰

Figure 1.

Regulatory mechanisms of WWOX in breast cancer. WWOX binds PPxY domain-containing proteins and sequesters them in the cytoplasm, suppressing their transcriptional functions, including ErbB4, AP-2γ, SMAD3, and WBP2. Tyr33 phosphorylation of WWOX promotes the WW–PPxY interaction. Tyr287 phosphorylation process is associated with WWOX polyubiquitination and degradation. (A color version of this figure is available in the online journal.)

As is mentioned earlier, ectopic WWOX suppresses the transcriptional functions of AP2γ, ErbB4, and SMAD3 by sequestering them in the cytoplasm of cultured cell lines. However, endogenous WWOX suppresses the transcriptional functions of CREB by binding to CREB in rats. Thus, multiple modes may be involved in WWOX regulation of transcription factors. Sequestering transcription factors in the cytoplasm may be specific for WWOX regulation of breast cancer-related transcription factors. Further studies are needed to prove this assumption.

Loss of WWOX correlates with resistance to endocrine therapy for breast cancer

Endocrine therapy for breast cancer has been used for more than a century. Endocrine therapy inhibits the accelerating effect of ER signaling pathway on cell proliferation by suppressing the estrogen production and restraining the ER activity.^41,42 Tamoxifen is one of the most widely used endocrine therapy drugs for breast cancer. It functions as a selective estrogen-receptor modulator by blocking estrogen from binding to its receptor in breast cancer cells. Tamoxifen reduces the risk of recurrence of early stage breast cancers in premenopausal and postmenopausal women.^43,44 However, five years of tamoxifen is presently established as the standard duration for endocrine therapy for breast cancer. Due to the development of drug resistance, prolongation of the therapy cannot enhance the treatment efficacy. Thus, it is important to investigate the mechanism underlying tamoxifen resistance in breast cancer.

Gothlin et al. first discovered the relationship between WWOX expression and tamoxifen treatment. They examined 912 paraffin-embedded breast cancer tissues by immunohistochemistry. The results indicated that high expression of WWOX was associated with better outcome of tamoxifen treatment.⁴⁵ In breast cancer cell lines, loss of WWOX expression induces the release of Ap2γ from the cytoplasma to the nucleus and the up-regulation of human epidermal growth factor receptor 2 (Her2). Increased Her2 promotes the growth of cancer cells against endocrine therapy for breast cancer and leads to tamoxifen resistance.^6,46,47 The WW domain binding protein-2 (WBP2), a co-activator of ERα, may be another key regulatory protein for tamoxifen resistance. WBP2 can activate the ER transactivation pathway and promote cell proliferation. WWOX physically interacts with WBP2 and suppresses ER transactivation pathways^48,49 (Figure 1). Thus, loss of WWOX expression can result in tamoxifen resistance by up-regulating ER and Her2 transcriptional activities.

Conclusion and future directions

Fragile site gene Wwox has a critical role in the pathogenesis and endocrine therapy of breast cancer. Wwox is consistently lost or reduced in the majority of breast cancers, and Wwox KO mice show rudimentary mammary epithelium, defects in ductal structure, and breast cancer.^17,22,24 Loss of WWOX expression leads to tumorigenesis, cancer progression, and resistance to endocrine therapy for breast cancer. WWOX may affect tumorigenesis and drug resistance by inhibiting activities of some transcription factors or transcription activators, including ErbB4, AP-2γ, SMAD3, and WBP2. Reduced WWOX expression is also significantly correlated with clinical stages and poor survival. Therefore, WWOX can be used as a biological marker for progression and prognosis of breast cancer.

The function of WWOX SDR domain is still obscure. Is the dehydrogenase/reductase activity of WWOX associated with tumorigenesis? Is WWOX SDR domain related to the functions of hormone receptors and endocrine therapy resistance? Exploration of the functions of WWOX SDR domain may provide more clues to the roles of WWOX in progression of breast cancer and resistance to endocrine therapy.

Authors’ contributions

All authors participated in the writing, review, and editing of this manuscript.