Phosphorylation/de-phosphorylation in specific sites of tumor suppressor WWOX and control of distinct biological events

Short abstract

Abnormal differentiation and growth of hematopoietic stem cells cause the development of hematopoietic diseases and hematopoietic malignancies. However, the molecular events underlying leukemia development are not well understood. In our recent study, we have demonstrated that calcium ionophore and phorbol ester force the differentiation of T lymphoblastic leukemia. The event involves a newly identified IκBα/WWOX/ERK signaling, in which WWOX is Ser14 phosphorylated. Additional evidence also reveals that pS14-WWOX is involved in enhancing cancer progression and metastasis and facilitating neurodegeneration. In this mini-review, we update the current knowledge for the functional roles of WWOX under physiological and pathological settings, and provide new insights regarding pS14-WWOX in T leukemia cell maturation, and switching the anticancer pY33-WWOX to pS14-WWOX for cancer promotion and disease progression.

Impact statement

WWOX was originally designated as a tumor suppressor. However, human newborns deficient in WWOX do not spontaneously develop tumors. Activated WWOX with Tyr33 phosphorylation is present in normal tissues and organs. However, when pY33-WWOX is overly induced under stress conditions, it becomes apoptotic to eliminate damaged cells. Notably, WWOX with Ser14 phosphorylation is upregulated in the lesions of cancer, as well as in the brain hippocampus and cortex with Alzheimer’s disease. Suppression of pS14-WWOX by Zfra reduces cancer growth and mitigates Alzheimer’s disease progression, suggesting that pS14-WWOX facilitates disease progression. pS14-WWOX can be regarded as a marker of disease progression.

Introduction

The skin and immune systems are the first and second lines of defense that protect human body from microbial or viral infection.¹ This highly cooperative system comprises the innate and adaptive immune systems. The mucosal system possesses the complement proteins, inflammatory cytokines, and immune cells of the innate immune system to protect from pathogen invasion. The adaptive immune system provides a long-lasting and highly specific immune reaction. In this system, T- and B-lymphocytes are activated by the antigen-presenting cells (APCs) and/or dendritic cells (DCs). Both cells recognize and process the specific foreign antigen and execute its immune functions, including T-cell mediated cytotoxicity and specific antibody-dependent elimination of the particular antigen or pathogen.

Normal and aberrant T cell development

In murine, T-cell development starts from the differentiation of the bone marrow pluripotent hematopoietic stem cells (HSCs). These unique HSCs differentiate into the precursor T-cells and migrate to the thymus for further maturation. In the special microenvironment of thymic cortex, the precursor T-cells (CD44^low) differentiate into the CD4⁻CD8⁻ double negative (DN) cells and then go through four stages: I, CD44⁺CD25⁻; II, CD44⁺CD25⁺; III, CD44⁻CD25⁺ with the presence of pre-T-cell receptor (pre-T-cell receptor) expression; IV, CD44⁻CD25⁻.² Post these differentiation processes, T-cells express CD4 and CD8 molecules in the cell membrane and become CD4⁺CD8⁺ double positive (DP) cells. Later, those DP T-cells are subjected to positive selection to eliminate TCR-deficient cells in the cortex and negative selection to eliminate self-reactive cells in the medulla.^3–6 The surviving T-cells become matured, including CD4⁺, CD8⁺ single positive (SP), regulatory T cells (Treg), and natural killer-like T cells (NKT). These mature T cells circulate in the blood stream or reside in the lymph nodes to execute their respective immune functions. In humans, T-cell development starts from the differentiation of the CD34⁺, CD7⁺, and CD5⁺ HPCs, followed by migrating to the thymus and going through the developmental processes similar to that of the murine system, thus leading to the production of functional CD4 or CD8 T-cells.^7–9 However, abnormal differentiation of T-cells causes leukemia, which is an aggressive hematologic tumor. Translocation and aberrant expression of transcription factors or recombinant kinases attribute to the development of the disease.^10–12 Participation of nuclear factor kappaB (NF-κB) has been implicated in the development of leukemia.^13–15

Role of NF-κB in T cell development

NF-κB participates in the T-cell differentiation and positive/negative selection.^3,16–18 The NF-κB protein family consists of c-Rel, p65 (RelA), RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). During activation, the highly conserved N-terminal Rel homology (RHD) domain mediates its binding to the κB elements in the DNA sequence. Based on the differences at the C-terminal domain, the class I NF-κB protein family includes p50/p105 and p52/p100. Each of them contains an ankyrin repeat-containing transrepression domain (TRD), and represses the transcription of κB elements. The class II members RelA, RelB, and c-Rel bear a tranactivation domain (TAD), which mediates active transcription.¹⁹ NF-κB activation is needed for cell survival and tissue inflammation and is regulated in a fine-tuned manner. IκBα is an inhibitor of NF-κB and belongs to the IκB protein family, including IκBβ, IκBγ, IκBε, and Bcl-3.^20,21 IκBα participates in immune cell development. Newborn IκBa^−/− mice survive for less than 10 days and possess an altered population of double-positive thymocytes and increased populations of granulocytes and macrophages.²² Thymocyte development toward maturation is severely impaired in the transgenic mice overexpressing truncated IκBα. This truncated form lacks 36 amino acids at the N-terminus, and yet possesses an increased inhibitory activity against NF-κB. Nevertheless, the CD8 lineage of thymocytes is most affected.^23,24 Together, IκBα is crucial in immune cell development, whereas the molecular event needs further elucidation.

Tumor suppressor WW-domain containing oxidoreductase WWOX

The rationale for focusing on WWOX is its control of immune cell maturation,²⁵ cancer progression,²⁶ and neuronal injury and degeneration.²⁷ We will update the current knowledge with immune cell maturation and mention the documented role of WWOX in cancer and neural diseases.²⁸ WWOX/Wwox gene encodes a tumor suppressor WW domain-containing oxidoreductase, known as human WWOX or FOR and murine WOX1.^29–31 Human WWOX gene is located on the common fragile site FRA16D on chromosome 16q23.2. This gene encodes approximately 1 million bases and contains nine exons and transcribes a spliced 2.2 kb mRNA. Most recently, multiple PARTICLE (Gene PARTICL ‘Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) triplex clusters are found in the WWOX gene.³² PARTICLE affects remote loci in the human genome and the ability of lncRNAs to regulate the expression of numerous genes. WWOX protein possesses 414 amino acids and has two conserved N-terminal WW-domains, a C-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, and a nuclear localization signal (NLS) in between the WW domains.^29–31 A proapoptotic D3 region is located at the C-terminal tail.^33,34

WWOX gene alterations in cancer, metabolic disorders, and neural diseases

Null mutations in the WWOX/Wwox gene lead to complete loss of protein expression. Loss of WWOX expression has been reported in breast,^35–38 esophageal,^39–41 lung,^42,43 ovarian,^44,45 colon,⁴⁶ prostate,^47,48 and gastric⁴⁹ carcinomas. Alternative transcripts of WWOX are found in breast cancer.⁵⁰ However, truncated proteins of less than 41 kDa are not stable. They tend to be readily subjected to proteosomal degradation. Epigenetic modification of the CpG island of WWOX gene by methylation that leads to null or altered expression has been shown in breast cancer and other types of cancer.⁵¹ Loss of WWOX gene also contributes the development of osteosarcomas in Wwox heterozygous mice and lung papillary carcinoma in the Wwox knocked out mice.⁵² Heterozygous Wwox mice are more susceptive to chemical mutagen-induced gastric cancer and lymphoma,^49,52 implying the crucial role of WWOX in the anti-tumor response. The mutagens include N-nitrosomethylbenzylamine and ethyl nitrosourea. Conditional Wwox knockout mice suffer severe metabolic defect, growth retardation, reduced bone volume, hypocapnia, impaired hematopoiesis, leukopenia, and splenic atrophy.⁵³ Altered or loss expression of WWOX protein has been found in 51% of leukemia patients and 55% of leukemic cell lines.⁵⁴

Null mutations of WWOX/Wwox in humans, mice, and rats lead to severe neural diseases (e.g. epilepsy, microcephaly, retinal degeneration, ataxia, and etc.), metabolic disorders associated with lipid, cholesterol and glucose metabolism, and early death.^27,28,55,56 However, no spontaneous tumor growth is shown in the newborns of humans and rats. Studies have been limited using WWOX/Wwox-deficient animals due to death of embryos in utero. WWOX loss leads to alterations in cancer cell adhesion to the extracellular matrix, and this affects cell migration and metastasis.⁴⁴ Also, under WWOX-deficiency, normal protein conformation may be subjected to changes, which results in tumor growth inhibition. Indeed, when WWOX is downregulated, a cascade protein aggregation occurs for leading to amyloid β formation and tau tangle generation.^{27,28,57–60} The protein cascade includes TRAPPC6AΔ (TPC6AΔ; Trafficking protein particle complex 6A delta), TIAF1 (TGFβ1-Induced Anti-Apoptotic Factor 1), and SH3GLB2 (SH3 Domain Containing GRB2 Like, Endophilin B2).^57,60–62 Oddly, there is an inverse relationship between the occurrence of Alzheimer’s disease (AD) and cancer.^63,64 The greater the extent of protein aggregation in AD, the less likely the incidence of cancer. Cancer cell survival is not affected by intracellular protein aggregation.⁶¹ However, cancer cells in the brain induce neuronal death due to TIAF1 aggregation in the neurons.⁶¹ We have reported cancer-induced neurodegeneration in mice.⁶² However, cancer patients are unlikely to survive long enough to develop neurodegeneration.

In the lethal dwarfism and epilepsy lde/lde rat, there is a 13-bp deletion in the exon 9 of Wwox gene.⁶⁵ The deletion causes a frameshift of the Wwox gene and the produced WWOX protein has an altered amino acid sequence at the C-terminus. Wwox knockout mice survive for three weeks,^58,66 and the lde/lde rats die before maturation (within 3 to 12 weeks). No spontaneous tumor growth is shown in the lde/lde rat.⁶⁵ Moreover, these rats have accumulated the extracellular vacuoles in the hippocampus,^65,66 suggesting the occurrence of progressive neuronal degeneration and eventual death. WWOX is significantly downregulated in AD patients.^59,67 Downregulation of WWOX in the hippocampi starts to occur in the mid-aged humans, and this may lead to slow aggregation of TRAPPC6AΔ and TIAF1 for caspase activation and mitochondrial damage, followed by degradation of amyloid precursor protein (APP), amyloid β formation, and tau tangle formation as shown in AD patients at age 70 and greater.^57–59

lde/lde rats exhibit a significantly decreased number of spermatocytes, abnormal differentiation of Leydig cells, and low testosterone concentration in the plasma.⁶⁵ WWOX binds sex steroid hormones such as estrogen and androgen, and may act as a cytosolic hormone receptor.^68,69 Relocation of the estrogen/WWOX complex, along with p53, to the nucleus allows transcription of specific genes to support cell growth and differentiation. Without WWOX, it is not surprising to observe the defect in spermatogenesis.

Upregulation of WWOX in the early stage of cancer progression

Much less attention has been paid to the WWOX expression in the hyperplasia tissues prior to progression toward cancerous and metastatic stages. During the acute phase of UVB irradiation-induced skin squamous cell carcinoma (SCC) in hairless mice, WWOX is significantly upregulated and activated with Tyr33 phosphorylation in 24 h.⁷⁰ Presumably, activated WWOX struggles to block cancerous progression, eliminate damaged cells by apoptosis, and suppress the inflammatory functions of NF-κB.⁷⁰ Later, during the chronic phase, UVB-treated mice develop cutaneous SCCs in three months, with significant downregulation of WWOX and its activated form. Similarly, breast cancer progression to a premetastatic state is associated with WWOX upregulation and activation, followed by significant downregulation or absent expression during metastasis.⁶⁹ Estrogen participates in the upregulation and activation of WWOX.⁶⁹

WWOX structure, signaling networks, and physiological and pathological events

WW domain serves in protein/protein interactions in many signal pathways (e.g. Hippo pathway). WW domain is a compact protein module ranging from 35 to 40 amino acids and contains two conserved tryptophan (W) residues that are spaced apart by ∼20 amino acids.^71–75 There are at least 52 WW domain-containing proteins identified in the human proteome (Supplementary Table 1), and more than 10,000 among all species. Both WW domain and Src homology domain 3 (SH3) recognize the proline-rich region of protein ligands. However, no consensus sequence in the binding targets is shown by both domains.^72,73 There are at least four identified subgroups of WW domain. Each WW domain recognizes the specific proline-rich sequence.^74,75

WWOX interacts with one or more than one proteins in each signaling paths (Figure 1). The N-terminal WW domains of WWOX, belonging to the Group I WW domain, preferably bind PPxY- or PPPY-containing proteins.^26,74–77 PPxY-motif proteins include p73,⁶⁶ AP-2γ,⁷⁸ ErbB4,⁷⁹ cJun,⁸⁰ Runx2,⁴³ Ezrin,⁸¹ TMEM207,⁸² and others (Figure 1). The binding interaction involves the first WW domain with each target protein, in which the secondary WW domain assists the first WW domain for the binding.⁸³ When WWOX is transiently overexpressed in the cytoplasm, the ectopic WWOX strongly binds the aforementioned transcription factors and blocks their oncogenic activity for suppressing cancer cell survival. Unfortunately, this does not appear to be the case in vivo. Metastatic cancer cells frequently lose the expression of WWOX.^26,69,77 Also, when rats are subjected to sciatic nerve dissection, endogenous WWOX is rapidly upregulated during the acute phase and binds and relocates together with transcription factors (e.g. CREB and cJUN) to the nucleus to support neuronal survival or cause death.⁸⁰ WW domain-containing Yes-associated protein (YAP) interacts with tyrosine kinase ErbB4 and p73, and acts as a coactivator for transcription. Ectopic WWOX inhibits the nuclear translocation and transcriptional activity of both proteins.⁷⁹ To control YAP activation, the upstream tumor suppressors LATS1/2 kinases form complexes with the WW domain-containing N-terminus of YAP.⁸⁴ Together, WWOX participates in numerous physiological events due to its interactions with functional proteins.

Figure 1.

WWOX in signaling networks. (a) WWOX is composed of two N-terminal WW domains and a C-terminal SDR domain. In a native-folded conformation, the WW domains bind the SDR domain. Under stress condition, WWOX becomes Tyr33 phosphorylated, and the protein unfolded.⁵⁹ An NSYK motif in the SDR domain binds estrogen or androgen.^68,85 The C-terminal D3 tail is proapoptotic.³³ D3 binds TRAPPC6AΔ (TPC6AΔ) to prevent aggregation in the brain cortex such as in Alzheimer’s disease.^57–59 However, once activated, the unfolded WWOX utilizes its first WW domain to bind TPC6AΔ.^57–59 (b) A schematic pie graph shows the representative signaling networks that WWOX is involved in. WWOX interacts with many proteins in each signaling network. See text for details. (A color version of this figure is available in the online journal.)

WWOX expression, activation and degradation

WWOX expression is induced by hyaluronidases PH-20, HYAL1 and HYAL2,³¹ and apoptotic stresses.^31,85–88 Antisense mRNA, dominant negative, and small interfering RNA (siRNA) suppress WWOX and protect cells from apoptosis.^31,70,85,88 When cells are exposed to tumor necrosis factor (TNF), anisomycin, UV irradiation, transforming growth factor β (TGF-β), and other apoptotic stresses, WWOX is upregulated and readily becomes activated via Tyr33 phosphorylation.⁸⁷ Tyrosine kinase Src appears to mediate the Tyr33 phosphorylation.⁶⁶ Activated pY33-WWOX is upregulated in vivo during neuronal damage,²⁷ retinal degeneration,⁸⁹ traumatic brain injury,⁹⁰ initial stage of skin and breast cancer progression,^69,70 and pterygium progression and recurrence.⁹¹ However, pY33-WWOX is downregulated during the progression of AD.^27,67 In small cell lung cancer, Bmi1 (B Lymphoma Mo-MLV Insertion Region 1 Homolog) targets the expression of WWOX to facilitate cancer cell growth.⁹² Notably, a novel fusion protein of PVT1-WWOX, resulting from fusion of PVT1 exon 1 to WWOX exon 9, is present in the 8q24 rearrangement-positive multiple myeloma.⁹³

The activated pY33-WWOX gains an increased capability in binding a broad spectrum of proteins in various signaling pathways such as Hyal-2, p53, Smad4, ERK, JNK, GSK3β, IκBα, p73, and many others.^85,87,94 Anisomycin and UV light activate JNK1 for binding and functionally blocking pY33-WWOX.⁸⁷ Also, UV induces the binding of pY33-WWOX with pS46-p53, and both proteins cause apoptosis in a synergistic manner.^31,87 While p53 and WWOX are partners in apoptosis and cancer suppression,^85–88 loss of p53 and WWOX drives the development of osteosarcoma in a double knockout mouse model.⁹⁵ Estrogen also initiates the binding of pY33-WWOX with pS15-p53, and that apoptosis occurs if the concentration of estrogen is raised up to micromolar levels.⁶⁸ Intracellular activated Cdc42-associated kinase (ACK1) negatively regulates WWOX by phosphorylating Tyr287 in prostate cancer, which leads to WWOX polyubiquitination and proteosomal degradation.⁹⁶ Overall, activated WWOX with Tyr33 phosphorylation controls the progression of cancer and AD in vivo.

WWOX in signaling networks

Among others (Figure 1), WWOX induces human ovarian cancer cell detachment from the extracellular matrix and thus leads to apoptosis by reducing the expression of integrin α3.⁴⁴ The hepatocyte growth factor (HGF)/Met pathway is deregulated in advanced breast cancer. Ectopic WWOX inhibits the nuclear translocation and transcriptional activity of Met.⁹⁷ WWOX is also an inhibitor of the Wnt/β-catenin pathway. High β-catenin transcriptional activity correlates with poor prognosis of breast cancer patient. WWOX inhibits this pathway by preventing the nuclear translocation of Dishevelled and decreasing the stability of β-catenin.⁹⁸ In parietal cell, binding of Ezrin with WWOX is essential for the membrane-cytoskeleton remodeling and H,K-ATPase membrane translocation and insertion during cell activation.⁸¹ The small integral membrane protein of the lysosome/late endosome (SIMPLE) binds WWOX in the Golgi apparatus, whereas the functional implication is unknown.⁹⁹ Overall, the aforementioned studies showed that transiently overexpressed WWOX sequesters many transcription factors in the cytoplasm to render cancer growth inhibition.

Endogenous WWOX/MEK1 complex is a molecular switch for cancer cell death

Endogenous WWOX and its binding partners exhibit physiological effects in vivo. For example, WWOX physically binds MEK1 in the cytoplasm and, in part, in the lysosomes of leukemia T cells.³⁴ Dissociation of the WWOX/MEK1 complex by phorbol myristate acetate (PMA) leads WWOX to relocate to the mitochondria to induce apoptosis, and MEK1 to the lipid rafts.³⁴ Conceivably, WWOX/MEK1 is a switch for turning on/off death in cancer cells. Appropriate chemicals can be designed to dissociate the WWOX/MEK1 complex for causing cancer death in vivo.

WWOX in the bubbling cell death

We have recently discovered a novel type of cell death, termed bubbling cell death (BCD), caused by UV irradiation and cold shock.^100,101 This study concerns the devastating effect of frostbite. When ambient temperature surrounding dying cells drops from 37°C to 22°C or down to 4°C, apoptosis stops. Cells choose to die by BCD. During BCD, each cell generates “a nitric oxide-containing bubble” from the nucleus. The bubble relocates to the cell surface and is then released from the cell. Ultimately, the cell dies. Unlike apoptosis, there are no phosphatidylserine flip-over, caspase activation for mitochondrial apoptosis, damage to Golgi complex, and chromosomal DNA fragmentation in BCD. Rapid release of extracellular vesicle/exosome-like materials is found in cells undergoing BCD. When cells receive UV irradiation, followed by brief cold shock at 4°C, many cytosolic and membrane proteins rapidly relocate to the nucleus. Among these, UV-induced pY33-WWOX and pS46-p53 are essential for executing BCD. As a protective mechanism, UV activates TNF receptor-associated factor-2 (TRAF2) to antagonize cell death mediated by activated WWOX and p53. Detailed interactions among WWOX, p53, TRAF2, and other proteins are being determined.^28,100,101

Hyal-2/WWOX/Smad4 signaling for hyaluronan and TGF-β

WWOX is anchored, in part, in the membrane/cytoskeleton area by membrane Hyal-2,¹⁰² TMEM207,⁸² and Ezrin.⁸¹ WWOX binds the C-terminal PPxY motif in the transmembrane protein 207 (TMEM207) and this binding appears to nullify the tumor suppressor function of WWOX and facilitates the invasiveness of gastric signet-ring cell carcinoma. In our recent review,⁹⁴ we have described that both hyaluronan and TGF-β1 utilize the non-canonical Hyal-2/WWOX/Smad4 signaling to drive the SMAD promoter activity and control cell growth and death.^76,94,102 For example, during traumatic brain injury in rats, dramatic accumulation of the Hyal-2/WWOX complex in the nuclei of apoptotic neurons occurs.⁹⁰ In vitro analysis revealed the indispensable role of Hyal-2 and WWOX in conferring cell death.⁹⁰ Naturally occurring high molecular weight hyaluronan does not effectively induce cell death, whereas hyaluronan signals the ectopic Hyal-2/WWOX/Smad4 complex to cause BCD,^90,100,101 suggesting that when the level of endogenous Hyal-2/WWOX/Smad4 complex is upregulated, extracellular matrix hyaluronan may exert cell death via the Hyal-2/WWOX/Smad4 signaling.

C1q/WWOX in the non-inflammatory anticancer response

WWOX is responsive to activation by serum complement C1q of the humoral immune system to limit cancer growth.^103–105 The classical complement system is involved in the antibody-dependent inflammatory response against invading pathogens. When exogenous C1q interacts with its membrane receptor in prostate and neuroblastoma cancer cells, the binding transmits a signal to WWOX to carry out an untypical type of apoptosis in cancer cells in vitro.¹⁰³ In vivo experiments also showed the efficacy of complement C1q in curbing the growth of breast cancer cells via WWOX activation.^103–105 Presence of C1q in organs prevents them from carcinogenesis progression.¹⁰³ That is, downregulation of C1q in the prostate enhances the prostate hyperplasia toward cancerous formation due to lack of WWOX activation.¹⁰³ Whether C1q receptor binds Hyal-2 that triggers the downstream WWOX/Smad4 signaling for executing cancer cell death remains to be established.

WWOX in antiviral response

WWOX is involved in host defense against viral and bacterial infections.¹⁰⁶ Human T-Lymphotropic Virus 1 (HTLV-1) contributes to the development of adult T-cell leukemia. HTLV-1-encoded oncogenic Tax protein physically binds WWOX that regulates the NF-κB signaling.¹⁰⁷ WWOX suppresses the Tax-induced activation of canonical NF-κB signaling by blocking IKKα-mediated phosphorylation of NF-κB at the subunit RelA. However, Tax represses WWOX expression via the non-canonical NF-κB signaling mediated by p100/p52.

Epstein-Barr virus (EBV) participates in the development of nasopharyngeal carcinoma and B-cell lymphomas.^108,109 Oncoprotein latent membrane protein 2A (LMP2A) encoded by EBV supports cancer growth, survival, and metastasis. The first WW domain of WWOX physically binds the PPPPY motifs in LMP2A, and the binding facilitates the ERK-Fra-1-MMP9 signaling, increases MMP9 production, and promotes cancer metastasis.¹¹⁰ In the absence of LMP2A, WWOX exerts negative effects on the MEK1-ERK signaling and suppresses cancer growth and invasion, suggesting that LMP2A facilitates viral proliferation via inhibition of WWOX function.¹¹⁰

WWOX in chromosomal stability

Loss of WWOX frequently links to tremendous chromosome instability and gene deletion, which might provide a selective advantage for neoplasm.^111–113 There are at least three mechanisms that participate in repairing damaged DNA, namely non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). NHEJ uses a short nucleotide sequence as a guide to anneal with the other strand of DNA.¹¹⁴ Owing to this annealing which is not specific to the homologous sequence, NHEJ frequently causes nucleotide deletion or translocation. Similar to NHEJ, MMEJ trims the breaking ends in both strands and anneals both stands through the microhomologous region present in both ends. However, this mechanism causes more nucleotides loss and contributes to extend nucleotide deletion. In contrast, HR uses the homologous sequence in the sister chromatid as the template to repair the damaged strand.

When double-strand break (DSB) occurs, WWOX associates with phosphorylated ataxia telangiectasia-mutated (ATM), triggers the downstream damage responses, and repairs damaged chromosomes. While Lys63-ubiquitination promotes the stability and nuclear translocation of WWOX, ubiquitination at Lys274 plays a critical role in the activation of WWOX to associate with ATM in the MMEJ.^115,116 WWOX modulates the activity of ATM and Rad3-related protein (ATR) in response to single-strand break (SSB).¹¹⁷ Intriguingly, loss of WWOX enhances HR upon cisplatin and radiation treatment in breast cancer. Mechanistically, WWOX competes with Rad50, one of the components of MRN complex, for binding to Brac1 (Breast cancer 1).¹¹⁸ Therefore, WWOX hampers the HR as mediated by Brac1 and promotes NHEJ in WWOX-expressing cells.¹¹⁸

Inhibition of WWOX signaling by JNK1 and Zfra

Both JNK1 and Zfra are known inhibitors of WWOX signaling. JNK1 blocks the apoptotic function of p53 and WWOX.^85,86 Zfra, known as zinc finger-like protein that regulates apoptosis, is a 31-amino-acid protein capable of counteracting apoptosis mediated by WWOX and p53.³³ Synthetic Zfra peptides, Zfra1–31, and Zfra4–10, prevent and block the growth of many types of cancer cells,¹¹⁹ and restore memory loss in triple transgenic mice by blocking the aggregation of TRAPPC6AΔ (TPC6AΔ), SH3GLB2, tau and amyloid β, as well as inflammatory NF-κB activation.⁶² Zfra covalently interacts with intracellular proteins and accelerates their degradation independently of ubiquitination and proteosomal degradation.⁶² Zfra binds WWOX to specific sites in the WW and SDR domains at the N- and C-termini, respectively.³³ Whether Zfra accelerates WWOX degradation to abolish signaling remains to be established.

WWOX in forced T-cell maturation via a novel IκBα/WWOX/ERK signal pathway

Although WWOX loss is frequently found in neoplasm, clinical findings point out that WWOX loss in leukemia is not as frequent as other non-hematopoietic neoplasms. Indeed, loss of WWOX protein in acute lymphoblastic leukemia (ALL) patients is due to promoter hypermethylation of WWOX gene.^120,121 FHIT and p73 genes are also hypermethylated in ALL. Restoration of WWOX gene reduces leukemic tumorigenesis.¹²² Phorbol ester-mediated dissociation of WWOX/MEK1 complex leads to leukemia cell death.³⁴ WWOX gene is located on the chromosomal fragile site FRA16D, and alterations in this site lead to loss or alterations of WWOX protein as shown in leukemia patients and leukemic cell lines.^54,123 By gene trap technology, Wwox gene knockout mice are susceptible to develop B-cell lymphomas.¹²⁴ While WWOX is involved in maintaining chromosomal stability, loss of WWOX may contribute to DNA breaks and fusion of chromosomes such as PML/RARα for promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARα) genes in acute promyelocytic leukemia (APL).¹²⁵

We have recently identified a novel IκBα/WWOX/ERK signal pathway for forced T cell maturation (Figure 2(a)).²⁵ Upon exposure to calcium ionophore A23187 and phorbol ester (IoP), human ALL MOLT-4 cells undergo terminal maturation toward T cell phenotype via the IκBα/WWOX/ERK signaling (Figure 2(a)).²⁵ In resting MOLT-4, an endogenous pY33-WWOX/IκBα/ERK complex is present in the cytoplasm and mitochondria. Mapping by co-immunoprecipitation and yeast two-hybrid analysis reveals that pY33-WWOX binds ERK and non-PEST (domain rich in proline, glutamate, serine and threonine) area of IκBα.²⁵ IoP rapidly causes WWOX de-phosphorylation at Tyr33 and Tyr287. Meanwhile, phosphorylation of ERK and IκBα occurs in the complex in 5 min or less. WWOX then becomes Ser14 phosphorylated. pS14-WWOX causes instability of the complex. p-IκBα undergoes polyubiquitination/proteosomal degradation, and ERK de-phosphorylation occurs in the next 3–12 h. Later, a portion of WWOX and ERK re-associates and translocates to the nucleus to induce the expression of T cell maturation antigens CD3 and CD8 in 15–24 h. Inhibition of ERK phosphorylation by U0126 or restriction of IκBα degradation by MG132 abolishes the MOLT-4 maturation.²⁵ In addition to WWOX, specific site phosphorylation in the WW domain (e.g. in Pin1 and YAP1) that confers biological functions has been shown.^126,127 However, switches in phosphorylation and de-phosphorylation among specific sites in WW domain and non-WW domain areas that alter protein functions are largely unknown.

Figure 2.

pS14-WWOX in promoting T cell differentiation and cancer progression and neurodegeneration. (a) During forced leukemia T cell maturation by calcium ionophore and phorbol ester, WWOX becomes dephosphorylated at Tyr33 in the IκBα/WWOX/ERK complex, followed by phosphorylation at Ser14.²⁵ Translocation of WWOX and ERK to the nucleus results in induction of leukemia T cell maturation in 16–24 h.²⁵ (b) Activated WWOX with Tyr33 phosphorylation is proapoptotic.^85,87,88 WWOX with Ser14 phosphorylation can be found in the lesions of cancer and brain hippocampus and cortex with Alzheimer’s disease.⁶² Suppression of pS14-WWOX by Zfra reduces cancer growth and mitigates AD symptoms,^62,119 suggesting that pS14-WWOX facilitates disease progression. (A color version of this figure is available in the online journal.)

Potential role of pS14-WWOX in disease progression

Whether pS14-WWOX drives normal T cell differentiation and maturation is unknown. We have shown that pS14-WWOX is involved in disease progression. For example, nude mice receive subcutaneous inoculation with melanoma B16F10 cells in both flanks. Two months later, tumors grow up to 2000–3000 mm³ in the mice. There are occurrences of neurodegeneration in the hippocampus, amyloid plaque formation in the cortex, and melanoma infiltration in the lung.⁶² However, when nude mice receive Zfra peptide injections first, followed by inoculating B16F10 cells, cancer cell growth and metastasis are blocked.⁶² In the brain, no neurodegeneration and plaque formation are found. Zfra inhibits pS14-WWOX expression, thus resulting in suppression of cancer growth and neurodegeneration. That is, pS14-WWOX expression positively correlates with the melanoma metastasis to the lung and neurodegeneration in the brain. Zfra does not suppress the protein level of WWOX and its Tyr33 phosphorylation in control and Zfra-treated mice. Clearly, there is a positive correlation between the expression of pS14-WWOX and the progression of cancer growth and neurodegeneration in the hippocampus and plaque formation in the cortex.

Concluding remarks

When intracellular WWOX is Tyr33 phosphorylated, cells are self-protected. pY33-WWOX functions in blocking cancer growth and supporting normal neuronal physiology (Figure 2(b)).⁸⁶ Substantial evidence shows that pY33-WWOX inhibits tumor growth in vitro and in vivo. However, down-regulation of pY33-WWOX occurs in the hippocampi in AD patients.⁶⁷ This downregulation may start to occur in the mid-aged humans, which facilitates the activation of a cascade of protein aggregation that allows generation of amyloid β plaques and tau tangles in the AD brains.^27,57–60 Thus, pY33-WWOX runs against neurodegeneration. In stark contrast, pS14-WWOX supports cancer growth and AD progression (Figure 2). Whether pS14-WWOX works alone or together with other proteins to promote disease progression remains to be established. Overall, the tumor suppressor function of endogenous WWOX is abolished upon acquiring Ser14 phosphorylation. Suppression of endogenous pS14-WWOX expression by Zfra results in inhibition of tumor growth and restoration of memory loss in triple transgenic mice for AD.⁶² That is, one of the mechanisms by which Zfra prevents and blocks cancer growth is due to its suppression of WWOX phosphorylation at Ser14. Nevertheless, endogenous pS14-WWOX is needed for forcing maturation of T cell leukemia,²⁵ which is a conceptual advance for leukemia therapy.

Authors’ contributions

The manuscript supported, in part, SSH graduation for his Master degree. Both SSH and NSC contributed to manuscript writing and discussion and proof reading.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was supported by the Ministry of Science and Technology, Taiwan (MOST 105–2320-B-006–046, 105–2320-B-006–036, 106–2320-B-006 –017, and 106–2320-B-006 –061 for NSC), and the Department of Defense, USA (W81XWH-08–1–0682 for NSC). Publication cost was supported in part by the China Medical University.