WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners

Tumor suppressor WWOX inhibits cancer growth and retards Alzheimer’s disease (AD) progression. Supporting evidence shows that the more strongly WWOX binds intracellular protein partners, the weaker is cancer cell growth in vivo. Whether this correlates with retardation of AD progression is unknown. Two functional forms of WWOX exhibit opposite functions. pY33-WWOX is proapoptotic and anticancer, and is essential for maintaining normal physiology. In contrast, pS14-WWOX is accumulated in the lesions of cancers and AD brains, and suppression of WWOX phosphorylation at S14 by a short peptide Zfra abolishes cancer growth and retardation of AD progression. In parallel, synthetic Zfra4-10 or WWOX7-21 peptide strengthens the binding of endogenous WWOX with intracellular protein partners leading to cancer suppression. Indeed, Zfra4-10 is potent in restoring memory loss in triple transgenic mice for AD (3xTg) by blocking the aggregation of amyloid beta 42 (Aβ42), enhancing degradation of aggregated proteins, and inhibiting activation of inflammatory NF-κB. In light of the findings, Zfra4-10-mediated suppression of cancer and AD is due, in part, to an enhanced binding of endogenous WWOX and its binding partners. In this perspective review article, we detail the molecular action of WWOX in the HYAL-2/WWOX/SMAD4 signaling for biological effects, and discuss WWOX phosphorylation forms in interacting with binding partners, leading to suppression of cancer growth and retardation of AD progression.


Introduction
The protein-protein interaction network supports normal physiology for cell survival and death [1][2][3]. Alteration of a single component or multiple components in the signaling network may result in enhanced or reduced cancer cell survival. That is, the flow of the altered signaling pathway is redirected or shut down unexpectedly, or runs uncontrollably. The strength of protein-protein binding interactions during signaling in cells is affected by environmental factors such as local ionic strength and pH, amino acid compositions in the binding motif(s) or domain(s), competitors in the protein-binding cascade, and transient changes in the protein conformation upon interacting with another binding partner [1][2][3]. Under physiological conditions, a signaling pathway is initiated by an extracellular trigger or a ligand to allow the normal operation of protein-protein binding and progression of the signaling cascade. However, the process by which the protein-protein signaling is redirected from physiological to pathological conditions is largely unknown.

Protein Interaction Network in Normal Signaling and Diseases
Computational analysis may provide the prediction of the protein-protein interactions that link to cancer progression. Gene expression analysis from thousands of cancer samples at the Cancer Genome Atlas yields differential arrays or clusters of protein-protein signaling network among cancers. These aberrant signal pathways are specific for the progression of one or multiple cancer types [1][2][3]. Nonetheless, empirical determination for the dynamic protein-protein binding and dissociation in cell levels is needed, so as to build the big picture. For example, formation of an intracellular large protein complex may involve changes in protein conformations, enzymatic cleaves, energy requirement, heat release, and interactions with small molecules, ions, and/or amino acids [4][5][6][7][8][9][10].
In this review article, we will mainly detail the extracellular stimuli-mediated activation of WW domain-containing oxidoreductase (WWOX) signaling network. We will discuss how transforming growth factor beta (TGF-β1) and hyaluronan activate WWOX and downstream normal or aberrant protein partners and the potential consequences for the progression of cancer and Alzheimer's disease (AD) (Figure 1). In principle, we will address in a stepwise manner how WWOX signals interact with p53, HYAL-2 and SMAD4 to generate significant biological events. Our writing flow is as follows: (1) WWOX structure and domains involved in protein binding, controlling cell adhesion and migration, cell-cell recognition, and relevance to cancer metastasis and neuronal heterotopia, (2) reduction in Y33 phosphorylation and subsequent upregulation of S14 phosphorylation in WWOX during disease progression, (3) measuring WWOX signaling by real-time Förster resonance energy transfer (FRET) microscopy, (4) TGF-β1-regulated activation of WWOX and binding proteins (e.g., p53 and TIAF1) for cell death by time-lapse microscopy, and (5) competitive binding interactions leading to altered signaling. The following scheme illustrates the flow of this review article.

WW Domain-Containing Oxidoreductase (WWOX)
WWOX and its binding proteins form a complicated signaling network which is needed for normal physiology and/or disease progression [11][12][13][14][15][16][17][18][19][20][21]. WWOX was first discovered in 2000 [12]. Most recently, the human WWOX gene has been defined as a risk factor for AD [22]. Currently, there are many outstanding review articles documented in the PubMed database. In brief, WWOX protein possesses two N-terminal WW domains, a C-terminal short chain alcohol dehydrogenase/reductase (SDR) domain, and a nuclear localization signal present in between the WW domains [12] (Figure 2A). A mitochondria-targeting region is located in the SDR domain. Both WW and SDR domains have their own specific protein-binding partners, as reviewed in a recent article [19]. The first WW domain (WW1) possesses two tryptophans and binds PPXY (e.g., PPPY) or LPXY motif [12,23] motif in the target proteins, where X is any amino acid. However, when Y33 in the WW1 becomes phosphorylated, pY33-WWOX has an expanded binding capability with numerous proteins.

WWOX Controls Cell Migration, Cell-Cell Recognition, and Neuronal Heterotopia
Of utmost concern is that human newborns with WWOX gene deficiency suffer severe neural diseases such as seizure, encephalopathy, and early death [19][20][21]41,42]. There is no cure for the disease. Loss of WWOX enhances cell mobility [30,36]. WWOX gene deficiency in newborns results in neuronal migration disorders (or neuronal heterotopia) that lead to epileptic seizures [40,42]. Two types of cells based upon their expression of functional or dysfunctional WWOX have recently been identified [36,38]. Cells which are deficient in WWOX protein, or express dysfunctional WWOX, can be considered as metastatic cancer cells (designated as WWOXd).
At room temperature, WWOXd cells are less efficient in generating Ca 2+ influx and undergo non-apoptotic explosion in response to UV irradiation. In contrast, functional WWOX-expressing cells (designated WWOXf) exhibit non-apoptotic nucleus-dependent bubbling cell death (BCD) [17] and efficient Ca 2+ influx caused by UV or apoptotic stress at room temperature [36]. Formation of a nitric oxide (NO)-containing nuclear bubble per cell during BCD is due to UV-induced upregulation of NO synthase 2 (NOS2) [17]. WWOXf cells, which are mainly normal and benign cancer cells, migrate collectively and force the individually migrating WWOXd cells to undergo retrograde migration [36,37] ( Figure 2B,C). WWOXd cells, in return, induce WWOXf cells to undergo apoptosis from a distance without physical contact. The cytokines responsible for the induced apoptosis are unknown [36,37]. During cell-to-cell encounter, WWOXd cells exhibit activation of MIF, HYAL-2, Eph, and Wnt pathways, which converge to the MEK/ERK signaling and enables the cells to move away from WWOXf cells ( Figure 2B,C). Specific antibodies against MIF or HYAL-2, or inhibitors for WNT, cause WWOXf to greet WWOXd cells, and both cells merge eventually ( Figure 2B,C). Metastatic cancer cell-derived TGF-β1 allows merger of WWOXf with WWOXd cells [36,37]. A detailed signaling for the pathway of WNT, HYAL-2, EPH-Ephrin, EGF/EFGR, or MIF linking to ERK (or ERK1/2) is shown ( Figure 3). The CD44/HYAL-2 complex prevents the binding of TGF-β1 with HYAL-2. CD44 does not appear to be involved in the HYAL-2/WWOX/SMAD4 signaling [18] (Figure 3). Together, these observations suggest WWOX controls cell migration and cell-to-cell recognition and plays a crucial role in neuronal heterotopia that contributes to epileptic seizure [30,[35][36][37].   [36,37]. The role of HYAL-2/CD44 in controlling retrograde migration remains to be established.

WWOX Signaling Network
By STRING analysis (https://string-db.org/cgi/network?taskId=brQHTnXWILzL& sessionId=b73ctYMesgwG) (accessed on 19 June 2022), the first level of WWOX-binding protein network reveals that WWOX has connection with 5 interactors, namely TP53, ERBB4, DVL2, FAM189B, and WBP2 [11,25,[43][44][45][46][47][48]. The binding has been validated by experimental approaches (Figure 4). Further analysis expanding the interactors up to 26 and 136, respectively, shows many biological process and molecular functions, including betacatenin destruction complex assembly, response to metformin, ezrin [49], and many others. Again, the network can be expanded tremendously. The significance of this approach is that the data show the high complexity in the biological network and yet precise generation of functional machinery. The downside is that the analysis does not show domain/domainspecific binding interactions. Binding affinity-based interaction network is not clear. The computational approach also fails to prove our empirical observations that the stronger the binding of WWOX with intracellular proteins, the better the suppression of cancer growth and retardation of AD progression [19].

WWOX Functional Measurement by Time-Lapse FRET Microscopy
Förster resonance energy transfer (FRET) microscopy is a feasible approach to measure WWOX function in a real-time manner. FRET can be utilized to determine spatial proximity among proteins either at a single or multiple protein levels in cultured cells in a real-time mode [18,43,44,[55][56][57][58][59][60][61][62]. For example, binding of a CFP (cyan fluorescence protein)-tagged bait (or donor) protein with a YFP (yellow fluorescence protein)-tagged target (or acceptor) protein results in energy flow from the donor to the acceptor. In other words, an excitation wavelength is used to excite a donor protein (e.g., CFP tagged), and once excited the donor protein transfers the resulting energy to the acceptor (e.g., YFP tagged), which subsequently emits a longer wavelength with a lower energy. To be effective in signal transduction, protein proximity at 1-10 nm is needed for FRET detection. Furthermore, energy release from the first donor protein can excite two acceptors tagged with different fluorophores for determining parallel signaling pathways. We have first developed the technology for energy transfer from the first excited donor protein going directly to the second acceptor, and then the emitted energy from the second acceptor going to the third one [56]. This approach allows us to follow the signaling flow, the eventual outcome, and novel kinetics of protein binding [43,56,58]. We believe that the technology for signaling more than 3 partners in a pathway can be developed by tagging each protein with a small fluorescent probe. This is to prevent the self-aggregation of GFP or related fluorescent proteins.

TGF-β1 Induction of Initial Driving Force and then Execution Force for Protein-Protein Binding and Cell Death: TIAF1 Is a Blocker of TGF-β1/SMAD Signaling
Analysis of protein-protein binding kinetics by time-lapse FRET microscopy reveals the time-related changes in the binding force for a single protein or two to three binding protein partners, as well as alterations in cell morphology [44,56,58]. Transforming growth factor beta 1 (TGF-β1) induces the time-dependent self-polymerization of TIAF1 (TGFβ-induced antiapoptotic factor) in colon HCT116 cells [63][64][65]. Indeed, TGF-β induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid β plaques in Alzheimer's disease [63]. HCT116 is a WWOXf cell line [36]. WWOX binds TIAF1 to prevent the protein from undergoing self-polymerization [64][65][66]. TIAF1 aggregates can be found in the lesions of AD brains [50]. When TGF-β1-induced TIAF1 self-polymerization (using ECFP-TIAF1 and EYFP-TIAF1) reaches a maximal extent, cells start to undergo apoptosis [63][64][65]. Indeed, TGF-β1 stimulates aggregation of transiently overexpressed EGFP-TIAF1 to form intracellular green punctate prior to membrane blebbing and apoptosis [63]. When an equal amount of cDNA expression constructs for ECFP-SMAD4 and EYFP-TIAF1 is co-expressed in HCT116 cells, binding of SMAD4 with TIAF1 does not occur. No TGF-β1-induced aggregation of TIAF1 and apoptosis is observed [63], suggesting that SMAD4 prevents TIAF1 self-aggregation.
In contrast, when non-small cell lung cancer NCI-H1299 cells express a lower amount of ectopic EYFP -TIAF1 and a higher amount of ECFP-SMAD4, TGF-β1 increases the binding of EYFP-TIAF1 with ECFP-SMAD4, as determined by time-lapse FRET microscopy [63], NCI-H1299 is also a WWOXf cell line [36], Notably, an initial binding force between TIAF1 with SMAD4 is gradually increased followed by reduction, hereby designated as phase I. In phase II, the binding force between TIAF1 and SMAD4 becomes stronger than phase I by 30%. Meanwhile, there is a sharp decrease in cell volumes, indicating cells undergo apoptosis [63], In comparison, when cells express a greater amount of EYFP-TIAF1 than ECFP-SMAD4, TGF-β1-induced binding force for EYFP-TIAF1 and ECFP-SMAD4 is reduced in phases I and II. The extent of cell shrinkage and death is also retarded. While transiently overexpressed TIAF1 strongly binds endogenous SMAD2, 3 and 4 [35,[63][64][65][66], TIAF1 blocks the TGF-β1/SMAD signaling.
Overall, during signaling transduction, an initial force is needed for protein-protein binding in order to drive the signal pathway forward. Importantly, protein concentrations affect the binding status for two proteins, yielding distinct biological effects such as signaling moving forward or getting on hold. Furthermore, despite both HCT116 and NCI-H1299 cells possessing functional WWOX, their subcellular signaling pathways are likely to be different. Thus, under SMAD4 and TIAF1 overexpression, the biological outcome is expected to be different.

The Dynamics of WWOX/TIAF1/p53 Triad Formation and Functional Antagonism between p53 and WWOX for Enhancing the Progression of Cancer and Alzheimer's Disease
How WWOX deficiency (or downregulation) or pS14-WWOX upregulation contributes to disease progression is largely unknown [50,51,67]. Supporting evidence shows that the status of WWOX binding with p53 and TIAF1 may play a role in cancer and AD progression. Intracellular p53 and WWOX may counteract each other functionally, and thereby lead to cancer growth enhancement and development of AD pathologies in vivo [35]. WWOX physically binds and stabilizes wild type p53 from being degraded by the proteasomal system [48]. Under stress conditions, tumor suppressors p53 and WWOX form a complex with TIAF1, and the WWOX/TIAF1/p53 triad strongly inhibits cancer cell growth, migration, anchorage-independent transforming growth, and SMAD promoter activation, and ultimately causes cancer cell apoptosis [35]. The WWOX/TIAF1/p53 triad has been confirmed by co-immunoprecipitation and FRET microscopy.
There are 12 p53 isoforms [68]. Among these, ∆133p53γ isoform is the strongest in suppressing cancer cell migration, and this positively correlates with ∆133p53γ-mediated SMAD promoter activation [35]. We do not exclude the possibility that ∆133p53γ undergoes self-association and this provides a driving energy to cause SMAD promoter activation and inhibition of cell migration. Unlike the full-length p53, ∆133p53 isoforms α, β, and γ do not have the transactivation domains and the beginning of the DNA-binding domain [68,69]. Additionally, ∆133p53 isoforms play a critical role in cancer, aging, neurodegeneration and immunity [68,69].
Most interestingly, ectopic WWOX inhibits lung cancer NCI-H1299-mediated inflammatory splenomegaly and cancer cell growth in nude mice, and that p53 counteracts the effect of WWOX in these mice. When mice have ongoing growth of p53/WWOX-expressing lung cancer cells, the mice tend to have BACE (β-secretase 1) upregulation, APP (amyloid precursor protein) degradation, tau tangle formation, and amyloid β generation in the brain and lung [35]. That is, functional antagonism between p53 and WWOX leads to enhanced cancer growth and accelerated neurodegeneration in vivo [35]. We do not exclude the possibility that p53/TIAF1/WWOX triad becomes aggregated in the brain and contributes to aggregation of tau and amyloid beta of the AD pathologies.
Whether the p53/TIAF1/WWOX triad induces cancer cell death in vivo is unknown. When phosphorylation of WWOX in Y33 is downregulated and S14 upregulated, the binding strength between WWOX and p53 is expected to be reduced. This facilitates the growth of cancer and the progression of Alzheimer's disease [50,51]. By the same token, binding of pS14-WWOX with other intracellular proteins is weakened and thereby favors the growth of cancer and the progression of AD [19,53]. In parallel, when WWOX is downregulated or Y33 phosphorylation is blocked, UV-induced p53 protein expression is suppressed, even though p53 mRNA levels are stable [47]. Thus, the proteomic profiles of intracellular protein-binding partners for pS14-and pY33-WWOX, respectively, remains to be established.

A WWOX7-21 Epitope Peptide Drives the HYAL-2/WWOX/SMAD4 Signaling
The mechanisms by which WWOX-mediated cancer suppression and inhibition of neurodegeneration take place are largely unknown. There are two surface exposed epitopes in WWOX, which are at amino acids #7-21 and #286-299 [36,53,54]. Synthetic WWOX7-21 peptide, or truncation down to 5-amino acid WWOX7-11, strongly blocks and prevents the growth and metastasis of melanoma and skin cancer cells in mice [54]. WWOX286-299 also inhibits cancer cell growth, whereas it fails to block cancer metastasis to the lung and liver [54].
By time-lapse microscopy, we determined that antibody against WWOX7-21 suppresses ceritinib-mediated breast 4T1 cell sphere explosion and death ( Figure 6A,C; Supplementary Videos S1-S3). 4T1 cell spheres express many makers of stem cells (e.g., Sox2, Oct4 and Nanog). Ceritinib is an antineoplastic kinase inhibitor for treating anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) [73]. In contrast, WWOX7-21 peptide potently enhances the function of ceritinib in causing the Figure 5. Binding of cytosolic WWOX by SMAD4 reduces WWOX interaction with membrane-bound HYAL-2 and thereby inhibits yeast cell growth at 37 • C. (A) By Ras rescue-based yeast two-hybrid analysis [11,25,26,38,48,[70][71][72]. Binding of WWOX with p53 or MafB self-interaction allows the growth of yeast at 37 • C using a selective agarose plate containing galactose. No yeast growth at 37 • C is seen for the empty pSos and pMyr vectors. SMAD4 binds the N-terminal first WW domain of WWOX (WW1) and the C-terminal SDR domain binds SMAD4 or HYAL-2 [44]. HYAL-2 binds WW1 in a pY33-dependent manner [44]. WW1(Y33R) fails to bind SMAD4 or HYAL-2. HYAL-2 and SMAD4 fail to bind each other [44]. (B) In competitive binding assay, ectopic HYAL-2 (target; green) is designed for anchoring onto the cell membrane of yeast. Various amounts of cytosolic SMAD4 (competitor; red) are used to bind a constant amount of cytosolic WWOX (bait; dark blue) and reduce WWOX binding to membrane HYAL-2. (C) When an increased amount of SMAD4 is overly expressed, SMAD4 binds WWOX to prevent the signaling for HYAL-2/WWOX and thereby leads to growth suppression of yeast. (D) When WWOX undergoes Y33 phosphorylation, it becomes unfolded to let its binding with HYAL-2 and SMAD4. WW domain physically interacts with SDR domain in non-activated WWOX [36,37]. (data adapted from Reference [44] with major modifications and new interpretations; permission not required from Oncotarget).
HYAL-2 and SMAD4 competitively interact with the WW1 domain of WWOX [64] ( Figure 5B). Increasing amounts of ectopic SMAD4 block the binding between WWOX and HYAL-2, and thereby inhibit the yeast cell growth ( Figure 5C). We reported that WW domain physically binds SDR domain in WWOX as a non-activated form, and WW or SDR domain can undergo self-polymerization as determined by FRET microscopy [36]. In response to TGF-β1, UV, hyaluronan, or other stress stimuli, folded WWOX opens up and becomes Y33 phosphorylated as an activated form to interact with HYAL-2 and SMAD4 [26,44] (Figure 5C). Similarly, activated pY33-WWOX strongly binds pS46-p53 to induce cell death in the nucleus [48].

A WWOX7-21 Epitope Peptide Drives the HYAL-2/WWOX/SMAD4 Signaling
The mechanisms by which WWOX-mediated cancer suppression and inhibition of neurodegeneration take place are largely unknown. There are two surface exposed epitopes in WWOX, which are at amino acids #7-21 and #286-299 [36,53,54]. Synthetic WWOX7-21 peptide, or truncation down to 5-amino acid WWOX7-11, strongly blocks and prevents the growth and metastasis of melanoma and skin cancer cells in mice [54]. WWOX286-299 also inhibits cancer cell growth, whereas it fails to block cancer metastasis to the lung and liver [54].

Zfra4-10 or WWOX7-21 Activates the HYAL-2/WWOX/SMAD4 Signaling for Z Cell Activation and Suppression of Disease Progression In Vivo
An additional mechanism for the peptide function in vivo is that Zfra peptides, including Zfra4-10 and Zfra1-31, become polymerized in the circulation and then trapped in the spleen as the polymers emit red and green fluorescence [51]. Zfra binds membrane HYAL-2 of spleen Z cells, but not T and B cells. This binding leads to initiation of the HYAL-2/WWOX/SMAD4 signaling that results in activation of memory cytotoxic Z cells [50][51][52][53]. The activated Z cell, which is HYAL-2+ CD3− CD19−, is highly potent in killing cancer cells both in vitro and in vivo. The Zfra-activated Z cells have never encountered cancer cell antigens, and yet they effectively recognize and kill cancer cells [50][51][52][53]. Unlike the generation of chimeric antigen receptor T-cells (CAR-T), Z cell activation does not require pre-exposure to cancer antigens. Polymerized Zfra peptide probably possesses certain motifs or domains similar to those in the cancer antigens. Transfer of activated Z cells to naïve mice or cancer-growing mice confers suppression of cancer growth [50,51,53]. Activated Z cells also kill cancer cells in vitro [51,53]. Additionally, Zfra4-10 peptide is capable of restoring memory loss and inhibiting neurodegeneration in triple transgenic mice for AD [50], suggesting that activated Z cells are involved in memory restoration in the AD mice.
As mentioned above, WWOX7-21 peptide enhances the efficacy of ceritinib-mediated cancer cell death in vitro, and activates Z cells in mice and thereby blocks tumor growth [54]. WWOX7-21 peptide colocalizes with membrane type II TGF-β receptor (TβRII) [36]. Both WWOX7-21 peptide and TβRII simultaneously undergo internalization in response to TGF-β1, suggesting that WWOX7-21 complexes with TβRII. Actually, presence of a HYAL-2/WWOX/TβRII complex is found in the lipid raft of cells in many organs (e.g., liver, spleen and brain) of control mice [36]. The amino acid sequence of WWOX7-21 peptide is located in the N-terminal leader sequence and a small part of the first WW domain. WW domain and SDR domain may undergo self-polymerization or hetero-polymerization at the intramolecular or intermolecular level [36]. WWOX7-21 peptide may undergo selfpolymerization in phosphate-buffered saline and binds the WW domain area [36]. Binding of WWOX7-21 peptide with membrane HYAL-2 is unknown. Importantly, WWOX7-21 peptide strengthens the binding of WWOX with intracellular proteins for blocking cancer and AD progression. Again, WWOX7-21 peptide antibody pulls down the full-length WWOX, membrane HYAL-2 and TGFβRII, further validating the concept that WWOX7-21 peptide is able to initiate the HYAL-2/WWOX/SMAD4 signaling [36].

Zfra-Induced Spleen Z Cell Activation Requires De-Phosphorylation at S14, Y33 and Y61 in WWOX In Vivo
When lymphocytic cells are stimulated with calcium ionophore A23187 and phorbol ester (IoP) in vitro, endogenous WWOX undergoes dephosphorylation at Y33 and Y61 and acquires S14 phosphorylation during the course of T cell differentiation ( Figure 7A) [56]. When mice receive pS14-WWOX7-21 peptide via tail vein injections, dramatic upregulation of cytotoxic CD8α+ T and CD19+ B cells, but not Foxp3+ T regulatory (Treg) cells, is observed in the spleen [54]. The induced T and B cells fail to kill cancer cells. Although pS14-WWOX7-21 peptide assists melanoma and breast cancer cells to grow even faster and bigger in mice, this peptide may be of therapeutic value in bringing up T/B cells from immunodeficient patients [53]. Without S14 phosphorylation, WWOX7-21 peptide fails to induce T/B cell expansion [54]. and acquires S14 phosphorylation during the course of T cell differentiation ( Figure 7A) [56]. When mice receive pS14-WWOX7-21 peptide via tail vein injections, dramatic upregulation of cytotoxic CD8α+ T and CD19+ B cells, but not Foxp3+ T regulatory (Treg) cells, is observed in the spleen [54]. The induced T and B cells fail to kill cancer cells. Although pS14-WWOX7-21 peptide assists melanoma and breast cancer cells to grow even faster and bigger in mice, this peptide may be of therapeutic value in bringing up T/B cells from immunodeficient patients [53]. Without S14 phosphorylation, WWOX7-21 peptide fails to induce T/B cell expansion [54]. and pY61 in the WWOX. However, S14 phosphorylation is needed for T cell differentiation [56]. Whether Ionophore/PMA induces Z cell differentiation is unknown. Zfra peptide (e.g., Zfra4-10) induces Z cell activation via dephosphorylation of pS14, pY33 and pY61 in WWOX. Membrane HYAL-2 is rapidly upregulated in Z cells [54]. (B) Agonists, including Zfra4-10, WWOX7-21, WWOX7-11 and 8 hr-sonicated hyaluronan (HAson8), induce Z cell activation via HYAL-2/WWOX/SMAD4 signaling for blocking cancer growth and retarding AD progression [54].
The differentiation of Z cells is different from that of T/B cells, in which S14, Y33 and Y61 are dephosphorylated ( Figure 6A). When mice receive Zfra4-10 peptide to build anticancer response, Zfra4-10 peptide in circulation becomes polymerized and is trapped in the spleen [51]. The polymerized Zfra4-10 peptide remains in the spleen for at least 2 months. It continuously stimulates the generation of activated Z cells. WWOX undergoes dephosphorylation at S14, Y33 and Y61 [54] ( Figure 7B). The activated spleen Z cells relocate to normal organs such as liver and lung, and cancer lesion sites in organs [51]. CD19+ or CD27+ B cells are not involved in Zfra-mediated cancer suppression [54].
In stark contrast, when mice receive both Zfra4-10 and WWOX7-21 peptides in combination via tail vein injections, both peptides nullify each other's function, which leads to enhanced tumor growth [53]. That is, reduced binding of endogenous WWOX with target proteins in organs and tumor lesions occurs, which allows enhanced cancer growth [53]. Both Zfra4-10 and WWOX7-21 peptides tend to undergo aggregation in phosphate-buffered saline [51,53]. We do not exclude the possibility that both Zfra4-10 and WWOX7-21 peptides covalently bind each other and thereby lose their function in signaling and anticancer activity [53].
A critical question is whether activated Z cells retard AD progression. Zfra4-10 and Zfra1-31 peptides are potent in restoring memory loss in triple transgenic 3xTg mice [50,51]. Zfra-mediated suppression of S14 phosphorylation in WWOX (>90%) is needed for Z cell activation, which positively correlates with prevention and blocking of AD progression in 3xTg and Wwox heterozygous mice [50]. If activated Z cells are involved in preventing neurodegeneration, then the activated Z cells are likely to secrete cytokines to support neuronal survival. Alternatively, activated Z cells may have acquired capability in traveling through the blood-brain barrier. Indeed, certain populations of inflammatory immune cells are capable of passing through the blood-brain barrier with the assistance of IL17-induced reactive oxygen species (ROS) to damage brain endothelial cells [76].
To examine the signaling flow of a trimolecular complex, cells are electroporated with ECFP-SMAD4, EGFP-WWOX and DsRed-p53 constructs for transient overexpression, and then treated with HA of 2 to 4 million Daltons, followed by time-lapse FRET microscopy. Membrane hyaluronidase HYAL-2 binds and degrades HA, and meanwhile induces the formation of a signaling complex of ectopic ECFP-SMAD4, EGFP-WWOX and DsRed-p53 [44,58]. In the initial driving phase (Phase I), there is an increased binding energy of WWOX with both p53 and SMAD4, which lasts 7 h and then jumps to a greater extent of execution force (Phase II) for 11 h. The cells undergo membrane blebbing without apoptotic death [44,58]. When ectopic p53 is replaced by an intracellular form of ectopic HYAL-2(-sp), the signaling of SMAD4/HYAL-2(-sp)/WWOX leads to nuclear-based bubbling cell death. The resulting driving force in phase I is shortened to 3 h and the execution phase to 20 h [58]. Taken together, replacing p53 with HYAL-2 allows the switching of membrane blebbing to bubbling cell death, suggesting the machinery of bubbling cell death emanating from the nucleus is switched on.

Discussion and Perspectives
In summary, the biological functions of the HYAL-2/WWOX/SMAD4 signaling pathway have been thoroughly described in this perspective article. Both TGF-β1 and hyaluronan are able to bind membrane HYAL-2, and thereby drive the downstream signaling with WWOX and SMAD4 [26]. This pathway controls cell growth, differentiation and death [26], spleen Z cell differentiation and activation [50,51], and traumatic brain injury [44]. During the hyaluronan/HYAL-2 signaling, hyaluronan initiates the ectopic HYAL-2/WWOX/SMAD4 pathway in cells, which results in bubbling cell death [44]. Agonist Zfra4-10 or WWOX7-21 peptide, HYAL-2 antibody or sonicated hyaluronan HAson8, also activate the HYAL-2/WWOX/SMAD4 signaling for spleen Z cell activation in order to kill cancer cells in vitro and in vivo.
Whether the HYAL-2/WWOX/SMAD4 signaling limits cancer and neurodegeneration in a balanced manner is unknown. When the WWOX/TIAF1/p53 triad is accumulated in cancer cells, cancer cell growth suppression and death are likely to occur (Figure 8) [55]. This protein triad strongly inhibits cell migration, anchorage-independent transforming growth, and SMAD promoter activation. Notably, when there is a functional antagonism between p53 and WWOX in vivo, this event favors cancer growth and enhanced AD progression. Another interesting finding is that WWOX7-21 or Zfra4-10 peptide increases the strength of endogenous WWOX binding with its protein partners in vivo. The stronger the binding, the better the cancer suppression [53]. Whether this correlates with inhibition of AD progression is being determined. phosphorylated. The effect of the triad on normal brain cells is unknown. (B) pY33-WWOX can be converted to pS14-WWOX. pS14-WWOX is gradually accumulated in the lesions of cancer and AD brain during disease progression. pS14-WWOX supports cancer growth and enhances neurodegeneration such as in AD. Zfra4-10 peptide suppresses phosphorylation of WWOX at S14 and thereby restores memory loss in AD mice and inhibits cancer growth [50,51].
Outstanding questions that remain to be resolved are: (1) How can we enhance the binding of WWOX with its intracellular protein partners, thereby leading to inhibition of cancer growth? Appropriate peptides or small chemicals will be designed and used to enhance the binding strength of WWOX with its protein partners, so as to suppress cancer growth and meanwhile inhibit AD progression. Indeed, either Zfra4-10 or WWOX7-21 peptides increase the binding strength of WWOX with its partners [53]. (2) How can activated Z cells exert cytotoxicity for leading to cancer cell death? Is this via cytokines or direct physical contact with cancer cells? Z cells need to be primed for activation by Zfra or WWOX peptides or sonicated hyaluronan via the signaling of HYAL-2/WWOX/SMAD4 [53]. Thus, utilization of either Zfra4-10 or WWOX7-21 peptide for Z cell activation in cancer treatment is feasible and is being tested in animals. 3) Can HAson8 provide a better strength in Z cell activation for cancer treatment? HAson8 is generated by sonicating hyaluronan at a sufficient kilohertz, timing and temperature [53]. Although all the designed drugs utilize the HYAL-2/WWOX/SMAD4 signaling to control cancer growth, it is necessary to compare the efficacy of HAson8, Zfra4-10, WWOX7-21 and other available drugs in treating cancer in vivo.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Video S1: Ceritinib induces individual breast 4T1 cell apoptosis and cell sphere explosion and death, Video can be converted to pS14-WWOX. pS14-WWOX is gradually accumulated in the lesions of cancer and AD brain during disease progression. pS14-WWOX supports cancer growth and enhances neurodegeneration such as in AD. Zfra4-10 peptide suppresses phosphorylation of WWOX at S14 and thereby restores memory loss in AD mice and inhibits cancer growth [50,51].
Outstanding questions that remain to be resolved are: (1) How can we enhance the binding of WWOX with its intracellular protein partners, thereby leading to inhibition of cancer growth? Appropriate peptides or small chemicals will be designed and used to enhance the binding strength of WWOX with its protein partners, so as to suppress cancer growth and meanwhile inhibit AD progression. Indeed, either Zfra4-10 or WWOX7-21 peptides increase the binding strength of WWOX with its partners [53]. (2) How can activated Z cells exert cytotoxicity for leading to cancer cell death? Is this via cytokines or direct physical contact with cancer cells? Z cells need to be primed for activation by Zfra or WWOX peptides or sonicated hyaluronan via the signaling of HYAL-2/WWOX/SMAD4 [53]. Thus, utilization of either Zfra4-10 or WWOX7-21 peptide for Z cell activation in cancer treatment is feasible and is being tested in animals. 3) Can HAson8 provide a better strength in Z cell activation for cancer treatment? HAson8 is generated by sonicating hyaluronan at a sufficient kilohertz, timing and temperature [53]. Although all the designed drugs utilize the HYAL-2/WWOX/SMAD4 signaling to control cancer growth, it is necessary to compare the efficacy of HAson8, Zfra4-10, WWOX7-21 and other available drugs in treating cancer in vivo.