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.

Overall, WWOX participates in many ligand/receptor-mediated signaling pathways such as WNT/β-catenin [24], JNK [25], TGFβ1/HYAL-2 [26], TGFβ/SMADs [26,27], HIPPO [28], HCG/MET [29], ], JAK2/STAT3 [30,31,32], p73 [33], and others. WW2 domain has only one tryptophan and its function is largely unknown. WW2 is proposed to line up with WW1 in facilitating the binding of WWOX with its interacting partners [34]. The SDR domain has an NSYK (Asn-Ser-Tyr-Lys) motif, which binds sex steroid hormones androgen and estrogen [12]. The SDR domain also exhibits redox activity [35,36,37], and binds tau and GSK-3β to prevent tau hyperphosphorylation [19,38,39,40]. Whether the redox activity of SDR domain limits GSK-3β-mediated tau hyperphosphorylation and subsequent tau aggregation is unknown [38,40].

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 Ca²⁺ 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 Ca²⁺ 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].

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 beta-catenin 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/domain-specific binding interactions. Binding affinity-based interaction network is not clear. The computational approach also fails to prove the 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].