Role of WW Domain-containing Oxidoreductase WWOX in Driving T Cell Acute Lymphoblastic Leukemia Maturation

Abstract

Whether tumor suppressor WWOX (WW domain-containing oxidoreductase) stimulates immune cell maturation is largely unknown. Here, we determined that Tyr-33-phosphorylated WWOX physically binds non-phosphorylated ERK and IκBα in immature acute lymphoblastic leukemia MOLT-4 T cells and in the naïve mouse spleen. The IκBα·ERK·WWOX complex was shown to localize, in part, in the mitochondria. WWOX prevents IκBα from proteasomal degradation. Upon stimulating MOLT-4 with ionophore A23187/phorbol myristate acetate, endogenous IκBα and ERK undergo rapid phosphorylation in <5 min, and subsequently WWOX is Tyr-33 and Tyr-287 de-phosphorylated and Ser-14 phosphorylated. Three hours later, IκBα starts to degrade, and ERK returns to basal or non-phosphorylation, and this lasts for the next 12 h. Finally, expression of CD3 and CD8 occurs in MOLT-4 along with reappearance of the IκBα·ERK·WWOX complex near 24 h. Inhibition of ERK phosphorylation by U0126 or IκBα degradation by MG132 prevents MOLT-4 maturation. By time-lapse FRET microscopy, IκBα·ERK·WWOX complex exhibits an increased binding strength by 1–2-fold after exposure to ionophore A23187/phorbol myristate acetate for 15–24 h. Meanwhile, a portion of ERK and WWOX relocates to the nucleus, suggesting their role in the induction of CD3 and CD8 expression in MOLT-4.

HYAL-2–WWOX–SMAD4 Signaling in Cell Death and Anticancer Response

“…Presumably, binding of Zfra to WWOX leads to Tyr33 phosphorylation and Ser14 de-phosphorylation, and the open form has an exposure of WWOX7-21 motif for Z cell activation. We have established that Tyr33-phosphorylated WWOX is an activated form for cancer suppression (Chang et al, 2001, 2003, 2007; Chiang et al, 2013; Abu-Remaileh et al, 2015; Huang et al, 2016). Nevertheless, how the extracellular polymerized Zfra peptide regulates the specific site phosphorylation in WWOX remains to be established.…”

“…When both WWOX and p53 are dysfunctional, osteosarcoma occurs in a double knockout mouse model, suggesting that both proteins participate in normal bone development (Del Mare et al, 2016). In another functional aspect, calcium ionophore/phorbol ester induces terminal maturation of MOLT-4 T cell acute lymphoblastic leukemia, which is due to Ser14 phosphorylation of WWOX, IκBα–ERK–WWOX signaling, and expression of CD3 and CD8 (Lin et al, 2011; Huang et al, 2016). …”

“…Second, functional deficiency or defect of the HYAL-2-WWOX-SMAD4 signaling complex may occur in cancer cells. For example, tumor suppressor WWOX is frequently missing in cancer cells (Bednarek et al, 2000; Chang et al, 2001, 2003, 2007; Iliopoulos et al, 2005; Aqeilan and Croce, 2007; Chiang et al, 2013; Gardenswartz and Aqeilan, 2014; Abu-Remaileh et al, 2015; Schrock and Huebner, 2015; Huang et al, 2016). Lastly, the CD44-HYAL-2 complex provides spatial hindrance for the binding of TGF-β1 with Hyal-2.…”

“…Relocation of the complex to the nucleus occurs in 20–40 min post stimulation. (B) As determined by time-lapse tri-molecular FRET microscopy (Hsu et al, 2016; Huang et al, 2016), HA activates the endogenous HYAL-2–WWOX–SMAD4 signaling and increases the complex formation of ectopic ECFP-SMAD4–EGFP-HYAL-2–DsRed-WWOX to undergo nuclear accumulation. Cells undergo bubbling cell death (Chen et al, 2015; Chang, 2016).…”

“…This allows cancer cells gain growth advantage and degraded HA-increased metastasis. Calcium ionophore/phorbol ester induces the signaling of IκBα–ERK–WWOX for maturation of T cell leukemia, whereas HA has no effect in inducing the signaling event (Hsu et al, 2016; Huang et al, 2016). …”