Transforming growth factor ␤ (TGF-␤) initiates multiple signal pathways and activates many downstream kinases. Here, we determined that TGF-␤1 bound cell surface hyaluronidase Hyal-2 on microvilli in type II TGF-␤ receptor-deficient HCT116 cells, as determined by immunoelectron microscopy. This binding resulted in recruitment of proapoptotic WOX1 (also named WWOX or FOR) and formation of Hyal-2⅐WOX1 complexes for relocation to the nuclei. TGF-␤1 strengthened the binding of the catalytic domain of Hyal-2 with the N-terminal Tyr-33-phosphorylated WW domain of WOX1, as determined by time lapse fluorescence resonance energy transfer analysis in live cells, co-immunoprecipitation, and yeast twohybrid domain/domain mapping. In promoter activation assay, ectopic WOX1 or Hyal-2 alone increased the promoter activity driven by Smad. In combination, WOX1 and Hyal-2 dramatically enhanced the promoter activation (8 -9-fold increases), which subsequently led to cell death (>95% of promoter-activated cells). TGF-␤1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-␤1-induced apoptosis. Together, TGF-␤1 invokes a novel signaling by engaging cell surface Hyal-2 and recruiting WOX1 for regulating the activation of Smad-driven promoter, thereby controlling cell growth and death. Transforming growth factor ␤ (TGF-␤)4 plays a dual role in cell growth and tumorigenesis (1, 2). TGF-␤ inhibits mammary epithelial cell growth. In contrast, invasive cancer cells frequently overproduce TGF-␤ to promote growth and metastasis (1, 2). The underlying mechanism is largely unknown. TGF-␤ induces the development of metastatic phenotypes, i.e. stimulation of epithelial-mesenchymal transitions in cancerous mammary epithelial cells (1, 2). These cells are normally devoid of functional type II TGF-␤ receptor (T␤RII), suggesting that TGF-␤ binds to an alternative receptor for signaling.Hyaluronan is the major components of pericellular coat and plays a key role in affecting cell morphology, communication, and behavior (3-5). Up-regulation of hyaluronan and hyaluronidases Hyal-1, Hyal-2, and PH-20 is associated with cancer metastasis (3-5). Hyaluronidases counteract the activity of TGF-␤1 (6 -8). TGF-␤1 suppresses the proliferation of normal epithelial cells, whereas PH-20 blocks the TGF-␤1 effect (6). Hyal-1 and Hyal-2 enhance the cytotoxic function of TNF and block TGF-␤1-mediated protection of murine L929 fibroblasts from TNF cytotoxicity (6 -8).Hyaluronidases PH-20, Hyal-1, and Hyal-2 induce the expression of tumor suppressor WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1 (8 -11). Human WWOX gene is located on a chromosomal fragile site 16q23 and encodes WWOX/FOR/WOX1 and isoforms (9, 10, 12-16). The full-length 46-kDa WOX1 possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence between the WW domains, and a C-terminal short chain alcohol dehydrogenase/reductase domain. Numerous exogenous stimuli, including sex stero...

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Zfra is a 31-amino-acid zinc finger-like protein, which participates in the tumor necrosis factor signaling. Here, we determined that when nude mice and BALB/c mice were pre-injected with nanogram levels of a synthetic Zfra1–31 or truncated Zfra4–10 peptide via tail veins, these mice became resistant to the growth, metastasis and stemness of melanoma cells, and many malignant cancer cells. The synthetic peptides underwent self-polymerization in phosphate-buffered saline. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra aggregation and its-mediated cancer suppression in vivo. Injected Zfra peptide autofluoresced due to polymerization and was trapped mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naïve mice conferred resistance to cancer growth. Zfra-binding cells, designated Hyal-2+ CD3− CD19− Z cells, are approximately 25–30% in the normal spleen, but are significantly downregulated (near 0–3%) in tumor-growing mice. Zfra prevented the loss of Z cells caused by tumors. In vitro stimulation or education of naïve spleen cells with Zfra allowed generation of activated Z cells to confer a memory anticancer response in naïve or cancer-growing mice. In particular, Z cells are abundant in nude and NOD-SCID mice, and can be readily activated by Zfra to mount against cancer growth.

BackgroundTumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats.Methodology/Principal FindingsSciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-κB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-κB (40–65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-κB. WOX1 directly activated NF-κB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons.Conclusions/SignificanceRapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-κB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-κB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury.

Self-aggregation of transforming growth factor β (TGF-β)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid β (Aβ) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aβ in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aβ aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aβ. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aβ formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.

Purpose:We investigated the role of candidate tumor suppressor and proapoptotic WOX1 (also named WWOX, FOR, or WWOXv1) in UVB-induced apoptosis and formation of cutaneous squamous cell carcinomas (SCC). Experimental Design: Expression of WOX1and family proteins (WWOX) in human primary cutaneous SCCs was examined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. UVB irradiation^induced WOX1 activation (Tyr 33 phosphorylation and nuclear translocation), apoptosis, and cutaneous SCC formation were examined both in vitro and in vivo. Results: Up-regulation of human WOX1, isoform WOX2, and Tyr 33 phosphorylation occurred during normal keratinocyte differentiation before cornification and death. Interestingly, significant reduction of these proteins and Tyr 33 phosphorylation was observed in nonmetastatic and metastatic cutaneous SCCs (P < 0.001), but without down-regulation of WWOX mRNA (P > 0.05 versus normal controls), indicating a translational blockade of WWOX mRNA to protein.During acute exposure of hairless mice to UVB, WOX1was up-regulated and activated in epidermal cells in 24 hours. In parallel with the clinical findings in humans, chronic UVB-treated mice developed cutaneous SCCs in 3 months, with significant reduction of WOX1 and Tyr 33 phosphorylation and, again, without down-regulation of WWOX mRNA. Human SCC-25 and HaCaT cells were transfected with small interfering RNA^targeting WOX1 and shown to resist UVBinduced WOX1 expression, activation, and apoptosis. Conclusions: WOX1 is essential for UVB-induced apoptosis and likely to be involved in the terminal differentiation of normal keratinocytes. During UVB-induced cutaneous SCC, epidermal cells have apparently prevented the apoptotic pressure from overexpressed WOX1 by shutting down the translation machinery for WWOX mRNA.

When COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10–30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause “bubbling death”. Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

The family of WW domain-containing proteins contains over 2000 members. The small WW domain module is responsible, in part, for protein/protein binding interactions and signaling. Many of these proteins are located at the membrane/cytoskeleton area, where they act as adaptors to receive signals from the cell surface. In this review, we provide molecular insights regarding recent novel findings on signaling from the cell surface toward WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1. More specifically, transforming growth factor beta 1 utilizes cell surface hyaluronidase Hyal-2 (hyaluronoglucosaminidase 2) as a cognate receptor for signaling with WWOX and Smad4 to control gene transcription, growth and death. Complement C1q alone, bypassing the activation of classical pathway, signals a novel event of apoptosis by inducing microvillus formation and WWOX activation. Deficiency in these signaling events appears to favorably support cancer growth.