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...

Hyaluronidases and hyaluronan are important mediators of tissue remodeling and cancer cell metastasis. Metastatic and malignant breast and prostate cancers frequently overexpress hyaluronidases and hyaluronan (1, 2). Hyaluronidases PH-20, Hyal-1, and Hyal-2 are known to induce the expression of a candidate tumor suppressor WW domaincontaining oxidoreductase WOX1 (also known as WWOX, FOR, or WWOXv1) (3-5).The human WWOX gene, which comprises nine exons encoding the WWOX/WOX1 family proteins, is located on a fragile site on the chromosome ch16q23.3-24.1 (6 -9). Eight mRNA transcripts of the WWOX gene have been found so far (7). However, it is still not known whether all of the mRNA transcripts are translated successfully into proteins. Isoforms WWOXv1 (46 kDa), WWOXv2 (42 kDa), and WWOXv8 (60 kDa) and several other small proteins can be found in normal and several types of cancer cells (3, 8 -11). 3 Genetic alterations of the WWOX gene and disappearance of WWOX/WOX1 family proteins have been shown in multiple types of cancers, especially at an invasive or a metastatic stage (8,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Hypermethylation of the WWOX gene may inactivate its expression (21). In contrast, significant up-regulation of WWOX/WOX1 family proteins has been shown during progression of breast, prostate and other cancers to a premetastatic state (10,22,23). Also, absent expression of these family proteins in metastatic cancer cells is not necessarily due to disruption of the WWOX gene. We have recently determined that post-transcriptional blockade of the fulllength mRNA translation to protein may account for the disappearance of the WWOX/WOX1 family proteins in cutaneous squamous cell carcinoma cells in patients and in UVB-treated mice (24).Wild type WOX1 possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence and a C-terminal short-chain alcohol dehydrogenase/reductase domain (which contains a mitochondria-targeting sequence) (3). Sex steroid hormones such as estrogen and androgen activate WOX1 via Tyr 33 phosphorylation (p-WOX1) and nuclear translocation (11). Importantly, p-WOX1 is located in the mitochondria during benign prostatic hypertrophy (11). Nuclear translocation of p-WOX1 occurs when prostate cells progress toward cancerous and metastatic states (11), suggesting a critical role of WOX1 phosphorylation during prostate cancer development.We determined that WOX1 enhances the cytotoxic function of tumor necrosis factor (TNF) 4 and induces apoptosis when overexpressed (3). We also showed that in response to stress or apoptotic stimuli, WOX1 becomes phosphorylated at Tyr 33 , which allows its complex formation with activated p53 and JNK1 (25). The p53-WOX1 complex translocates to the mitochondria and nuclei to mediate apoptosis (25,26). WOX1 induces apoptosis synergistically with p53 (3,25). In contrast, JNK1 may block WOX1-induced cell death (25). Src is known to phosphorylate WOX1 at Tyr 33 (27). We also determined that Tyr 33 -phosphorylated WO...

The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.

Lung cancer is the leading cause of cancer‐related death worldwide. In China, the incidence of lung cancer has grown rapidly, resulting in a large social and economic burden. Several researchers have devoted their studies to lung cancer and have demonstrated that there are many risk factors for lung cancer in China, including tobacco use, environmental pollution, food, genetics, and chronic obstructive pulmonary disease. However, the lung cancer incidence is still growing rapidly in China, and there is an even higher incidence among the younger generation. One explanation may be the triple‐neglect situation, in which medical policies that neglect prevention, diagnosis, and supportive care have increased patients' mortality and reduced their quality of life. Therefore, it is necessary to enhance the efficiency of prevention and early diagnosis not only by focusing more attention on treatment but also by drawing more attention to supportive care for patients with lung cancer. Cancer 2015;121:3080‐8. © 2015 American Cancer Society.

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

BackgroundTissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells.Methodology/Principal FindingsDU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues.Conclusions/SignificanceWe conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.

Tumor suppressor WWOX is involved in the progression of cancer and neurodegeneration. Here, we examined whether protein aggregation occurs in the brain of nondemented, middle-aged humans and whether this is associated with WWOX downregulation. We isolated an N-terminal internal deletion isoform, TPC6AΔ, derived from alternative splicing of the TRAPPC6A (TPC6A) gene transcript. TPC6AΔ proteins are present as aggregates or plaques in the extracellular matrix of the brain such as in the cortex. Filter retardation assays revealed that aggregate formation of TPC6AΔ occurs preceding Aβ generation in the hippocampi of middle-aged postmortem normal humans. In a Wwox gene knockout mouse model, we showed the plaques of pT181-Tau and TPC6AΔ in the cortex and hippocampus in 3-week-old mice, suggesting a role of WWOX in limiting TPC6AΔ aggregation. To support this hypothesis, in vitro analysis revealed that TGF-β1 induces dissociation of the ectopic complex of TPC6AΔ and WWOX in cells, and then TPC6AΔ undergoes Ser35 phosphorylation-dependent polymerization and induces caspase 3 activation and Aβ production. Similarly, knockdown of WWOX by siRNA resulted in dramatic aggregation of TPC6AΔ. Together, when WWOX is downregulated, TPC6AΔ is phosphorylated at Ser35 and becomes aggregated for causing caspase activation that leads to Tau aggregation and Aβ formation.

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