Volume 1 Issue 3 - September 7, 2007
WW domain-containing oxidoreductase: A tumor suppressor and beyond
Nan-Shan Chang, Ph.D., Professor, Institute of Molecular Medicine, National Cheng Kung University, Taiwan

Abstract – In this short communication, I´ll briefly describe the discovery of a tumor suppressor WWOX/WOX1 and its role in cancer and neurodegeneration. Proudly, I herein proclaim the outstanding effort from my collaborators in the NCKU medical campus in contributing to the early pioneering work.

History – Fragile sites are present in the human chromosomes. Mitotic pressure may break apart these fragile sites. Damaged chromosomes may lead to oncogenesis. Identifi cation of genes surrounding the chromosomal fragile sites is critical in understanding the etiology and progression of cancer. In year 2000, my research group in the Guthrie Research Institute (Sayre, PA, USA), together with the groups of Aldaz and Richards, has independently cloned a candidate tumor suppressor WW domain-containing oxidoreductase (literally named WWOX, FOR or WOX1) (1,2; reviews). WWOX gene encoding the WOX1 protein is located on a chromosomal common fragile site Ch16q23. Th is gene is altered in many types of cancers. Strikingly, we found that this gene is also involved in neurodegeneration (e.g. Alzheimer ´s disease). In the past few years, the heat has been on in the scientifi c community over the research for WWOX gene and protein.

WOX1 development in NCKU – Prior to my association with NCKU last November (2006), WOX1 research has been actively ongoing in the medical campus. My collaboration with researchers in NCKU started in 2001, proudly first with neuroscientist Dr. Sue Chen (陳淑姿) at the Anatomy and Cell Biology Department, and then with the most enthusiastic Drs. Yee-Shin Lin (林以行) and Li-Jin Hsu (徐麗君) at the Microbiology and Immunology Department, who have dynamically stepped up the collaboration eff ort. Drs. Hamm-Ming Sheu (許漢銘) and Feng- Jie Lai (賴豐傑), dermatologists at NCKU, have brought us to a new direction to the skin cancer. Dr. Chun-I Sze (司君一), now associated with the Pathology Department, has focused on neurodegeneration. Recently, our tentacles have reached colleagues in the Engineering School for developing innovative imaging. While our tentacles have kept growing globally, I am proud to say that Taiwan has the highest population of scientists working on the WOX1 project in the world. Together, we have been and will continue to work on the genetics, biology, and the signaling network that links this protein to the pathogenesis of cancer and neurodegeneration. As one of the leaders in the field, I earnestly hope that this is the landmark research to be established in NCKU.

WOX1 is considered as tumor suppressor – What is “tumor suppressor"? Every normal live cell possesses specific proteins to prevent itself from undergoing uncontrollable proliferation or becoming cancerous. Th ese proteins precisely orchestrate cell division, DNA replication and other molecular events, so that the cell will grow normally. Invasive cancer cells produce significantly elevated amounts of complex carbohydrates and glycosidases (e.g. hyaluronan and hyaluronidases) and proteinases (e.g. matrix metalloproteinases). Th ese cellular products are necessary for cancer cells to spread or metastasize to target organs. To investigate how cancer cells spread, we isolated WOX1 by functional cloning – a technique used to hunt novel genes possessing specifi c functions (1).

Figure 1. WOX1 and its splice variants. The predicted amino acid sequence (414 amino acids) of WOX1 (WWOX or FOR) possesses two N-terminal WW domains, a nuclear localization signal sequence (NLS), and a C-terminal short-chain alcohol dehydrogenase/reductase (ADH/SDR) domain. A mitochondria-targeting sequence in WOX1 was mapped within the ADH domain (amino acid #209-273). The structure of WOX2 (41 kDa) is also depicted. The C-terminal amino acid sequence of WOX2 is different from that of WOX1 (in red). A simulated structure of the first WW domain is shown. 1= 1st tryptophan; 2= Try33 phosphorylation site; 3= 2nd tryptophan. (These residues marked in yellow)
Human WWOX gene is considered as a tumor suppressor gene (1,2), and that this function is mediated through the encoded WOX1 protein and small size isoforms (if present). Functionally, WOX1 suppresses cancer growth and induces cancer cell death. A recent study shows that whole body knockout of murine Wwox gene induces spontaneous multiple tumor formation in vivo (3). Human WWOX gene is mapped to a common fragile site on chromosome ch16q23.3-24.1 (1,2). Numerous studies have shown alterations of WWOX gene occur in many types of cancer cells (1,2). Nonetheless, majority of the studies failed to stage the occurrence of genetic alterations during cancer development. A general consensus is that WOX1 protein disappears when cancer cells become invasive or start to metastasize. WWOX gene alterations, including epigenetic modifi cation and translational blockade, contribute to the disappearance of WOX1 protein in cancer cells.

How does WOX1 work? – Invasive cancer cells are devoid of WOX1. Restoration of WOX1 in these cells blocks their growth both in culture and in nude mice. WOX1 possesses specific structural units so that it can cause cell death. WOX1 possesses a nuclear localization sequence (NLS), two N-terminal WW domains (containing conserved tryptophan residues) and a C-terminal shortchain alcohol dehydrogenase/reductase (SDR) domain (Figure 1). Presence of NLS allows translocation of WOX1 to the nuclei. WW domain is a short stretch of amino acid residues (~40 amino acids) that forms a conserved protein module so that it can interact with proteins containing proline-rich motifs. SDR domain is a structural unit of an enzyme that normally metabolizes steroid hormones and small substrates. However, the function of SDR domain in WOX1 is largely unknown.

Figure 2. WWOX/WOX1 binding partners and actions. Route 1, Stress-induced activation of WOX1 involves Tyr33 phosphorylation and translocation to the mitochondria and nuclei. Phosphorylated WOX1 (p-WOX1) binds Ser46-phosphorylated p53 and translocates to the mitochondria. Route 2, Ectopic WOX1 binds and triggers redistribution of nuclear p73 and AP-2 to the cytoplasm, and compete with YAP compete for interaction with ErbB-4 to relocate to the nuclei. Route 3, In neurons, WOX1 prevents enzyme-mediated Tau hyperphosphorylation and tangle formation (see other Routes in Ref. #1).
The WW domains bind transcription factors such as p53, p73, AP-2 and ErbB-4 (1). Both the N-terminal WW and C-terminal SDR domains of WOX1 are involved in binding interactions. WW domain-containing proteins normally interact with proline-rich motifs such as PPPXY (P= proline, X= any amino acid; Y= tyrosine). p73, AP-2 and ErbB-4 possess this motif. Under stress conditions, WOX1 undergoes phosphorylation at Tyr33 and translocates to the mitochondria and nuclei to induce cell death both in vivo and in vitro (1). The Tyr33-phosphorylated WOX1 interacts with a large number of proteins mainly in the signal pathways. This includes signal transducers (e.g. TRADD, TRAF2, Smads, Hyal-2, p53, and JNK1) in the tumor necrosis factor (TNF), transforming growth factor beta (TGF-), hyaluronan and stress signal pathways (Figure 2). These signals are mainly for cell growth inhibition. These findings reveal a critical role of WOX1 in controling signal transduction and maintaining cell homeostasis.

More than a tumor suppressor!! – The presumptive tumor suppressor role of WOX1 has been raised into question. WOX1 supports cell growth and differentiation during embryonic development. For instance, in murine fetuses WOX1 is present prevalently in the heart, skin, brainstem, spinal cord and many tissues and organs, but its expression is decreased after birth (1). WOX1 also participates in the normal skin epithelial cell proliferation and eventual cornification. The whole body Wwox gene knockout mice could not survive beyond 40 days, probably due to defective organ differentiation (3). Intriguingly, during the early stages of cancer development (e.g. in breast, prostate, skin and brain) upregulation of WOX1 and isoform WOX2, along with phosphorylation at tyrosine 33 (Tyr33), occurs (Figure 1) (1). The increased levels of phosphorylated WOX1 and WOX2 are essential for cancer cell survival. Thus, these observations run against the tumor suppressor role of WOX1. We believe that WOX1 supports cell survival under physiological conditions, whereas it may exert cell death or control tumor growth under environmental stress.

WOX1 in neurodegeneration – One of the hallmarks of Alzheimer´s disease (AD) is the presence of neurofibrillary tangles (NFT) in the neuronal cells. Accumulation of NFT in neurons induces cell death. Deregulated and excessive phosphorylation of cytoplasmic Tau protein induces its polymerization and subsequent generation of fibrous structures. Numerous cytoplasmic enzymes hyperphosphorylate Tau in the AD brains, thereby generating NFT in the AD neurons. We found that the protein levels of WOX1, WOX2, and their phosphorylated forms are significantly reduced in the neurons of AD hippocampi, which is in contrast to the increased phosphorylation of Tau in the hippocampal neurons of AD (1). We were able to reproduce the in vivo finding in vitro. We introduced small interfering (siRNA) to suppress the expression of WOX1 protein in cultured neuronal cells, and found that there is a spontaneous induction of Tau phosphorylation and NFT formation in these cells. Enzymes responsible for hyperphosphorylating Tau are GSK-3, cdk5, JNK, ERK and several others. Their activities are increased in the neuronal cells when the levels of WOX1 are low. Accordingly, WOX1 is likely to play a critical role in regulating Tau phosphorylation in vivo.

Concluding Remarks – Endogenous WOX1 supports embryonic development and differentiation, and probably plays a homeostatic role in normal cell cycle progression in concert with p53, p73, ErbB4, and other transcription factors. However, environmental stress can turn WOX1 into a proapoptotic protein. It is intriguing how WOX1 supports cell growth at one time but becomes apoptotic under exogenous stress conditions. For cancer cells, however, they are defective in producing WOX1 at the invasive stage. Favorable clinical outcome in patients is associated with elevated expression of WOX1 in cancer specimens. Thus, a novel strategy to fight cancer is developing small molecular chemicals for upregulating WOX1 expression in cancer cells.

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