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Clinical Cancer Research Vol. 11, 5769-5777, August 15, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

WOX1 Is Essential for UVB Irradiation–Induced Apoptosis and Down-Regulated via Translational Blockade in UVB-Induced Cutaneous Squamous Cell Carcinoma In vivo

Feng-Jie Lai1, Ching-Li Cheng4,5, Shur-Tzu Chen2,4, Chin-Han Wu1,4, Li-Jin Hsu3, J. Yu-Yun Lee1, Sheau-Chiou Chao1, Maw-Chang Sheen6, Ching-Liang Shen5, Nan-Shan Chang7 and Hamm-Ming Sheu1

Authors' Affiliations: Departments of 1 Dermatology, 2 Anatomy, 3 Microbiology and Immunology, and 4 Institute of Basic Medical Sciences, National Cheng Kung University; 5 National Tainan Institute of Nursing, Tainan, Taiwan; 6 Department of Surgery, Kaohsiung Medical University, Kaohsiung, Taiwan; and 7 Laboratory of Molecular Immunology, Guthrie Research Institute, Sayre, Pennsylvania

Requests for reprints: Feng-Jie Lai and Hamm-Ming Sheu, Department of Dermatology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 70403, Taiwan. Phone: 886-6-2004326; Fax: 886-6-2004326; E-mail: laifj{at}mail.ncku.edu.tw and hmsheu{at}mail.ncku.edu.tw.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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 WOX1 and family proteins (WWOX) in human primary cutaneous SCCs was examined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. UVB irradiation–induced WOX1 activation (Tyr33 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 Tyr33 phosphorylation occurred during normal keratinocyte differentiation before cornification and death. Interestingly, significant reduction of these proteins and Tyr33 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, WOX1 was 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 Tyr33 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 UVB-induced 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.

Both skin basal cell carcinoma and squamous cell carcinoma (SCC) represent the first and second most common malignancies in humans (1). The incidence of these types of skin cancer has increased greatly for the past decade. However, the etiology is largely unknown (1). Unlike basal cell carcinoma, cutaneous SCC is malignant and tends to spread or metastasize (2). Cutaneous SCC is believed to originate from the squamous cells located at the suprabasal epidermis (3).

Human WWOX gene, encoding the WWOX/FOR/WOX1 family proteins, is mapped to a fragile site on chromosome 16q23.2 (4, 5). Loss of heterozygosity of WWOX gene has been shown in numerous types of cancers, including breast, ovarian, esophageal, lung, pancreatic, gastric, and other carcinomas (4–13). Whether this gene is altered in cutaneous SCC has not been documented. Also, the role of WOX1 family proteins in the development of SCC is unknown.

WOX1 is considered as a candidate tumor suppressor protein. There are at least eight spliced variants of human WWOX mRNA (14). Presence of a 46 kDa WOX1 and a 41 kDa WOX2 has been shown in human brains (15). Small-sized, aberrant transcripts of WWOX gene have been found in several types of tumors (9, 10) and cell lines (7, 16). This seems to correspond to the presence of small-sized proteins in different types of tumors.8

The full-length WOX1 possesses two NH2-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence and a COOH-terminal short-chain alcohol dehydrogenase/reductase domain (4, 5, 17). Ectopically expressed WOX1 induces apoptosis in numerous cultured cancer cells (17, 18) and suppresses tumor growth both in vitro and in vivo (19). Under stress conditions, there is an increased association between WOX1 and p53 in vivo and both proteins induce apoptosis synergistically (17, 18).

In this study, we investigated WWOX gene expression and protein levels in epidermal tissues of patients with primary and metastatic cutaneous SCCs by in situ hybridization, reverse transcription-PCR (RT-PCR) and immunohistochemistry. By using pan- and species-specific antibodies, we assessed the expression levels of WOX1, WOX2, total family proteins (WWOX), and Tyr33 phosphorylation in the first WW domain (p-WOX1) in cutaneous SCC samples. UV light has been shown to activate WOX1 via Tyr33 phosphorylation and nuclear translocation in vitro (18). Nonetheless, Tyr33-phosphorylated WOX1 seems to play a critical role in the progression of breast and prostate cancers in patients (20). We determined whether WOX1 participates in UVB irradiation–induced apoptosis of cultured skin cells and formation of cutaneous SCC in hairless mice.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Clinical cases. Consecutive cases of cutaneous SCC samples were retrospectively retrieved from the tissue bank of the Department of Dermatology, National Cheng Kung University Hospital, Taiwan. They were 25 well-differentiated, 6 moderately differentiated, 9 poorly differentiated, and 13 metastatic SCCs. Clinicopathologic data were collected from medical records. All cases were independently reviewed by two of the authors (J.Y-Y. Lee and H-M. Sheu). Histologic typing and grading of cutaneous SCCs were evaluated according to Lever's classification (2).

UVB irradiation on mouse skin. Twelve hairless SKH-hr1 female mice, 6-week-old, were purchased from Charles River Laboratories (Wilmington, MA) and housed in individual cages in a room with a constant temperature and humidity and an alternating 12-hour light and dark cycle. The mice were fed ad libitum with a commercial diet and water. We followed an approved protocol for animal use from the Institutional Animal Care and Use Committee of this university.

In acute exposure, three mice were exposed to UVB (2.16 kJ/m2; 312 nm) once and sacrificed 1 day later. A BLE-8T312 UV lamp was from Spectronics (Westbury, NY) and housed in a BIO-LINK BLX chamber (Vilber Lourmat, France). In chronic exposure, mice were exposed to UVB thrice per week (Monday, Wednesday, and Friday) starting with 0.36 kJ/m2, respectively, for 1 and 5 months (n = 3), followed by increasing 100% weekly. After week 10, a consistent dose of UVB irradiation (2.16 kJ/m2) was given over the next 8 weeks. In a control group, mice received no UVB irradiation.

Cell lines. Human SCC-25 cells, a well-differentiated tongue SSC, grown in a 50:50 mixture of DMEM and F12, containing 10% fetal bovine serum and 1% antibiotic-antimycotic (Life Technologies/Invitrogen, Carlsbad, CA). Immortalized human keratinocyte HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL). These cells were from American Type Culture Collection (Manassas, VA).

WOX1 knockdown by small interfering RNA. A plasmid construct for generating small interfering RNA (siRNA)–targeting human and mouse WOX1 (WOX1si) was made in pSuppressorNeo (Imgenex, San Diego, CA) as described (15). Also, a negative control vector with a scrambled sequence was from Imgenex. SCC-25 and HaCaT cells were cultured overnight in 6- or 24-well plates and transfected with these constructs by FuGENE 6 (Roche, Indianapolis, IN) according to the protocol of the manufacturer. Twenty-four hours later, these cells were treated with or without UVB irradiation (600 J/m2), followed by culturing for 2 hours and fixing with 4% paraformaldehyde in PBS for immunocytochemistry. Where indicated, the cells were harvested and processed for Western blotting (15). Apoptotic nuclei were assessed morphologically using Hoechst staining.

Antibodies and immunohistochemistry. Two pan-specific antibodies, recognizing human, mouse, and rat WOX1 and other family proteins, were against (a) an NH2-terminal region between the first and second WW domains of murine WOX1 (17) and (b) the first WW domain of murine WOX1 (21). Also, we used species-specific antibodies against the unique COOH termini of human WOX1 (WWOXv1; ref. 15) and WOX2 (WWOXv2; ref. 15), and Tyr33 phosphorylation in the first WW domain (18). The quality of these antibodies has been tested repeatedly (15, 17, 18, 20, 21). Antibody against a mutant p53 peptide was from BioGenex (San Ramon, CA).

Immunohistochemistry of human skin tissue sections (5 µm), using the above-mentioned antibodies, was done as described (15). In negative controls, antisera were preabsorbed with 100 µmol/L specific synthetic peptides for immunization (15). Assessment of the expression of WOX1 and other indicated family protein in each human tissue section was as follows: –, negative (0%); +, focal positive (<5% of tumor areas); ++, moderate positive (5-50% tumor areas); and +++, diffuse positive (>50% tumor areas).

Generation of complementary RNA probes and in situ hybridization. Total RNAs were isolated from five cases of the primary cutaneous SCCs and adjacent normal epidermal tissues by TRIzol reagent (Invitrogen). Two micrograms of total RNA from the specimens were reverse-transcribed (22). Single-stranded, digoxigenin-labeled WOX1 cRNA probes were generated using a two-step PCR amplification and in vitro RNA transcription (22). Briefly, amplifications for 35 cycles under standard conditions were done using the following primer pairs: WOX1 (sense) 5'-AAAACGACTATTGGGCGATG-3'; T7 + WOX1 (antisense) 5'-ACTCACTATAGGGAGAGTGTTGGAGGGACATTTGGA-3'. The purified PCR product was further amplified using the above sense primer of WOX1 and another composite primer (universal T7; 5'-TAAGCTTTAATACGACTCACTATAGGGAGA-3') to extend and complete the full-length 23 bp T7 promoter. One microgram of the secondary PCR products served as templates for RNA transcription using a DIG RNA Labeling Kit (Roche). A sense cRNA probe was made for negative controls. The generated antisense or sense cRNA was used for in situ hybridization and the signals were detected with an enzyme-linked immunoassay kit (Roche; refs. 22, 23). The extent of WWOX mRNA expression was analyzed as indicated above.

Reverse transcription-PCR. To quantify the relative WWOX mRNA levels in cutaneous SCCs and adjacent normal tissues, both nested and multiplex RT-PCR were done. Nested RT-PCR was done to amplify the coding region of WWOX mRNA (8). The primer pairs were as follows: forward 5'-AGTTCCTGAGCGAGTGGACC-3' and reverse 5'-TTACTTTCAAACAGGCCACCAC-3' for the first amplification, and forward 5'-AGGTGCCTCCACAGTC-3' and reverse 5'-GTGTGTGCCCATCCGCTCT-3' for the secondary amplification. In comparison, multiplex RT-PCR was carried out to amplify the exon 9 of WWOX mRNA transcripts using specific primers as follows: WOX1 forward, 5'-AAAACGACTATTGGGCGATG-3'; WOX1 reverse, 5'-GTGTTGGAGGGACATTTG GA-3'; ß-actin forward, 5'-AGCGGGAAATCGTGCGTG-3'; ß-actin reverse, 5'-CAGGGTACATGGTGGTG-3'. The presence of exons 8 to 9 in the mRNA indicates presence of a full-length transcript. The PCR products were separated in 1.5% agarose gels, analyzed with a UV transilluminator, and scanned with a densitometer (ONE-Dscan 1.33 software, Scanalytics, Fairfax, VA; ref. 24). The ratios of relative mRNA densities between controls and SCC groups were calculated.

Statistical analysis. Appropriate statistical analyses were used as follows: (a) Fisher's exact test was used to determine the correlation of each patient's gender relative to the protein expression, including p53 and WOX1. (b) Also, the extent of WOX1 expression in cutaneous SCC versus normal epidermis was analyzed by {chi}2 test. (c) Paired Student's t test was used to compare the levels of WWOX mRNA in cutaneous SCC and the adjacent normal epidermis. All data were analyzed using SigmaStat software (Jandel Scientific, San Rafael, CA). P values <0.05 were considered as statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patients and tumor characteristics. Cutaneous SCC samples were from 53 patients, including 29 males and 24 females, with a mean age of 69.6 and a median age of 71 (between 44 and 96 years). Based on Lever's classification (2), these cases were 25 well-differentiated, 6 moderately differentiated, 9 poorly differentiated, and 13 metastatic SCCs.

Down-regulation of human WOX1, WOX2, total WWOXs, and Tyr33phosphorylation in cutaneous squamous cell carcinoma. To assess the expression of WOX1 and family proteins (if present) in cutaneous SCC, immunohistochemistry was done. Normally, the 46 kDa WOX1 is the major species found in the extracts of human tissues, organs, and cell lines. By using a pan-specific WOX1 antibody (against the first WW domain; ref. 21), we show that in normal epidermis WOX1 (and other family proteins) is expressed in the cytoplasm of the deepest suprabasal cells, and the expression is increased in intensity toward the superficial layers (Fig. 1A). WOX1 expression seems to reach a maximal extent in the nuclei of differentiated granular layers, before cornification and death (Fig. 1A). In negative controls, antiserum was preabsorbed with a corresponding synthetic peptide used for immunization. No immunoreactivity was observed (Fig. 1B). Alternatively, normal rabbit serum was used and produced no immunoreactivity (data not shown).



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  		Fig. 1. Down-regulation of WOX1 and family proteins (WWOXs) in cutaneous SCC. A specific antibody against an inter-WW domain region, recognizing mainly the 46 kDa WOX1 (and family members if present), was used for the following experiments (A-H). Similar results were observed using a specific antibody against the first WW domain (data not shown). A, all cell layers (except basal layer) are positive for expression of WOX1 in normal epidermis by immunostaining and the expression is gradually increased toward the outer layer of the epidermis. Nuclear localization of WOX1 is shown in the terminally differentiated stratum granulosa cells before cornification (arrow). B, in a negative control, no immunoreactivity was observed when WOX1 antibodies were preabsorbed with a corresponding synthetic peptide for immunization. C, in a well-differentiated SCC, WOX1 is expressed surrounding highly keratinized areas (horn pearls; *), but significantly reduced in the marginal areas (arrowheads). WOX1 is strongly expressed in nontumor infiltrating normal epidermis (arrow). D, WOX1 protein level is markedly decreased in a moderately differentiated SCC (top right; arrowhead) compared with the adjacent normal epidermis (top left; arrow). E and F, similarly, little or no WOX1 expression is shown in a poorly differentiated SCC (arrowheads; see F for a high magnification) versus the adjacent normal epidermis (arrow). G, again, lack of WOX1 expression is shown in an infiltrated poorly differentiated SCC (arrowheads) overlying with intraepidermal SCC in situ (Bowen's disease). The infundibulum of a hair follicle is WOX1 positive (arrow). H, WOX1 expression is barely detectable in a metastatic SSC (arrowheads) as opposed to normal epidermis (arrow). I, little or no Tyr33 phosphorylation in WOX1 (p-WOX1) is shown in a poorly differentiated SCC (arrowheads; see J for a high magnification) versus an adjacent normal epidermis (arrow; see K for a high magnification and nuclear translocation, arrow). L, in a well-differentiated SCC, p-WOX1 is present mainly in the cytoplasm (arrowhead) rather than in the nuclei. Similarly, down-regulation of WOX1 (M) and WOX2 (N) is observed in a poorly differentiated SCC as determined by using species-specific antibodies (see Materials and Methods). O, nuclear localization of mutant p53 was found in ~50% of SCC cases.

 
In parallel with the above observations, similar expression profiles of WOX1, WOX2, and total WWOXs were observed in normal skin tissues when we used species-specific antibodies against human WOX1 (15), WOX2 (15), and another pan-specific antibody (against an inter-WW domain region; ref. 17; data not shown).

Of all the cutaneous SCC cases analyzed, we found that expression of WOX1 (and other family proteins if present) was significantly down-regulated compared with the adjacent normal epidermal tissues (first WW domain antibody used; Fig. 1C-F and H; Table 1). In well-differentiated primary cutaneous SCC, WOX1 expression was relatively strong surrounding highly keratinized areas (horn pearls), but decreased gradually in the marginal areas (Fig. 1C). Significant down-regulation or absent expression of WOX1 was observed in SCC with moderate and poor differentiation (Fig. 1D-G). In metastatic SCC, WOX1 expression was barely detectable (Fig. 1H). By using human WOX1- and WOX2-specific antibodies, we showed that these proteins were significantly down-regulated in the poorly differentiated SCC (Fig. 1M and N).


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  		Table 1. Grading of WWOXs expression in SCC and adjacent normal epidermis

 
Tyr33 phosphorylation in WOX1 (p-WOX1) plays an important role in the development of breast and prostate cancers before reaching metastasis (20). We determined that during the progression of cutaneous SCC from a well-differentiated status down to metastasis, Tyr33 phosphorylation was gradually decreased compared with the adjacent normal epidermal tissues (Fig. 1I-K). Nuclear translocation of p-WOX1 was observed along the line of keratinocyte differentiation (before cornification and death), particularly in the granular cell layers of normal skin (Fig. 1K). In contrast, nuclear translocation of p-WOX1 was less effective in poorly differentiated and well-differentiated cutaneous SCC (Fig. 1J and L).

The presence of mutant p53 proteins has been shown in cutaneous SCCs (25). WOX1 binds wild-type p53, and both proteins are capable of inducing apoptosis in a synergistic manner (17, 18). Whether WOX1 interacts with p53 mutants and whether this interaction affects cancer progression are not known and remain to be elucidated. In agreement with other reports (25), we showed the presence of mutant p53 proteins in cutaneous SCCs (Fig. 1O; data not shown for moderate and poorly differentiated SCCs).

Histologic grading and down-regulation of WOX1 expression in cutaneous squamous cell carcinomas. We verified our findings by histologic grading and showed a significant reduction of expression of WOX1 and family proteins (or WWOXs) in SCCs, compared with neighboring normal epidermis from the same patient (P < 0.001; n = 53; Table 1). For example, ~100% expression for WOX1 (and family proteins), grading at 1+, 2+, and 3+, was observed in the normal epidermis, as opposed to 58.5% in SCCs (31 of 53).

Alternatively, we examined the extent of tumor differentiation relative to the expression of WOX1 (and family proteins). Less differentiated and metastatic SCCs have a diminished or absent expression of WOX1 (and family proteins; P < 0.01; Table 2). In parallel with other reports (6–13, 20, 26), poorly differentiated and metastatic tumors normally have reduced levels of WOX1 and family proteins. Finally, there was no positive correlation for WOX1 (and family proteins) protein expression among gender groups (P > 0.05; data not shown). Presence of p53 mutant proteins was ~50% of SCC cases (26 of 53). However, there was no positive correlation for the expression of mutant p53 relative to WOX1 (and family proteins) in cutaneous SCCs (P > 0.05; Table 2).


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  		Table 2. Correlation between WWOXs or p53 immunoreactivity and histologic grades

 
WWOX mRNA is not down-regulated in cutaneous squamous cell carcinoma. Next, we examined whether down-regulation of WOX1 and family proteins correlates positively with reduced levels of WWOX mRNA in tissue sections. To maximize the specificity for the full-length WWOX mRNA by in situ hybridization, we selected exons 8 to 9 of human WWOX gene for designing both sense and antisense probes. Hybridizing with the antisense probe showed that WWOX mRNA was clearly present from the suprabasal layer to the granular layer of normal epidermis (Fig. 2A, a), which is consistent with the results from immunohistochemistry (Fig. 1A). In negative controls, the sense probe could not generate signals (Fig. 2A, b), thus confirming the specificity of the antisense probe. Remarkably, there was no significant reduction for WWOX mRNA expression in cutaneous SCC with well (Fig. 2A, c), moderate (Fig. 2A, d), or poor (Fig. 2A, e-g) differentiation, or in metastatic cutaneous SCC (Fig. 2A, h), compared with adjacent normal epidermis (Fig. 2A, c-h).



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  		Fig. 2. No significant down-regulation of WWOX mRNA expression in cutaneous SCCs. Shown in (A) are representative photomicrographs from in situ hybridization of sections of normal epidermis and SCCs. a, WWOX mRNA is expressed mainly in spinous and granular layers of normal epidermis (arrow). b, in a negative control, a sense probe was used for in situ hybridization. c to e, no significant differences in WWOX mRNA expression are shown in normal epidermis (arrow) and SCCs (arrowheads) with well, moderate, and poor differentiation, respectively. f, cytoplasmic localization of WWOX mRNA is shown (a high magnification from e). g, similarly, WWOX mRNA is strongly expressed in an infiltrated poorly differentiated SCC (arrowheads) overlying with SCC in situ (Bowen's disease). The infundibulum of a hair follicle is also WWOX mRNA positive (arrow; see Fig. 1G for comparison). h, WWOX mRNA is not down-regulated in metastatic SCC (arrowheads) compared with normal epidermis (arrow). In (B), no significant down-regulation in the WWOX mRNA levels were observed in primary cutaneous SCCs. a, semiquantitative, nested RT-PCR for the full-length coding region of WWOX mRNA showed that there were no significant difference between the adjacent normal skin and cutaneous SCCs (n = 5). b and c, multiplex RT-PCR was done for WWOX (exon 9) and ß-actin mRNAs from five representative samples of cutaneous SCCs and adjacent normal epidermal tissues. As normalized to controls, the relative expression of WWOX mRNA is shown (P > 0.05; n = 5; paired Student's t tests). M, marker; N, normal epidermis; T, tumor.

 
We further verified the above observations by RT-PCR. By semiquantitative, nested RT-PCR amplification, there were no significant differences in the levels of WWOX mRNA (full-length coding region) between cutaneous SCC and adjacent normal epidermis (n = 5; Fig. 2B, a). By using the same samples, we amplified the exons 8 to 9 of WWOX gene transcripts and ß-actin (Fig. 2B, b). As normalized to ß-actin, WWOX mRNA expression was not significantly reduced in the SCC versus normal epidermis (Fig. 2B, c).

Effects of acute and chronic UVB exposure on WOX1 protein expression and Tyr33 phosphorylation in hairless mouse skin. Control unirradiated mice did not develop skin lesions (Fig. 3A, a). In contrast, in the acute irradiation group, multiple sunburn cells appeared in the epidermis at day 1 (Fig. 3A, b). In the chronic irradiation group, initial tumors appeared at week 15 and continued to develop. These mice were sacrificed at week 20. Six large-sized skin lesions were isolated, analyzed by routine histologic method and shown to be cutaneous SCCs (Fig. 3A, c).



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  		Fig. 3. Acute and chronic UVB exposures on hairless mouse skin. A, representative photomicrographs (H&E stain) of skin sections: a, normal epidermis; b, presence of discrete sunburn cells (arrows) at day 1 after acute UVB exposure; c, formation of well-differentiated cutaneous SCC at month 5. B, at day 1 after acute UVB irradiation, up-regulation of WOX1 and family proteins (WWOX) is shown in the epidermis, especially in the sunburn cells (b, arrow), compared with the unirradiated normal epidermis (a). WWOXs expression was further up-regulated in the hyperplastic epidermis at month 1 (data not shown). In contrast, at month 5, expression of WWOXs was significantly down-regulated in well-differentiated SCCs and SCC in situ (c, *). Nonetheless, up-regulated WWOXs were still present in the peripheral hyperplastic skin (c, arrows). C, a similar profile is shown regarding WOX1 activation via Tyr33 phosphorylation (p-WOX1). UVB-activated WOX1 in sunburn cells at day 1 (b, arrow) and in hyperplastic epidermis at month 1 (c). By month 5, WOX1 activation is down-regulated in a well-differentiated SCC (d, arrowheads; see e for a high magnification), except in peripheral hyperplastic skin areas (f). D, representative photomicrographs from in situ hybridization of sections of SCCs and epidermal hyperplasia. a, no significant differences in WWOX mRNA expression are shown between SCCs (arrowheads) and adjacent epidermis (arrow). b, cytoplasmic localization of WWOX mRNA is shown (a high magnification from a). c, similarly, WWOX mRNA is strongly expressed in peripheral hyperplastic skin areas.

 
After day 1 of irradiation, significant increases in the immuno-intensity of WOX1 (and family proteins if present; Fig. 3B, b) and p-WOX1 (Fig. 3C, b) on the whole epidermis were observed, including discrete cells with high levels of expression in the cytoplasm and nuclei. After 1 month of UVB irradiation, formation of epidermal hyperplasia was observed, with up-regulation of WOX1 and p-WOX1 protein (Fig. 3C, c; data not shown for WOX1). Following 5 months of UVB irradiation, tumors were present in all hairless mice. Of all the cutaneous well-differentiated SCC cases analyzed, expression of WOX1 and p-WOX1 was significantly down-regulated (Fig. 3C, d and e; data not shown for WOX1), compared with the adjacent hyperplastic epidermis (Fig. 3C, f). A reduced level of WOX1 expression was also observed in the UVB-induced SSC in situ (Fig. 3B, c, asterisk), compared with the adjacent hyperplastic epidermis (arrows).

Notably, by in situ hybridization, we found that there was no significant reduction in the levels of WWOX mRNA in the UVB-induced SCC (Fig. 3D, a and b) versus the adjacent hyperplastic epidermis (Fig. 3D, c). These observations are in parallel with our clinical findings (Fig. 2).

As summarized in Fig. 4, we illustrate the levels of WOX1 (and family proteins) expression along the line of normal keratinocyte differentiation and cutaneous SCC formation in both human and hairless mouse. Briefly, there is a gradual increase in the expression of WOX1 during keratinocyte differentiation and significant down-regulation of WOX1 and WOX2 (and others) in human SCCs, which is in contrast to the relatively stable levels of WWOX mRNA. In comparison, UVB-induced epidermal hyperplasia and SCC formation in hairless mice show an initial up-regulation of WOX1 protein levels followed by down-regulation in cutaneous SCC. Again, in parallel with the clinical findings in humans, essentially little or no reduction of WWOX mRNA expression is shown in these samples from hairless mice.



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  		Fig. 4. A schematic model of expression of WOX1 protein and mRNA in human and hairless mice. A gradual increase in the expression of WOX1 (and family proteins if present) is shown during normal keratinocyte differentiation in both human and mice. Significant down-regulation of WOX1 and WOX2 (and others) in human SCCs, which is in contrast to the relatively stable levels of WWOX (or WOX1) mRNA. In comparison, UVB-induced epidermal hyperplasia and SCC formation in hairless mice show an initial up-regulation of WOX1 protein levels, followed by down-regulation in cutaneous SCC. Again, in parallel with the clinical findings in humans, there is essentially little or no reduction of WWOX mRNA expression in these samples from hairless mice.

 
WOX1 knockdown cells resist UVB-induced apoptosis. Finally, we determined the role potential of WOX1 in UVB-induced apoptosis in vitro and SCC formation in vivo. UV light is known to activate WOX1, and this activation seems to play an important role in cell death both in vivo and in vitro (18, 19, 27). Both SCC-25 and HaCaT cells were transfected with a siRNA construct targeting WOX1 expression (WOX1si). In controls, cells were transfected with a control construct expressing a scrambled siRNA. These cells express very low levels of WOX1. Transient expression of WOXsi in SCC-25 cells suppressed UVB-induced WOX1 protein expression by >70%, compared with control cells expressing a scrambled siRNA, as determined by cell immunostaining and Western blotting (Fig. 5A). In these WOX1-knockdown cells, phosphorylation of WOX1 was significantly decreased (Fig. 5B). Transient expression of WOXsi in SCC-25 cells significantly suppressed UVB-mediated cell death, compared with control cells expressing a scrambled siRNA (Fig. 5C). The extent of apoptotic cells was determined according to nuclear morphology from Hoechst stain. Similar results were observed with HaCaT cells (Fig. 5D).



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  		Fig. 5. WOX1 is essential for UVB-induced apoptosis. A, transient expression of siRNA-targeting WOX1 (WOXsi; in pSuppressorNeo plasmid) in SCC-25 cells suppressed WOX1 protein expression in 24 hours. WOX1si inhibited UVB-induced WOX1 protein expression by >70% compared with scrambled siRNA-expressing cells (see both immunostaining and Western blotting). B, similarly, Tyr33 phosphorylation of WOX1 was significantly decreased (>70%) in the WOX1 knockdown SCC-25 cells after UVB exposure. C, UVB-induced apoptosis in the scrambled siRNA-expressing SCC-25 cells (>80%), but not in the WOX1si-expressing cells (30%<, n = 3; determined by nuclei morphology from Hoechst staining). D, similar results were observed when testing keratinocyte HaCaT cells (30%<, n = 3).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown for the first time that the proapoptotic WOX1 is essential for UVB-induced cell death. Knockdown of WOX1 by siRNA abolishes epidermal cell death caused by UVB. Previously, we have shown that WOX1 induces apoptosis in a caspase-independent manner (17). WOX1 can mediate cell death synergistically with p53 (18). Without WOX1, p53 apoptosis is abolished (18). Nonetheless, WOX1 can act alone in causing cell death without p53. Furthermore, we cannot show a positive correlation between the expression of mutant p53 with that of WOX1 in SCCs.

Sun-exposed body parts, such as face, shoulders, hands, back, and others, are vulnerable to develop SCCs. Chronic and long-term exposure to sunlight is believed to cause most cases of SCCs. Epidermal cells undergo proliferation, maturation, and death to maintain a homeostatic status of the skin (28). Dysregulation of the apoptotic process may lead to uncontrollable growth of epithelial cells, thus leading to skin cancer (28). WOX1 is strongly expressed in cells of epithelial origin, not only in the skin but also in mucous linings of digestive, mammary, reproductive, and respiratory systems. We propose that WOX1 plays a role in homeostasis and turnover of proliferating epidermal cells. In normal epidermis, WOX1 expression is gradually increased toward the outer layer of epidermis. That is, the level of WOX1 seems to reach maximally in keratinocytes at stratum granulosa of the epidermis before their cornification and death. Up-regulation of WOX1 occurs along the line of keratinocyte differentiation, suggesting that WOX1 is involved in terminal differentiation and death of these cells. Accordingly, a likely scenario for the development of cutaneous SCCs is due to successful prevention of the apoptotic pressure from overexpressed WOX1 in skin cells. Shutting down the translation machinery for WWOX mRNA allows continuous growth of skin cells to a cancerous state.

Our clinical findings support the above assumption. We showed significant down-regulation of the protein levels of WOX1 and family proteins in patients with cutaneous SCCs. This down-regulation is not due to suppression of WWOX mRNA (full length) expression in these samples, thus indicating that WWOX gene is not altered in SCC and the expressed mRNA is relatively stable. Apparently, a translational blockade of mRNA to protein occurs in these cancer cells that accounts for the suppression of WOX1, WOX2, and other member proteins in cutaneous SCCs. Our observations are evidenced by studies using immunohistochemistry, statistical analyses, in situ hybridization, and RT-PCR.

Furthermore, we showed that UVB induces WOX1 expression and activation in mouse epidermal cells, accompanied by cell death, during acute exposure. However, during chronic exposure, we found that UVB-induced hyperplasia of skin cells is associated with up-regulation of WOX1 expression and activation. Down-regulation of WOX1 is observed when these cells reach cancerous and metastatic states. Recently, we have shown that WOX1 supports the progression of breast and prostate cancers to a premetastatic state (20). Thus, development of SCC seems to follow a similar pattern as those of breast and prostate cancers regarding their WOX1 expression. That is, an initial up-regulation and activation of WOX1 is essential to support skin cell hyperplasia, followed by down-regulation of WOX1 for cancer progression.

WW domain–containing proteins are known to bind proline-rich motifs (29). Nonetheless, phosphorylation of the conserved Tyr33 in the first WW domains enables WOX1 to interact with numerous proteins independently of the proline binding. For example, WOX1 binds p53 and JNK1 in a Tyr33 phosphorylation–dependent manner (17, 18). During the progression of prostate and breast cells from hyperplasia to a premetastatic cancerous state, Tyr33-phosphorylated WOX1 is mainly located in the mitochondria (20), suggesting that WOX1 functions through its COOH-terminal alcohol dehydrogenase/reductase domain to maintain mitochondrial homeostasis and cell survival. Subcellular localization of WOX1 during the progression of SCC remains to be established. However, we believe that WOX1 is likely to be present in the mitochondria during skin hyperplasia.

Numerous genetic changes are known during the malignant progression of cutaneous SCC, including genes coding for p16INK4a, p53, and RAS (30). Furthermore, activation of telomerase and constitutive activation of epidermal growth factor receptor in malignant SCC have been shown (30). Nonetheless, the molecular pathways, which lead to progression and malignancy of SCC, are largely unknown. Our study has provided a novel mechanism regarding the participation of WOX1 in the progression of cutaneous SCC. Functional interactions between WOX1 and other proteins in SCC cells remain to be established.

Finally, dysregulation of the machinery for transcription and translation may account for generation of malignant phenotype (31). p53 participates in controlling the translation of specific mRNAs, such as those encoding cdk4, FGF-2, and p53 itself (31). Nonetheless, p53 does not regulate the transcription of WWOX mRNA, as determined in p53-knockout mice (21). Whether p53 affects the translation of WWOX mRNA remains to be established.


   Acknowledgments
 
We thank Ming-Shih Li, Yu-Hsiu Lee, Ya-Chuan Su, Pei-Jung Huang, Shih-Shih Chang, and Jui-Chu Tseng for technical assistance; Chu-Miao Chen for sample collection; the laboratories of Dr. Nan-Shan Chang (Guthrie Research Institute, Sayre, PA) and Dr. Robert I. Richards (University of Adelaide, Adelaide, Australia) for the production of species-specific antibodies against human WOX1 and WOX2; and Terri Zimmer (Guthrie Research Institute) for antibody production in rabbits.


   Footnotes
 
Grant support: National Science Council grants 91-2314-B-006-116 and 93-2314-B-006-029 (H-M. Sheu) and 91-2320-B-439-001 (C-L. Shen), Taiwan, ROC, and Department of Defense grant DAMD17-03-1-0736 (N-S. Chang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: N-S. Chang and H-M. Sheu contributed equally as senior authors.

8 N-S. Chang et al., unpublished data. Back

Received 11/15/04; revised 5/ 9/05; accepted 5/27/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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