Basal cell carcinomas (BCC), which are the most common form of skin malignancy, are invariably associated with the deregulation of the Sonic Hedgehog (Shh) signalling pathway. As such, BCC represent a unique model for the study of interactions of the Shh pathway with other genes and pathways. We constructed a tissue microarray (TMA) of 75 paired BCC and normal skin and analysed the expression of β-catenin and RUNX3, nuclear effectors of the wingless-Int (Wnt) and bone morphogenetic protein/transforming growth factor-β pathways, respectively. In line with previous reports, we observed varying subcellular expression pattern of β-catenin in BCC, with 31 cases (41%) showing nuclear accumulation. In contrast, all the BCC cases tested by the TMA showed RUNX3 protein uniformly overexpressed in the nuclei of the cancer cells. Analysis by Western blotting and DNA sequencing indicates that the overexpressed protein is normal and full-length, containing no mutation in the coding region, implicating RUNX3 as an oncogene in certain human cancers. Our results indicate that although the deregulation of Wnt signalling could contribute to the pathogenesis of a subset of BCC, RUNX3 appears to be a universal downstream mediator of a constitutively active Shh pathway in BCC.
Cutaneous basal cell carcinomas (BCC) comprise approximately 80% of non-melanoma skin cancers and have been reported as the most common human malignancy in the United States (Rubin et al., 2005). The molecular pathogenesis of BCC bears the distinction of being almost entirely associated to the deregulation of the Sonic Hedgehog (Shh) signalling pathway (for a recent review on this topic, see Daya-Grosjean and Couve-Privat, 2005). Originally identified as a determinant of segment polarity in Drosophila, Shh is a secreted glycoprotein that plays a major role in vertebrate development. The binding of Shh to its receptor, the 12-pass transmembrane protein patched homologue 1 (PTCH1), relieves the suppression of another transmembrane protein, the G-protein-coupled receptor, smoothened (SMO). SMO in turn initiates a signalling cascade that leads to the activation of the Ci-like (GLI) family of transcription factors, which are the primary effectors of the Shh signal. Mutations in Shh, PTCH1, SMO and GLI have all been identified in BCC cases, with loss-of-function mutation in PTCH1 identified in 100 and 12–38% of familial and sporadic BCC cases, respectively (Dahmane et al., 1997; Unden et al., 1997; Daya-Grosjean and Couve-Privat, 2005).
Similar to the Shh pathway, the wingless-Int (Wnt) and bone morphogenetic protein/transforming growth factor-β (BMP/TGF-β) pathways play central developmental roles that are conserved from the fruitfly to human (Chen and Meng, 2004; Logan and Nusse, 2004). Mutations and deregulation of the Wnt pathway are the characteristic features of many human cancers, most notably in intestinal malignancies (Gregorieff and Clevers, 2005). Several lines of evidence hint at a role for the Wnt pathway in BCC. Firstly, the Wnt pathway is involved in hair follicle morphogenesis and its ligands are known targets of Shh signalling (Reddy et al., 2001; Andl et al., 2002). Secondly, the Wnt pathway has been linked to other skin malignancies: 75% of human pilomatricomas cases bear activating mutations of β-catenin (Chan et al., 1999), and the deregulation of Wnt pathway is linked to the progression of human melanomas (Rubinfeld et al., 1997; Weeraratna, 2005). For these reasons, the involvement of Wnt signalling in BCC pathogenesis has been keenly evaluated. Specifically, efforts have been focused on the incidence of nuclear accumulation of β-catenin, a hallmark of constitutive activation of Wnt signalling, in BCC tumor samples (Behrens et al., 1996; Huber et al., 1996). However, several such attempts to detect nuclear accumulation of β-catenin have yielded conflicting observations (Boonchai et al., 2000; Yamazaki et al., 2001; El-Bahrawy et al., 2003; Saldanha et al., 2004). To further verify the involvement of Wnt pathway in BCC, we constructed a tissue microarray (TMA) of 75 BCC tumors and corresponding normal skin samples, and analysed these samples for β-catenin expression and subcellular localization. Our study detected nuclear accumulation of β-catenin in 31 of 75 (41%) BCC tumor samples. Whereas the expression of β-catenin is uniformly restricted to the cell membrane in normal epidermis (Figure 1a and b), its expression varied considerably between BCC samples. Expression ranged from being undetectable (Figure 1c) to predominant expression in the nucleus (Figure 1d), cytoplasm (Figure 1e) or cytoplasm and membrane (Figure 1f). In certain cases, combined expression in the cytoplasm and nucleus, or membrane was observed (Figure 1d and f, respectively). Together, these observations suggest that activation of Wnt signalling is evident in a subset of BCC, but the subcellular localization of β-catenin varies significantly between tumor samples.
In a previous study, Boonchai et al. (2000) reported that nuclear β-catenin was undetectable in all 195 cases of BCC analysed. However, several subsequent studies using different antibodies were able to detect nuclear β-catenin in 23–70% of BCC samples (Yamazaki et al., 2001; El-Bahrawy et al., 2003; Saldanha et al., 2004). Although the precise reasons for such discrepancies is not clear, it has been suggested that this could be caused by differences in tissue fixation and processing (Yamazaki et al., 2001; Saldanha et al., 2004). Our findings are consistent with the latter reports and serve to validate the effectiveness and robustness of our TMA methodology.
In addition to Wnt ligands, Shh is also known to upregulate ligands of the BMP pathway, specifically BMP4 and BMP7 (Kawai and Sugiura, 2001; Gianakopoulos and Skerjanc, 2005). To investigate the involvement of this pathway in BCC, we studied the expression profile of RUNX3, a nuclear effector of the BMP/TGF-β pathways, and a key tumor suppressor gene in the gastric epithelium (Li et al., 2002; Ito and Miyazono, 2003; Bae and Choi, 2004). The runt-related (RUNX) family of transcription factors share homology with the Drosophila segmentation gene runt and encode the DNA-binding subunit of the heterodimeric transcription factor polyomavirus enhancer binding protein 2/core-binding factor complex (PEBP2/CBF) (for classification and nomenclature, see van Wijnen et al., 2004). RUNX1 is critical for the generation and maintenance of hematopoietic stem cells and is frequently targeted by chromosomal translocations and point mutations in human leukemia. Although RUNX2 is yet to be directly associated with human tumors, its oncogenicity has been demonstrated in mouse models, in cooperation with ectopically expressed c-myc (Blyth et al., 2005). RUNX3 was initially described as a candidate tumor suppressor in the gastric epithelium and is epigenetically silenced in greater than 50% of gastric cancer cell lines (Li et al., 2002). More recently, we have demonstrated that RUNX3 is not detectable in 43 of 97 (44%) cases of gastric cancer, and a further 38% showed mislocalization in the cytoplasm, therefore suggesting that RUNX3 is inactivated in >80% of gastric cancers (Ito et al., 2005). Reduced expression of RUNX3 has now been observed in numerous human malignancies, including bladder (Kim et al., 2005), liver (Mori et al., 2005), colorectal (Ku et al., 2004) and lung cancers (Yanada et al., 2005). Furthermore, RUNX3 point mutations have been discovered in human gastric and bladder cancers (Li et al., 2002; Kim et al., 2005).
Immunohistochemical staining of our TMA samples showed that RUNX3 protein is expressed in normal skin (Figure 2a), with distinct nuclear, mild-to-moderate positivity (grade 1–2) in approximately 75% of epidermal cells (Figure 2b). The expression is present in all the epidermal layers. The number of RUNX3-expressing cells is particularly prominent in the basal cell layer, but patchy in the prickle cell, granular cell and keratin layers. The expression is also prominent in the hair shaft, being more prominent in the outer root sheath of the pilosebaceous unit and the associated eccrine sweat glands (data not shown). The analysis of BCC samples showed that there is a strong, uniform (grade 3) nuclear expression of the RUNX3 in all 75 cases (Figure 2c and d). The expression was present in virtually 100% of the BCC neoplastic cell, irrespective of the histological subtype. Furthermore, expression of RUNX3 within the well-demarcated ‘islets’ of neoplastic cells is distinctly higher than that of the adjacent normal epidermal tissue. Taken together, RUNX3 appears overexpressed in BCC compared to the expression levels observed in normal epidermis.
We next sought to investigate the nature of the RUNX3 proteins expressed in BCC. Firstly, we performed Western blot analysis on normal skin (ATCC-CRL-7761) and BCC-derived (ATCC-CRL-7762) cell lines, using a RUNX3-specific monoclonal antibody R3-5G4 (Ito et al., 2005). Figure 3 shows that the RUNX3 proteins expressed in CRL-7762 cells are of the same length as that of ectopically expressed, full-length RUNX3 in transfected COS7 cells, suggesting that functional RUNX3 is expressed in BCC cells. Importantly, RUNX3 expression is markedly stronger in CRL-7762 compared to the normal CRL-7761, confirming the observation of RUNX3 overexpression in BCC TMA samples. Two RUNX3-related bands were detected in CRL-7762 and SNU5 cell lysates, which may represent different RUNX3 isoforms, although the doublet could also be caused by phosphorylated or proteolytically degraded forms of RUNX3.
To provide direct evidence that the RUNX3 proteins overexpressed in BCC are full-length and intact, we analysed the RUNX3 gene for point mutations. The entire RUNX3-coding region of CRL-7761 (normal), CRL-7762 (BCC-derived) and selected BCC tumors was sequenced. However, these analyses showed no evidence of mutation in the RUNX3-coding region. Therefore, our data indicate that the overexpressed RUNX3 in BCC is without structural alteration or mutation, and fully functional.
Although it is well established that deregulation of the Shh pathway plays a central role in the molecular pathogenesis of BCC, the specific targets and mechanisms through which tumorigenesis is effected remain elusive. Of equal interest is the potential involvement of other, well-characterized pathways, implicated in other human cancers, that cooperate with the breakdown in Shh signalling. A significant player appears to be the tumor suppressor gene p53, which is mutated in approximately 50% of sporadic BCC (Ziegler et al., 1993). Our data show that, in line with previous studies, β-catenin is targeted to the nucleus in some BCC samples. However, the frequency at which this occurs indicates that although Wnt signalling is involved in the progression of some BCC, it is not likely to be a primary mechanism by which the Shh signal is mediated. In contrast, overexpression of RUNX3 in the nucleus of neoplastic cells is observed in all of the 75 samples in our TMA, therefore strongly implicating a role for RUNX3 as an oncogene downstream of the Shh pathway. This striking observation is made all the more significant in view of the current understanding of RUNX3 as a tumor suppressor in several human malignancies, most notably in gastric cancer. Moreover, RUNX3 is also a downstream target of the TGF-β tumor suppressor pathway. Although all members of the RUNX family, most notably RUNX2, are known to promote tumorigenecity in mouse models (Cameron and Neil, 2004; Yanagida et al., 2005), our study implicates RUNX3 acting as a putative oncogene in human cancer.
An important implication of our findings is the possibility of cooperation between the Shh pathway and BMP/TGF-β pathways in BCC, which warrants a thorough investigation. It is well established that the Shh pathway is modulated via a negative feedback loop through PTCH1, as PTCH1 itself is a positive target of GLI3 (Goodrich et al., 1996; Marigo and Tabin, 1996). In BCC, this intrinsic circuitry control is broken and although the genetics may differ, they invariably lead to the constitutive activation of the Shh pathway. Importantly, in addition to PTCH1, GLI transcription factors are also known to transcriptionally regulate several of the BMPs (Kawai and Sugiura, 2001; Gianakopoulos and Skerjanc, 2005). Whether this then leads to the nuclear accumulation and overexpression of RUNX3 in BCC must now be a subject for rigorous examination. If substantiated, then understanding the involvement of RUNX3 in BCC will avail a new avenue of unraveling the complex pathogenesis of this highly frequent malignancy.
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This work was supported by the Agency for Science, Technology and Research (A*STAR), Singapore. M Salto-Tellez and SH Tan are recipients of Singapore Cancer Syndicate Grants MN005 and BS002, Agency for Science, Technology and Research, Singapore.
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Salto-Tellez, M., Peh, B., Ito, K. et al. RUNX3 protein is overexpressed in human basal cell carcinomas. Oncogene 25, 7646–7649 (2006) doi:10.1038/sj.onc.1209739
- basal cell carcinoma
- Sonic Hedgehog
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