Human cutaneous malignant melanoma is the most serious type of skin cancer, which is derived from epidermal melanocytes. Epidemiological studies estimate a doubling of melanoma incidence every 10–20 yrs (Garbe et al., 2000). Although early diagnosed melanomas are curable with surgical excision, up to 20% of patients will develop metastatic tumors because of its high capability of invasion and rapid metastasis to other organs (Balch et al., 2001). Despite many advances in cancer treatment over the last several decades, the prognosis for patients with advanced melanoma remains poor. The 5-yr survival rate for patients with distant metastases is <10% (Trinh, 2008).
The ubiquitin–proteasome system controls the abundance of a number of cellular proteins and plays a crucial role in maintaining and regulating cellular homeostasis (Paul, 2008). Dysfunction of E3 ubiquitin ligases that target substrates for polyubiquitination and degradation, contributes abnormal cell growth and differentiation (Reinstein and Ciechanover, 2006). Cullin1 (Cul1) serves as a rigid scaffold in SCF (Skp1/Cullin/Rbx1/F-box protein) complex, the largest family of ubiquitin-protein E3 ligases, and aberrant expression of Cul1 is involved in dysfunction of SCF E3 ligases (Zheng et al., 2002). It has been reported that the loss of Cul1 results in early embryonic lethality and deregulation of cyclin E (Dealy et al., 1999). In addition, c-Myc activates Cul1 gene expression and promotes ubiquitin-dependent proteolysis (O’hagan et al., 2000). However, little is known about the expression of Cul1 in cancers.
In this study, we first compared Cul1 protein level in nine melanoma cell lines with that in normal human melanocytes. Eight melanoma cell lines (MMAN, MEWO, MMRU, PMWK, RPEP, RPM-MC, Sk-mel-2 and Sk-mel-3) had higher Cul1 expression in comparison with normal human melanocytes, whereas only one cell line MMLH showed similar Cul1 level to melanocytes (Figure 1A and B). We next investigated Cul1 staining in 43 cases of dysplastic nevi, 89 primary and 47 metastatic melanomas using tissue microarray technology and immunohistochemistry and analyzed the correlation between Cul1 expression and clinicopathologic variables and patient survival. For the 89 primary melanoma cases, there were 54 men and 35 women, with age ranging from 21 to 93 (median = 57). For primary melanoma staging, we used Breslow thickness as our criteria for evaluating Cul1 expression, 58 tumors ≤2.0 mm and 31 >2.0 mm. Seventy-two melanomas were located in sun-exposed sites (head and neck), and 17 were located in sun-protected sites (other locations). Ulceration was observed in 18 cases (Table 1).
|Negative-weak (%)||Moderate-strong (%)||Total||P valuea|
|≤57||7 (15)||38 (85)||45||>0.05|
|>57||13 (29)||31 (71)||44|
|Male||12 (22)||42 (78)||54||>0.05|
|Female||8 (23)||27 (77)||35|
|Tumor thickness (mm)|
|≤2.0||13 (22)||45 (78)||58||>0.05|
|>2.0||7 (23)||24 (77)||31|
|Present||2 (11)||16 (89)||18||>0.05|
|Absent||18 (25)||53 (75)||71|
|Nodular||1 (7)||13 (93)||14||>0.05|
|Superficial spreading||11 (29)||27 (31)||38|
|Lentigo maligna||4 (25)||12 (75)||16|
|Othersb||4 (14)||17 (86)||21|
|Sun-protected||17 (7)||55 (93)||72||>0.05|
|Sun-exposed||3 (24)||14 (76)||17|
As shown in Figure 2, various levels of cytoplasmic and nuclear Cul1 staining were observed in nevi and melanoma biopsies (Figure 2A–D). Among the groups, there are differences in the pattern of Cul1 expression, with increased levels of expression from dysplastic nevi to melanoma. Significant differences for Cu1 staining were observed between dysplastic nevi and primary melanoma (P < 0.01, χ2 test), or metastatic melanoma (P < 0.01, χ2 test). However, there was no significant difference for Cul1 staining between primary and metastatic melanoma (P = 0.76, χ2 test) (Figure 2E).
We also assessed the correlation between Cul1 expression and clinicopathological parameters of the patients and found that Cul1 expression did not correlate with age, gender, tumor thickness, ulceration, tumor subtype and sites. In addition, we constructed Kaplin–Meier survival curves using either overall or disease-specific 5-yr survival of primary, metastatic or all the melanoma patients and found that Cul1 expression did not correlate with melanoma patient survival (Figure S1).
Previously, it was reported that Cul4A, another cullin protein, was commonly expressed in different cancers, and Cul4A protein expression was increased in breast and hepatocellular cancers (Chen et al., 1998; Yasui et al., 2002). In this study, we for the first time demonstrated overexpression of Cul1 in melanoma. However, it remains unclear why Cul1 is highly expressed in melanoma. The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells (Cole, 1986). O’hagan et al. (2000) have verified that Cul1 is a direct Myc-responsive gene target, and c-Myc promotes ubiquitin-dependent proteolysis by directly activating the expression of Cul1 gene. As an oncogenic transcription factor, c-Myc is frequently upregulated in human melanoma (Ross and Wilson, 1998) although its role in melanomagenesis has not been addressed. It is possible that upregulated c-Myc may contribute to overexpression of Cul1 in melanomas. Future studies should be conducted to elucidate the relationship between c-Myc and Cul1 expression in human melanoma progression.
In summary, Cul1 overexpression is significantly correlated with melanoma development at early stages, implying targeting Cul1 may serve as a potential therapy for melanoma.