Guangdi Chen

Role of Tip60 in Human Melanoma Cell Migration, Metastasis, and Patient Survival

The tumor suppressor Tip60 regulates gene transcription, DNA damage response, apoptosis, and cancer development, but its role in melanoma is unknown. In this study, we investigated the expression pattern of Tip60 in melanoma and assessed its prognostic value. Using tissue microarrays consisting of 448 cases of melanomas (201 for the training set and 247 for the validation set) and 105 cases of nevi, we found that Tip60 expression was significantly reduced in metastatic melanoma compared to common nevi (P=0.045), dysplastic nevi (P=0.047), and primary melanoma (P=0.001). Kaplan-Meier survival curve and univariate Cox regression analyses showed that reduced Tip60 expression was associated with a poorer 5-year disease-specific survival in primary melanoma (P=0.016) and metastatic melanoma patients (P=0.027). Multivariate Cox regression analyses indicated that Tip60 expression was an independent prognostic marker for primary (P=0.024) and metastatic melanomas (P=0.035). In vitro wound healing assay showed that enforced Tip60 expression inhibited but Tip60 knockdown enhanced melanoma cell migration, suggesting that Tip60 might regulate melanoma metastasis. Finally, we showed that overexpression of Tip60 in melanoma cells resulted in significantly increased chemosensitivity. Our data indicate that Tip60 may serve as a potential biomarker for melanoma patient outcome as well as a potential therapeutic target.

Prognostic significance of RUNX3 expression in human melanoma.

RUNX3 is a tumor suppressor that plays important roles in cell proliferation, apoptosis, and metastasis. The authors investigated the role of RUNX3 in melanoma pathogenesis and analyzed the prognostic impact of RUNX3 expression in a large series of melanoma patients.

Prognostic Significance of Cytoplasmic p27 Expression in Human Melanoma

Background: The cyclin-dependent kinase inhibitor p27 plays important roles in cell proliferation, cell motility, and apoptosis. Interestingly, the nuclear and cytoplasmic p27 exert opposite biological functions. In this study, we investigated the prognostic impact of subcellular p27 expression. Methods: We constructed melanoma tissue microarrays in a large series of melanoma patients, including 29 normal nevi, 52 dysplastic nevi, 270 primary melanomas, and 148 metastatic melanomas. The expression level of subcellular p27 in different stages of melanocytic lesions and its prognostic significance were evaluated. Results: Compared with dysplastic nevi, nuclear p27 expression was remarkably reduced in primary melanomas and further reduced in metastatic melanoma (P < 0.001 for both), whereas cytoplasmic p27 expression is significantly increased from dysplastic nevi to primary melanomas (P = 0.032) and further increased in melanoma metastases (P = 0.037). Although loss of nuclear p27 expression is correlated with a worse 5-year survival of primary melanoma patients in Kaplan–Meier analysis (P = 0.046), it is not a prognostic factor by multivariate Cox regression analysis. On the contrary, Kaplan–Meier analysis showed that gain of cytoplasmic p27 was associated with a poor 5-year survival of metastatic melanoma patients (P < 0.001). Multivariate Cox regression analysis revealed that positive cytoplasmic p27 expression is an independent prognostic factor to predict metastatic melanoma patient outcome. Conclusion: Cytoplasmic p27 may serve as a promising prognostic marker for metastatic melanoma. Impact: Because there is no reliable prognostic marker for metastatic melanoma, our finding may have important clinical implications using cytoplasmic p27 as a prognostic biomarker for advanced melanoma. Cancer Epidemiol Biomarkers Prev; 20(10); 2212–21. ©2011 AACR.

Cytoplasmic Skp2 Expression Is Increased in Human Melanoma and Correlated with Patient Survival

Background S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation. Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival. In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial. Methods To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas. The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry. The Kaplan-Meier method was used to evaluate the patient survival. The univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up. Results Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas. Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II–IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (Pu200a=u200a0.005). Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (Pu200a=u200a0.018) but not metastatic melanoma (P>0.05). Conclusion This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival. Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease.

Cul1 expression is increased in early stages of human melanoma.

Dear Editor, 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). 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

Prognostic Significance of Fbw7 in Human Melanoma and Its Role in Cell Migration

The E3 ubiquitin ligase Fbw7 (F-box and WD repeat domain-containing 7) is broadly considered as a tumor suppressor because of its role in the turnover of several well-known oncoproteins. However, the role of Fbw7 in melanomagenesis is not clear. To investigate the expression profile and biological functions of Fbw7 in melanoma, we examined Fbw7 expression level using melanoma tissue microarray and immunohistochemistry. Our data showed that Fbw7 expression is significantly reduced in primary melanoma compared with dysplastic nevi (P=0.020) and further reduced in metastatic melanoma compared with primary melanoma (P=0.011). Furthermore, we observed a strong correlation between negative Fbw7 expression and a worse 5-year survival of melanoma patients (P=0.015). We also found that both Fbw7 protein and mRNA expression was significantly reduced in nine melanoma cell lines compared with normal melanocytes. Moreover, our in vitro studies showed a remarkable increase of cell migration and stress fiber formation in Fbw7 knockdown cells, and treatment of selective MEK (MAPK/ERK kinase) inhibitor abrogated Fbw7α knockdown-induced melanoma cell migration. Taken together, our findings indicate that Fbw7 has an important role in melanoma progression, and it inhibits melanoma cell migration through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway and may serve as a prognostic marker.

Abstract 3430: Role of Tip60 in human melanoma cell migration, melanoma metastasis and patient survival

The tumor suppressor Tip60 plays a major role in transcription, DNA damage response, apoptosis and cancer development, but its role in melanoma is unknown. In this study, we investigated the role of Tip60 in melanoma pathogenesis and assessed its prognostic value. Using tissue microarrays consisting of 448 cases of melanomas (201 for the training set and 247 for the validation set) and 105 cases of nevi, we found that Tip60 expression was significantly reduced in metastatic melanoma compared to normal nevi (P = 0.045), dysplastic nevi (P = 0.047) and primary melanoma (P = 0.001). Kaplan-Meier survival curve and univariate Cox regression analyses showed that reduced Tip60 expression was associated with a poorer five-year disease-specific survival in primary melanoma (P = 0.016) and metastatic melanoma patients (P = 0.027). Multivariate Cox regression analyses indicated that Tip60 expression was an independent prognostic marker for primary (P = 0.002) and metastatic melanomas (P = 0.035). In vitro wound healing assay showed that enforced Tip60 expression inhibited melanoma cell migration suggesting that Tip60 might regulate melanoma metastasis. Finally, we showed that overexpression of Tip60 in melanoma cells resulted in significantly increased chemosensitivity. Our data indicate that Tip60 may serve as a potential biomarker for melanoma patient outcome as well as a potential therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3430. doi:1538-7445.AM2012-3430

Abstract 2103: Reduced Tip60 expression as a predictive biomarker for advanced melanoma and patient outcome

The tumor suppressor Tip60 plays a major role in transcription, DNA damage response, apoptosis and cancer development. We investigated the role of Tip60 in melanoma pathogenesis and assessed the prognostic value of Tip60 expression on melanoma patient survival. Two sets of tissue microarrays were constructed consisting of 448 cases of melanomas (201 for the training set and 247 for the validation set) and 105 cases of nevi. The TMA was assessed for Tip60 expression by immunohistochemistry. The Kaplan-Meier method was used to evaluate the patient survival, and univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up. We found that Tip60 expression was significantly reduced in metastatic melanoma compared to normal nevi (P = 0.045), dysplastic nevi (P = 0.047) and primary melanoma (P = 0.001). Tip60 expression correlated with AJCC stages (I-II vs III-IV, P = 0.001) and tumor thickness (≤4.00 vs. >4.00 mm, P = 0.037). Reduced Tip60 expression was associated with a poor five-year disease-specific survival in primary melanoma (P = 0.016) and metastatic melanoma patients (P = 0.027). Furthermore, Cox regression analyses indicated that Tip60 expression was an independent prognostic marker for primary (P = 0.002) and metastatic melanomas (P = 0.035). In conclusion, reduced Tip60 expression is significantly associated with thick primary melanoma and metastatic melanoma, as well as poor patient prognosis, suggesting that Tip60 may serve as a potential biomarker for advanced melanoma and patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2103. doi:10.1158/1538-7445.AM2011-2103

Abstract 1075: Cul1 Expression Is Increased in Early Stages of Human Melanoma

As the largest family of ubiquitin-protein E3 ligases, the SCF (Skp1/Cullin/Rbx1/F-box protein) complexes ubiquitinate a broad range of proteins involved in cell cycle progression, signal transduction and transcription. Cullin1 (Cul1) serves as a rigid scaffold in SCF complex for Rbx1, Skp1 and F-box protein subunits assembly and aberrant expression of Cul1 is involved in dysfunction of SCF E3 ligases. To investigate the role of Cul1 in the development of melanoma, we examined the expression of Cul1 in melanocytic lesions at different stages and analyzed the correlation between Cul1 expression and clinicopathologic parameters by tissue microarray and immunohistochemistry. The result showed that Cul1 expression was significant increased in primary and metastatic melanoma compared with dysplastic nevi, while there was no significant difference of Cul1 expression between primary and metastatic melanoma, which suggested that Cul1 plays an important role in the initiation stage of melanoma development. We found knockdown of Cul1 inhibited melanoma cell growth and overexpression of Cul1 promoted melanoma cell growth through regulating CDK inhibitor p27Kip expression. Knockdown of Cul1 abrogated Skp2-induced p27 degradation in melanoma cells. These results suggested that Cul1 is involved in cell cycle regulation of melanoma cells via Cul1-dependent ubiquitination and degradation of the p27kip1 by SCFSkp2 complex. In conclusion, our data indicate that Cul1 may serve as a potential marker for human melanoma initiation and early diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1075.