Yabin Cheng

Targeting MAPK pathway in melanoma therapy

New drugs targeting the mitogen-activated protein kinase (MAPK) pathway have generated striking clinical response in melanoma therapy. From the discovery of BRAF mutation in melanoma in 2002, to the approval of first BRAF inhibitor vemurafenib for melanoma treatment by the US Food and Drug Administration in 2011, therapies targeting the MAPK pathway have been proven effective in less than a decade. The success of vemurafenib stimulated more intensive investigation of the molecular mechanisms of melanoma pathogenesis and development of new treatment strategies targeting specific molecules in MAPK pathway. Although selective BRAF inhibitors and MEK inhibitors demonstrated improved overall survival of metastatic melanoma patients, limited duration or development of resistance to BRAF inhibitors have been reported. Patients with metastatic melanoma still face very poor prognosis and lack of clarified therapies. Studies and multiple clinical trials on more potent and selective small molecule inhibitory compounds to further improve the clinical effects and overcome drug resistance are underway. In this review, we analyzed the therapeutic potentials of each member of the MAPK signaling pathway, summarized important MAPK-inhibiting drugs, and discussed the promising combination treatment targeting multiple targets in melanoma therapy, which may overcome the drawbacks of current drugs treatment.

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 (P = 0.005). Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) 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 KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4

KAI1/CD82 is a member of the transmembrane 4 superfamily, which was first identified as a metastasis suppressor for prostate cancer. The expression of KAI1 was found to be reduced in many types of cancers, including prostate, breast, ovarian, cervical and endometrial cancer. However, the role of KAI1 in melanoma pathogenesis is not known. In this study, we investigated the expression level of KAI1 in a large set of melanocytic lesions at different stages. We found that the expression of KAI1 is significantly decreased during melanoma progression. In fact, KAI1 expression is drastically reduced in primary melanoma compared with dysplastic nevi (P = 1.8×10(-4)) and further reduced in metastatic melanoma compared with primary melanoma (P = 9.4 × 10(-15)). Furthermore, decreased KAI1 staining is strongly correlated with a worse 5 year and 10 year patient survival. Multivariate Cox regression analysis showed that KAI1 is also an independent prognostic factor for both 5 year and 10 year survival. Moreover, we found that overexpression of KAI1 significantly inhibited melanoma cell migration through suppression of Rho-associated kinase-mediated formation of stress fiber. Our data also suggested that overexpression of KAI1 significantly inhibited melanoma cell invasion by reducing the activity of metalloproteinase-2. In addition, we found that suppression of melanoma cell migration by KAI1 is mediated by another tumor-suppressor protein called inhibitor of growth 4 through the regulation of p65. Taken together, our data suggest that KAI1 may be used as a promising prognostic marker and a possible therapeutic target for human melanoma.

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.

JWA inhibits melanoma angiogenesis by suppressing ILK signaling and is an independent prognostic biomarker for melanoma

Melanoma is the deadliest cutaneous malignancy because of its high incidence of metastasis. Melanoma growth and metastasis relies on sustained angiogenesis; therefore, inhibiting angiogenesis is a promising approach to treat metastatic melanoma. JWA is a novel microtubule-associated protein and our previous work revealed that JWA inhibited melanoma cell invasion and metastasis. However, the role of JWA in melanoma angiogenesis and the prognostic value are still unknown. Here, we report that JWA in melanoma cells significantly inhibited the tube formation of endothelial cells. In addition, JWA regulated integrin-linked kinase (ILK) through integrin αVβ3 and such regulation was achieved through the transcription factor Sp1. Notably, both in vitro and in vivo angiogenesis assays revealed that JWA dramatically suppressed melanoma angiogenesis by inhibiting ILK signaling. Furthermore, we examined the expression of JWA protein in a large set of melanocytic lesions (n = 505) at different stages by tissue microarray and found an inverse correlation between JWA expression and melanoma progression (P = 5 × 10(-6)). Importantly, reduced JWA expression was correlated with a poorer overall, and disease-specific 5 year survival of patients (P = 0.001 and 0.007, respectively). Multivariate Cox regression analyses indicated that JWA was an independent prognostic marker for melanoma patients. Moreover, we found a significant negative correlation between JWA and ILK in melanoma biopsies, and their concomitant expression was closely correlated with melanoma patient survival (P = 0.004), further indicating the regulation of ILK expression by JWA is critical in melanoma. Taken together, our data highlight the function of JWA in melanoma angiogenesis and reveal the clinical prognostic value of JWA.

Therapeutic efficacy of combined BRAF and MEK inhibition in metastatic melanoma: a comprehensive network meta-analysis of randomized controlled trials.

Background Several recent randomized clinical trials have preliminarily demonstrated that initial targeted therapy with combined BRAF and MEK inhibition is more effective in metastatic melanoma (MM) than single agent. To guide therapeutic decisions, we did a comprehensive network meta-analysis to identify evidence to robustly support whether combined BRAF and MEK inhibition is the best initial targeted therapeutic strategy for patients with MM. Methods The databases of PubMed and trial registries were researched for randomized clinical trials of targeted therapy. Data of outcome were extracted on progression-free survival (PFS), objective response rate (ORR), and overall survival (OS). Network meta-analysis using a Bayesian statistical model was performed to evaluate relative hazard ratio (HR) for PFS and OS, odds ratio (OR) for ORR. Results Finally, 16 eligible trials comprising 5976 participants were included in this meta-analysis. PFS were significantly prolonged in patients who received combined BRAF-MEK inhibition compared with those who received BRAF inhibition (HR: 0.58, 95%CI: 0.51-0.67, P < 0.0001) or MEK inhibition alone (HR: 0.29, 95%CI: 0.22-0.37, P < 0.0001). Combined BRAF-MEK inhibition also improved the OS over BRAF inhibition (HR: 0.67, 95%CI: 0.56-0.81, P < 0.0001) or MEK inhibition alone (HR: 0.48, 95%CI: 0.36-0.65, P < 0.0001). The ORR was superior in combined BRAF and MEK inhibition comparing with BRAF inhibition (OR: 2.00, 95%CI: 1.66-2.44, P < 0.0001) or MEK inhibition alone (OR: 20.66, 95%CI: 12.22-35.47, P < 0.0001). Conclusions This study indicates that concurrent inhibition of BRAF and MEK improved the most effective therapeutic modality as compared as single BRAF or MEK inhibition for patients with MM.