Tumour suppressor ING1b maintains genomic stability upon replication stress.

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.

The ING family tumor suppressors: from structure to function.

The Inhibitor of Growth (ING) proteins belong to a well-conserved family which presents in diverse organisms with several structural and functional domains for each protein. The ING family members are found in association with many cellular processes. Thus, the ING family proteins are involved in regulation of gene transcription, DNA repair, tumorigenesis, apoptosis, cellular senescence and cell cycle arrest. The ING proteins have multiple domains that are potentially capable of binding to many partners. It is conceivable, therefore, that such proteins could function similarly within protein complexes. In this case, within this family, each function could be attributed to a specific domain. However, the role of ING domains is not definitively clear. In this review, we summarize recent advances in structure-function relationships in ING proteins. For each domain, we describe the known biological functions and the approaches utilized to identify the functions associated with ING proteins.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33(ING1b).

The tumour suppressor p33(ING1b) ((ING1b) for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33(ING1b) phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33(ING1b) with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33(ING1b). We found that p33(ING1b) is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33(ING1b). Furthermore, ultraviolet irradiation induces p33(ING1b) phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.

Myeloid leukemia-1 expression in benign and malignant melanocytic lesions.

Myeloid leukemia-1 (Mcl-1) is an anti-apoptotic protein implicated in tumor progression. Its expression was found to be elevated in many types of human cancers and is correlated with tumor progression. The expression of Mcl-1 in melanoma is not fully understood. We investigated the expression of Mcl-1 in normal nevi, dysplastic nevi, primary melanoma and melanoma metastases by tissue microarray and immunohistochemistry. We found that Mcl-1 expression was significantly increased in dysplastic nevi, primary melanoma and melanoma metastases when compared to normal nevi, though the expression of Mcl-1 was decreased in metastatic melanoma when compared to dysplastic nevi. We did not find any correlation between Mcl-1 expression and melanoma patient survival. Our data suggest that Mcl-1 may play a critical role in the initiation of melanoma development.

The role of integrin-linked kinase in melanoma cell migration, invasion, and tumor growth.

Melanoma is a life-threatening disease with a high mortality rate due to rapid metastasis. Currently, there is no effective treatment for metastatic melanoma. Integrin-linked kinase (ILK) is a serine/threonine kinase and has its role implicated in connecting cell-extracellular matrix interaction and growth factor signaling to cell survival, cell migration, invasion, anchorage-independent growth, angiogenesis, and epithelial-mesenchymal transition. However, the functional role of ILK in melanoma progression is not completely understood. We have previously shown that strong ILK expression was significantly associated with melanoma thickness. In this study, we further elucidate the role of ILK in melanoma cell migration, invasion, anchorage-independent growth, and tumor growth in vivo by specific ILK knockdown using small interfering RNA and short hairpin RNA. We found that ILK knockdown impeded melanoma cell migration, which was associated with reduced stress fiber formation, cell spreading, and cell adhesion. Furthermore, ILK knockdown decreased the invasion ability of melanoma cells and the formation of anchorage-independent colonies in soft agar. Moreover, ILK knockdown significantly impaired the growth of melanoma xenografts in severe combined immunodeficient mice. This study highlights the importance of ILK in melanoma progression and provides an attractive target for the treatment of melanoma.

Elevated expression of Rad18 regulates melanoma cell proliferation.

The E3 ligase Rad18 is a key regulator for the lesion bypass pathway, which plays an important role in genomic stability. However, the status of Rad18 expression in melanoma is not known. Using melanoma tissue microarray (TMA), we showed that nuclear Rad18 expression was upregulated in primary and metastatic melanoma compared to dysplastic nevi. Rad18 expression was significantly reduced in sun-exposed sites compared to the sun-protected sites. Strong Rad18 expression correlated with worse 5-year patient survival and was an independent prognostic factor for melanoma found in the sun-protected sites. Furthermore, we showed that melanoma cell proliferation and the expression of pAkt and cyclin D1 were reduced upon Rad18 knockdown. We, for the first time, showed that Rad18 is significantly increased in melanoma and predicts the poor outcome for melanoma in the sun-protected sites. Rad18 is involved in the regulation of melanoma cell proliferation, which can be exploited in designing new strategy for melanoma treatment.

The role of ING tumor suppressors in UV stress response and melanoma progression.

The INhibitor of Growth (ING) genes were discovered during the past decade and identified as type II tumor suppressor genes. Previous studies demonstrated that ING family members participate in various cellular stress responses and thus play important roles in the pathogenesis of various types of cancers, including melanoma. Epidemiological studies showed that UV radiation is the primary etiological factor in melanoma development. Here we review the studies on the role of ING proteins in cellular responses to UV irradiation, melanoma cell motility, and melanoma progression.

Loss of SNF5 expression correlates with poor patient survival in melanoma.

PURPOSE: Aberrant expression of SWI/SNF chromatin remodeling complex is involved in cancer development. The tumor suppressor SNF5, the core subunit of SWI/SNF complex, has been shown to regulate cell differentiation, cell cycle control, and apoptosis. To investigate the role of SNF5 in the development of melanoma, we examined the expression of SNF5 in melanocytic lesions at different stages and analyzed the correlation between SNF5 expression and clinicopathologic variables and patient survival. EXPERIMENTAL DESIGN: Using tissue microarray and immunohistochemistry, we evaluated SNF5 staining in 51 dysplastic nevi, 88 primary melanomas, and 48 metastatic melanomas. We studied chemosensitivity of melanoma cells with reduced SNF5 expression by siRNA using cell survival and apoptosis assays. RESULTS: SNF5 expression was reduced in metastatic melanoma compared with dysplastic nevi (P = 0.005), in advanced primary melanoma (Clark's level V) compared with low risk Clark's level II melanoma (P = 0.019), and in melanoma at sun-exposed sites compared with sun-protected sites (P = 0.044). Furthermore, we showed a strong correlation between negative SNF5 expression and a worse 5-year survival in melanoma patients (P = 0.016). Multivariate Cox regression analysis revealed that negative SNF5 expression is an independent prognostic factor to predict patient outcome in primary melanomas (P = 0.031). Finally, we showed that knockdown of SNF5 in melanoma cell lines resulted in significant chemoresistance. CONCLUSIONS: Our data indicate that SNF5 may be an important marker for human melanoma progression and prognosis as well as a potential therapeutic target.

The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA.

ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.