A Gene Expression Signature of Invasive Potential in Metastatic Melanoma Cells

BACKGROUND: We are investigating the molecular basis of melanoma by defining genomic characteristics that correlate with tumour phenotype in a novel panel of metastatic melanoma cell lines. The aim of this study is to identify new prognostic markers and therapeutic targets that might aid clinical cancer diagnosis and management. PRINCIPAL FINDINGS: Global transcript profiling identified a signature featuring decreased expression of developmental and lineage specification genes including MITF, EDNRB, DCT, and TYR, and increased expression of genes involved in interaction with the extracellular environment, such as PLAUR, VCAN, and HIF1a. Migration assays showed that the gene signature correlated with the invasive potential of the cell lines, and external validation by using publicly available data indicated that tumours with the invasive gene signature were less melanocytic and may be more aggressive. The invasion signature could be detected in both primary and metastatic tumours suggesting that gene expression conferring increased invasive potential in melanoma may occur independently of tumour stage. CONCLUSIONS: Our data supports the hypothesis that differential developmental gene expression may drive invasive potential in metastatic melanoma, and that melanoma heterogeneity may be explained by the differing capacity of melanoma cells to both withstand decreased expression of lineage specification genes and to respond to the tumour microenvironment. The invasion signature may provide new possibilities for predicting which primary tumours are more likely to metastasize, and which metastatic tumours might show a more aggressive clinical course

Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB-1 determines its cellular location

Critical Role for Cold Shock Protein YB-1 in Cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division

Critical Role for Cold Shock Protein YB-1 in Cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division

Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB-1 determines its cellular location

Prognostic association of YB-1 expression in breast cancers: a matter of antibody.

The literature concerning the subcellular location of Y-box binding protein 1 (YB-1), its abundance in normal and cancer tissues, and its prognostic significance is replete with inconsistencies. An explanation for this could be due in part to the use of different antibodies in immunohistochemical and immunofluorescent labeling of cells and tissues. The inconsistencies could also be due to poor resolution of immunohistochemical data. We analyzed two cohorts of breast tumours for both abundance and subcellular location of YB-1 using three different antibodies; two targeting N-terminal epitopes (AB-a and AB-b) and another (AB-c) targeting a C-terminal epitope. We also investigated stress-induced nuclear translocation of YB-1 in cell culture. We report that both AB-a and AB-c detected increased YB-1 in the cytoplasm of high-grade breast cancers, and in those lacking estrogen and progesterone receptors; however the amount of YB-1 detected by AB-a in these cancers is significantly greater than that detected by AB-c. We confirm our previously published findings that AB-b is also detecting hnRNP A1, and cannot therefore be used to reliably detect YB-1 by immunohistochemistry. We also report that AB-a detected nuclear YB-1 in some tumour tissues and stress treated cells, whereas AB-c did not. To understand this, cancer cell lines were analyzed using native gel electrophoresis, which revealed that the antibodies detect different complexes in which YB-1 is a component. Our data suggest that different YB-1 antibodies show different staining patterns that are determined by the accessibility of epitopes, and this depends on the nature of the YB-1 complexes. It is important therefore to standardize the protocols if YB-1 is to be used reproducibly as a prognostic guide for different cancers