Adele G. Woolley

YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

BACKGROUND Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. METHODS YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis-free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. RESULTS YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis-free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter-reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= -2.64, 95% confidence interval = -3.57 to -1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. CONCLUSION YB-1 expression is associated with the activity of E2F transcription factors and may control tumor cell growth by this mechanism.

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.

Genotoxic stress-induced nuclear localization of oncoprotein YB-1 in the absence of proteolytic processing

Y-box-binding protein 1 (YB-1) is an oncogenic transcription factor whose overexpression and nuclear localization is associated with tumor progression and drug resistance. Transcriptional activation of YB-1 in response to genotoxic stress is believed to occur in the cytoplasm through sequence-specific endoproteolytic cleavage by the 20S Proteasome, followed by nuclear translocation of cleaved YB-1. To study the proteolysis model, we developed a two-step affinity purification of endogenous YB-1 protein species and characterized the products using mass spectrometry. Whereas full-length YB-1 was readily identified, the smaller protein band thought to be activated YB-1 was identified as hnRNP A1. An antibody specific for YB-1 was generated, which revealed only one YB-1 species, even after genotoxic stress-induced nuclear YB-1 translocation. These findings warrant re-evaluation of the mechanism of YB-1 nuclear translocation and transcriptional activation. The relationship between nuclear YB-1 and tumor progression may also have to re-evaluated in some cases.

Developmental loss of NT-3 in vivo results in reduced levels of myelin-specific proteins, a reduced extent of myelination and increased apoptosis of Schwann cells

This work investigates the role of NT‐3 in peripheral myelination. Recent articles, based in vitro, propose that NT‐3 acting through its high‐affinity receptor TrkC may act to inhibit myelin formation by enhancing Schwann cell motility and/or migration. Here, we investigate this hypothesis in vivo by examining myelination formation in NT‐3 mutant mice. On the day of birth, soon after the onset of myelination, axons showed normal ensheathment by Schwann cells, no change in the proportion of axons which had begun to myelinate, and no change in either myelin thickness or number of myelin lamellae. However in postnatal day 21 mice, when myelination is substantially complete, we observed an unexpected reduction in mRNA and protein levels for MAG and P0, and in myelin thickness. This is the opposite result to that predicted from previous in vitro studies, where removal of an inhibitory NT‐3 signal would have been expected to enhance myelination. These results suggest that, in vivo, the importance of NT‐3 as a major support factor for Schwann cells (Meier et al., ( 1999 ) J Neurosci 19:3847‐3859) over‐rides its potential role as an myelin inhibitor, with the net effect that loss of NT‐3 results in degradation of Schwann cell functions, including myelination. In support of this idea, Schwann cells of NT‐3 null mutants showed increased expression of activated caspase‐3. Finally, we observed significant reduction in width of the Schwann cell periaxonal collar in NT‐3 mutant animals suggesting that loss of NT‐3 and resulting reduction in MAG levels may alter signaling at the axon‐glial interface.

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.

Neurotrophin‐3 null mutant mice display a postnatal motor neuropathy

This paper examines early postnatal development of the neuromuscular system in mice with a null mutation in the gene for neurotrophin‐3. We report that alpha‐motoneurons at first develop substantially normally, despite a known 15% deficit in their somal size [ Woolley et al. (1999)Neurosci. Lett., 272, 107–110.] and the absence of proprioceptive input [ Ernfors et al. (1994)Cell, 77, 503–512]. At birth, motor axons have extended into the muscle, forming normal‐looking neuromuscular junctions with focal accumulations of acetylcholine receptors. Detailed ultrastructural analysis does however, reveal subtle abnormalities at this time, particularly a decrease in the extent of occupancy of the postsynaptic site by nerve terminals, and a small but significant deficit in myofibre number. After the relative normality of this early neuromuscular development, there then occurs a catastrophic postnatal loss of motor nerve terminals, resulting in complete denervation of hindlimb muscles by P7. In systematic semi‐serial samples through the entire muscle endplate zones, no neuromuscular junctions can be found. Intramuscular axons are fragmented, as shown by both electron microscopic observations and neurofilament immunohistochemistry, and alpha‐bungarotoxin detection of acetylcholine receptors indicates dispersal of the junctional accumulation. At earlier times (postnatal days three and four) the terminal Schwann cells show ultrastructural abnormalities, and preliminary observations suggest marked disturbance of myelination. Based on comparison with other literature, the peripheral nerve degeneration seems unlikely to have arisen as a secondary effect of de‐afferentation. We discuss whether the neural degeneration is secondary to the disturbance of Schwann cell function, or due directly to a loss of neurotrophin‐3 based support of the motoneuron.

Alpha motoneurons are present in normal numbers but with reduced soma size in neurotrophin-3 knockout mice.

Neurotrophin-3 (NT-3) is essential for survival of proprioceptive and mechanoreceptor neurons, but its role in motoneuron development in vivo has seemed slight. Recent evidence however has suggested that NT-3 may be involved in motoneuron maturation. Here, we quantitatively assess the number and state of development of motoneurons within the lumbar lateral motor column (LLMC) of newborn NT-3 (-/-), (+/-) and (+/+) mutant mice. We find that the number of alpha motoneurons in the LLMC is the same in all genotypes, but soma size is significantly reduced in (-/-) mutants. This suggests that NT-3 is not required for normal production and survival of alpha motoneurons, but is essential for their full maturation and/or maintenance. We also confirm a previous report that gamma motoneurons are absent in NT-3 (-/-) mice.

Variable Expression of GLIPR1 Correlates with Invasive Potential in Melanoma Cells

GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms’ tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

Investigation of neuromuscular abnormalities in neurotrophin‐3‐deficient mice

Neurotrophin‐3 (NT‐3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT‐3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency‐induced reduction in NT‐3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1‐43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve‐mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1‐43 were impaired in mature NT‐3‐deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype‐dependent variation in the number of synaptic vesicles near the active zone. NT‐3+/− mice had normal soleus muscle fiber numbers but their fibers had smaller cross‐sectional areas and were more densely‐packed than wild‐type littermates. Moreover, the muscles of adult NT‐3‐deficient animals were weaker than those of wild‐type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT‐3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.

A novel EGR‐1 dependent mechanism for YB‐1 modulation of paclitaxel response in a triple negative breast cancer cell line

Chemotherapy with taxanes such as paclitaxel (PTX) is a key component of triple negative breast cancer (TNBC) treatment. PTX is used in combination with other drugs in both the adjuvant setting and in advanced breast cancer. Because a proportion of patients respond poorly to PTX or relapse after its use, a greater understanding of the mechanisms conferring resistance to PTX is required. One protein shown to be involved in drug resistance is Y‐box binding protein 1 (YB‐1). High levels of YB‐1 have previously been associated with resistance to PTX in TNBCs. In this study, we aimed to determine mechanisms by which YB‐1 confers PTX resistance. We generated isogenic TNBC cell lines that differed by YB‐1 levels and treated these with PTX. Using microarray analysis, we identified EGR1 as a potential target of YB‐1. We found that low EGR1 mRNA levels are associated with poor breast cancer patient prognosis, and that EGR1 and YBX1 mRNA expression was inversely correlated in a TNBC line and in a proportion of TNBC tumours. Reducing the levels of EGR1 caused TNBC cells to become more resistant to PTX. Given that PTX targets cycling cells, we propose a model whereby high YB‐1 levels in some TNBC cells can lead to reduced levels of EGR1, which in turn promotes slow cell cycling and resistance to PTX. Therefore YB‐1 and EGR1 levels are biologically linked and may provide a biomarker for TNBC response to PTX.