Biomedical subjects

Sandra Pavey

Publications and source records attributed to Sandra Pavey.

9 recordsLinked to original sources

PubMed2006

Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN.

The tumor suppressor PTEN antagonizes phosphatidylinositol 3-kinase (PI3K), which contributes to tumorigenesis in many cancer types. While PTEN mutations occur in some melanomas, their precise mechanistic consequences have yet to be elucidated. We sought to identify novel downstream effectors of PI3K using a combination of genomic and functional tests. Microarray analysis of 53 melanoma cell lines identified 610 genes differentially expressed (P<0.05) between wild-type lines and those with PTEN aberrations. Many of these genes are known to be involved in the PI3K pathway and other signaling pathways influenced by PTEN. Validation of differential gene expression by qRT-PCR was performed in the original 53 cell lines and an independent set of 18 melanoma lines with known PTEN status. Osteopontin (OPN), a secreted glycophosphoprotein that contributes to tumor progression, was more abundant at both the mRNA and protein level in PTEN mutants. The inverse correlation between OPN and PTEN expression was validated (P<0.02) by immunohistochemistry using melanoma tissue microarrays. Finally, treatment of cell lines with the PI3K inhibitor LY294002 caused a reduction in expression of OPN. These data indicate that OPN acts downstream of PI3K in melanoma and provides insight into how PTEN loss contributes to melanoma development.

Cell Line, Tumor↗

PubMed2006

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4R24C/R24C/TPras mice.

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4(R24C/R24C) mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4(R24C/R24C)/TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4(R24C/+)/TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4(R24C/R24C)/TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4(R24C/R24C)/TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4(R24C/R24C)/TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

Animals↗

PubMed2006

Increased p21-activated kinase-1 expression is associated with invasive potential in uveal melanoma.

Prognosis in patients with uveal melanoma is poor as approximately half of all tumors metastasize and currently there are no effective treatments for disseminated disease. Differences in invasiveness between uveal melanomas could therefore be of major significance regarding clinical outcome. To identify genes associated with invasive potential, we have used microarray expression profiling combined with phenotypic characterization of uveal melanoma and melanocyte cell lines to define a gene signature associated with cellular invasion. A panel of 14 uveal cell cultures was assessed using three assays of invasiveness: matrigel invasion chamber system, scratch wound closure and cell motility. We identified a set of 853 differentially expressed transcripts (Wilcoxon-Mann-Whitney test, P<0.01) that discriminated between samples with high or low invasive capacity based on a composite phenotype that takes into account behavior across all three assays. Of particular interest, expression of two members of the p21-activated kinase (PAK) family, PAK1 and PAK7, was elevated in the more invasive cultures. PAK1 has previously been shown to play a central role in regulating cell motility and invasiveness in other cell types, and increased expression has been observed in breast and colorectal carcinomas. Using small interfering RNA-mediated PAK1 knockdown, we showed up to a five-fold decrease in invasion through matrigel, indicating that elevated levels of PAK1 are associated with invasive potential in uveal melanoma. These data implicate PAK1 as a potential new target for therapy of these tumors.

Biomarkers, Tumor↗

PubMed2005

Melanocytes in conditional Rb-/- mice are normal in vivo but exhibit proliferation and pigmentation defects in vitro.

The function of the retinoblastoma tumour suppressor (Rb1), and the pocket protein family in general, has been implicated as an important focal point for deregulation in many of the molecular pathways mutated in melanoma. We have focused on the role of Rb1 in mouse melanocyte homeostasis using gene targeting and Cre/loxP mediated tissue-specific deletion. We show that constitutive Cre-mediated ablation of Rb1 exon 2 prevents the production of Rb1 and recapitulates the phenotype encountered in other Rb1 knockout mouse models. Mice with conditional melanocyte-specific ablation of Rb1 manifest overtly normal pigmentation and are bereft of melanocytic hyperproliferative defects or apoptosis-induced depigmentation. Histologically, these mice have melanocyte morphology and distribution comparable with control littermates. In contrast, Rb1-null melanocytes removed from their in vivo micro-environment and cultured in vitro display some of the characteristics associated with a transformed phenotype. They proliferate at a heightened rate when compared with control melanocytes and have a decreased requirement for mitogens. With progressive culture the cells depigment at relatively early passage and display a gross morphology which, whilst reminiscent of early passage melanocytes, is generally different to equivalent passage control cells. These results indicate that Rb1 is dispensable for in vivo melanocyte homeostasis when its ablation is targeted from the melanoblast stage onwards, however, when cultured in vitro, Rb1 loss increases melanocyte growth but the cells are not fully transformed.

Animals↗

PubMed2004

Microarray expression profiling in melanoma reveals a BRAF mutation signature.

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Amino Acid Substitution↗

PubMed2004

Analyzing checkpoint controls in human skin.

A short-term whole-skin organ culture model has been established to enable the investigation of cell cycle perturbations in epidermal layer cells following exposure to ultraviolet radiation (UVR). This model affords the opportunity to manipulate the growth and nutrient conditions, and to perform detailed biochemical and immunohistochemical analysis of skin cells in their normal epidermal layer microenvironment. The use of this model is described in this chapter.

Bromodeoxyuridine↗

PubMed2003

Expression profiling reveals that methylation of TIMP3 is involved in uveal melanoma development.

Uveal melanoma is associated with a high tumor-related mortality due to the propensity to develop metastases. The mechanisms that are responsible for malignant dissemination are largely unknown and need to be explored to facilitate diagnosis and treatment of metastases. To identify molecules involved in dissemination, we used cell lines derived from a primary uveal melanoma and 2 liver metastases from the same patient as a model for tumor progression. Using a microarray representing 1,176 genes, we identified 63 differentially expressed genes. Forty genes showed a higher expression and 23 showed a lower expression in the primary cell line compared to the metastasis cell lines. These genes are involved in processes like angiogenesis, apoptosis, macrophage stimulation, and extracellular matrix regulation. In contrast, the 2 liver metastasis cell lines produced nearly identical expression profiles. Demethylation of the primary melanoma cell line by 5-aza-2'deoxycytidine treatment revealed that 19 genes were suppressed by hypermethylation. An important finding was the 5-fold decreased expression of TIMP3 in the metastatic cell lines, a molecule involved in extracellular matrix degradation. We demonstrate in the cell lines that TIMP3 expression is regulated by methylation. These observations were confirmed in primary uveal melanoma and suggests a role for TIMP3 in uveal melanoma development.

Azacitidine↗

PubMed2002

Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma.

Germline mutations of the PTEN tumor-suppressor gene, on 10q23, cause Cowden syndrome, an inherited hamartoma syndrome with a high risk of breast, thyroid and endometrial carcinomas and, some suggest, melanoma. To date, most studies which strongly implicate PTEN in the etiology of sporadic melanomas have depended on cell lines, short-term tumor cultures and noncultured metastatic melanomas. The only study which reports PTEN protein expression in melanoma focuses on cytoplasmic expression, mainly in metastatic samples. To determine how PTEN contributes to the etiology or the progression of primary cutaneous melanoma, we examined cytoplasmic and nuclear PTEN expression against clinical and pathologic features in a population-based sample of 150 individuals with incident primary cutaneous melanoma. Among 92 evaluable samples, 30 had no or decreased cytoplasmic PTEN protein expression and the remaining 62 had normal PTEN expression. In contrast, 84 tumors had no or decreased nuclear expression and 8 had normal nuclear PTEN expression. None of the clinical features studied, such as Clark's level and Breslow thickness or sun exposure, were associated with cytoplasmic PTEN expressional levels. An association with loss of nuclear PTEN expression was indicated for anatomical site (p = 0.06) and mitotic index (p = 0.02). There was also an association for melanomas to either not express nuclear PTEN or to express p53 alone, rather than both simultaneously (p = 0.02). In contrast with metastatic melanoma, where we have shown previously that almost two-thirds of tumors have some PTEN inactivation, only one-third of primary melanomas had PTEN silencing. This suggests that PTEN inactivation is a late event likely related to melanoma progression rather than initiation. Taken together with our previous observations in thyroid and islet cell tumors, our data suggest that nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression.

Aged↗

PubMed2002

Alpha-melanocyte stimulating hormone potentiates p16/CDKN2A expression in human skin after ultraviolet irradiation.

The contribution of the UV component of sunlight to the development of skin cancer is widely acknowledged, although the molecular mechanisms that are disrupted by UV radiation (UVR) resulting in the loss of normal growth controls of the epidermal stem cell keratinocytes and melanocytes is still poorly understood. Alpha-melanocyte stimulating hormone (alpha-MSH), acting via its receptor MC1, has a key role in skin pigmentation and the melanizing response after exposure to UVR. The cell cycle inhibitor p16/CDKN2A also appears to have an important function in a cell cycle checkpoint response in skin after exposure to UVR. Both of these genes have been identified as risk factors in skin cancer, MC1R variants are associated with increased risk to both melanoma and nonmelanoma skin cancers, and p16/CDKN2A with increased risk of melanoma. Here we demonstrate that the increased expression of p16 after exposure to suberythemal doses of UVR is potentiated by alpha-MSH, a ligand for MC1R, and this effect is mimicked by cAMP, the intracellular mediator of alpha-MSH signaling via the MC1 receptor. This link between p16 and MC1R may provide a molecular basis for the increased skin cancer risk associated with MC1R polymorphisms.

Cyclin-Dependent Kinase Inhibitor p16↗