Automated Quantitative Analysis of Activator Protein-2α Subcellular Expression in Melanoma Tissue Microarrays Correlates with Survival Prediction

Berger, Aaron J.; Davis, Darren W.; Tellez, Carmen S.; Prieto, Víctor G.; Gershenwald, Jeffrey E.; Johnson, Marcella M.; Rimm, David L.; Bar‐Eli, Menashe

doi:10.1158/0008-5472.can-05-2300

Cited by 63 publications

(42 citation statements)

References 23 publications

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“…Quantitative analysis of a large retrospective cohort of patients suffering from melanoma reveals that a high cytoplasmic-to-nuclear ratio of AP2␣ correlates with a poor prognosis. In addition, a loss of AP2␣ expression has been associated with malignant transformation and melanoma progression (34,51). Also, a recent in vitro study demonstrated that AP2␣ transcription factor overexpression could reduce cell invasion (52).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Hypoxia-inducible Factor 2α Nuclear Interactome in Melanoma Cells Reveals Master Proteins Involved in Melanoma Development

Steunou

Ducoux-Petit

Lazar

et al. 2013

Molecular & Cellular Proteomics

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Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that play a key role in cellular adaptation to hypoxia. HIF proteins are composed of an ␣ subunit regulated by oxygen pressure (essentially HIF1␣ or HIF2␣) and a constitutively expressed ␤ subunit. These proteins are often overexpressed in cancer cells, and HIF overexpression frequently correlates with poor prognosis, making HIF proteins promising therapeutic targets. HIF proteins are involved in melanoma initiation and progression; however, the specific function of HIF2 in melanoma has not yet been studied comprehensively. Identifying protein complexes is a valuable way to uncover protein function, and affinity purification coupled with mass spectrometry and label-free quantification is a reliable method for this approach. We therefore applied quantitative interaction proteomics to identify exhaustively the nuclear complexes containing HIF2␣ in a human melanoma cell line, 501mel. We report, for the first time, a high-throughput analysis of the interactome of an HIF subunit. Seventy proteins were identified that interact with HIF2␣, including some well-known HIF partners and some new interactors. The new HIF2␣ partners microphthalmia-associated transcription factor, SOX10, and AP2␣, which are master actors of melanoma development, were confirmed via coimmunoprecipitation experiments. Their ability to bind to HIF1␣ was also tested: microphthalmia-associated transcription factor and SOX10 were confirmed as HIF1␣ partners, but the transcription factor AP2␣ was not. AP2␣ expression correlates with low invasive capacities. Interestingly, we demonstrated that when HIF2␣ was overexpressed, only cells expressing large amounts of AP2␣ exhibited decreased invasive capacities in hypoxia relative to normoxia. Hypoxia-inducible transcription factors (HIFs)1 are central mediators of cellular adaptation to hypoxia that control the expression of genes involved in anaerobic metabolism, intracellular pH, angiogenesis, and cell growth and survival (1). HIF proteins are heterodimers consisting of an oxygen-regulated ␣ subunit and a constitutively expressed ␤ subunit (also referred to as the arylhydrocarbon receptor nuclear translocator, or ARNT). Three isoforms have been described for each subunit; most HIF function, however, has been attributed to HIF1␣, HIF2␣, and HIF1␤ (2). HIF1␣ is detected in almost all tissues, whereas HIF2␣ (also known as EPAS1, for endothelial PAS protein 1) is restricted to certain tissues. HIF2 is expressed notably in hypoxic endothelium but also in distinct cell populations in the brain, liver, kidney, lung, and intestine (3). HIF␣ proteins are regulated post-translationally as a function of the partial pressure of oxygen. In normoxic conditions, the protein is rapidly hydroxylated by prolyl hydroxylases and then recognized by the von Hippel-Lindau protein, a component of an E3-ubiquitin ligase complex that targets the ␣ subunit for degradation by the proteasome. When the partial pressure of oxygen is low, prolyl hydroxylase activi...

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Section: Discussionmentioning

confidence: 99%

Identification of the Hypoxia-inducible Factor 2α Nuclear Interactome in Melanoma Cells Reveals Master Proteins Involved in Melanoma Development

Steunou

Ducoux-Petit

Lazar

et al. 2013

Molecular & Cellular Proteomics

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“…39 Reports by others have suggested that translocation of AP-2 from the cytoplasm to the nucleus is disrupted during melanoma progression and is a crucial event in the development of melanoma. 40 AP-2 may act to regulate not only Bcl-2 expression but also several other genes whose products are involved in tumor growth and metastasis of melanoma. AP-2 proteins were reported to bind with p53 to p53 target genes such as p21 and so act as a tumor suppressor.…”

Section: Discussionmentioning

confidence: 99%

Mcl-1, Bcl-XL and Stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of melanoma

et al. 2007

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Members of the Bcl-2 family of antiapoptotic proteins (Bcl-2, Bcl-XL and Mcl-1) are key regulators of apoptosis. The purpose of the present study was to examine and better define the role of Bcl-2, Bcl-XL and Mcl-1 in the progression of melanoma. Immunohistochemical staining for Bcl-2, Bcl-XL and Mcl-1 was performed on paraffin sections of 100 cases of benign nevi, primary melanoma and metastatic melanoma. Expression was correlated with histopathologic features, clinical progress and expression of transcription factors (AP-2, MITF and p-Stat3). Bcl-2 was expressed in 100% of benign nevi and thin melanoma (r1.0 mm) but was less in thick melanoma (41.0 mm) (88%), subcutaneous (62%) and lymph node metastases (35%). In contrast, Bcl-XL and Mcl-1 were expressed at lower levels in nevi and thin melanoma compared to Bcl-2 but their expression was much higher in thick melanoma and in subcutaneous and lymph node metastases (Po0.0001). Bcl-2 expression was negatively associated with tumor thickness (Po0.05) but Bcl-XL expression increased with increasing tumor thickness (Po0.05) and dermal tumor mitotic rate (Po0.05). Similarly Mcl-1 expression increased with increasing tumor thickness (Po0.09) and dermal tumor mitotic rate (Po0.17). Bcl-2 expression was positively correlated with expression of the transcription factors microphthalmia transcription factor (MITF) and nuclear AP-2 whereas Bcl-XL (and Mcl-1) expression were positively correlated with p-Stat3. This study is the first to show a clear dissociation between changes in Bcl-2 expression (downregulation) and Bcl-XL, Mcl-1 expression (upregulation) during progression of melanoma. The results were also consistent with a role for AP-2 and MITF in regulation of Bcl-2 and pStat3 in regulation of Bcl-XL. These findings have important implications for the development of treatments targeting antiapoptotic proteins in patients with melanoma. Modern Pathology (2007) 20, 416-426.

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“…This method calculates marker expression on a continuous scale by using pixel intensity and is shown to be widely applicable for biomarker analysis. [8][9][10][11][12][13][14] Previous measurements of ER by AQUA have correlated well with IHC analysis on tissue from two large clinical trials and have also predicted response to tamoxifen. 15,16 In an attempt to both quantify and standardize the measurement of ER in patient tissue, we first sought to define an ER cut point with biologic and clinical relevance.…”

Section: Journal Of Clinical Oncology O R I G I N a L R E P O R T V Omentioning

confidence: 99%

Standardization of Estrogen Receptor Measurement in Breast Cancer Suggests False-Negative Results Are a Function of Threshold Intensity Rather Than Percentage of Positive Cells

Welsh

Moeder

Kumar

et al. 2011

JCO

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A B S T R A C T PurposeRecent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance. MethodsAn assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods. ResultsTwo methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/g immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHCnegative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays. ConclusionAssessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.

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Automated Quantitative Analysis of Activator Protein-2α Subcellular Expression in Melanoma Tissue Microarrays Correlates with Survival Prediction

Cited by 63 publications

References 23 publications

Identification of the Hypoxia-inducible Factor 2α Nuclear Interactome in Melanoma Cells Reveals Master Proteins Involved in Melanoma Development

Identification of the Hypoxia-inducible Factor 2α Nuclear Interactome in Melanoma Cells Reveals Master Proteins Involved in Melanoma Development

Mcl-1, Bcl-XL and Stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of melanoma

Standardization of Estrogen Receptor Measurement in Breast Cancer Suggests False-Negative Results Are a Function of Threshold Intensity Rather Than Percentage of Positive Cells

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