An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays

INTRODUCTION: Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. RESULTS: Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples. The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues. CONCLUSIONS: With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type

The role of Sprouty-dependent inhibition of the Ras /ERK pathway in melanoma cells: A systems biology approach

Ras/ERK pathway signaling is frequently altered in melanoma. The discovery of mutations in the BRAF gene that lead to activation of the pathway suggested BRAF as a potential target for melanoma therapy. In order to design and optimize future therapeutic strategies for melanoma, it is important to identify additional factors that modulate the function of BRAF or cause activation of Ras/ERK signaling in the absence of a BRAF mutation. In order to identify and evaluate such factors, we used systems biology methodologies that enable the study of signaling networks with multiple components and interconnections. Towards this end, microarray-based gene expression profiling was performed for normal melanocytes and melanoma cell lines with or without a BRAF mutation. Data analysis of the Ras/ERK gene expression patterns revealed that the expression of the inhibitor SPRY2 was reduced in the cells without a BRAF mutation. This observation suggested that reduction in SPRY2 expression may be an alternative mechanism of activating the pathway in the absence of BRAF mutant. Subsequent functional analysis of SPRY2 using siRNA-mediated knockdown approaches in melanoma cells showed that upon SPRY2 knock-down by siRNA, ERK signaling is increased, but only in wild-type BRAF cells. The data suggested that SPRY2 is an inhibitor of the Ras/ERK pathway, but only when the BRAF mutation is absent. A potential mechanism of SPRY2 inhibition involves direct binding of SPRY2 to BRAF, as demonstrated by co-immunoprecipitation experiments. This interaction is disrupted by the presence of mutations in the kinase domain of BRAF, providing a potential explanation for the absence of SPRY2-dependent inhibition in the presence of BRAF mutations. The results of the functional analysis suggested that SPRY2 may play a role as a tumor suppressor being down-regulated in melanoma of wild-type BRAF status, and it is therefore of interest to understand its expression regulation. In this study, we constructed a computational model, which was based upon information provided by the functional analysis of SPRY2 mentioned above. The model was then used to explore SPRY2 expression regulation, by studying its responsiveness to an ERK-induced feedback loop. The computational model proposed that the Elk1-phosphatase PP2B modulates the ERK-induced regulation of SPRY2 expression, and predicted down-regulation of PP2B in a melanoma cell line. The model-driven hypotheses were then confirmed with biological experiments. The findings suggest that the behavior of the Ras/ERK pathway depends on alterations such as the BRAF mutation, differential SPRY2 levels and PP2B. Each of these pathway components may vary across different melanoma tumors, giving rise to complex pathway behaviors that can be evaluated by high-throughput profiling and computational approaches

The role of Sprouty-dependent inhibition of the Ras /ERK pathway in melanoma cells: A systems biology approach

Ras/ERK pathway signaling is frequently altered in melanoma. The discovery of mutations in the BRAF gene that lead to activation of the pathway suggested BRAF as a potential target for melanoma therapy. In order to design and optimize future therapeutic strategies for melanoma, it is important to identify additional factors that modulate the function of BRAF or cause activation of Ras/ERK signaling in the absence of a BRAF mutation. In order to identify and evaluate such factors, we used systems biology methodologies that enable the study of signaling networks with multiple components and interconnections. Towards this end, microarray-based gene expression profiling was performed for normal melanocytes and melanoma cell lines with or without a BRAF mutation. Data analysis of the Ras/ERK gene expression patterns revealed that the expression of the inhibitor SPRY2 was reduced in the cells without a BRAF mutation. This observation suggested that reduction in SPRY2 expression may be an alternative mechanism of activating the pathway in the absence of BRAF mutant. Subsequent functional analysis of SPRY2 using siRNA-mediated knockdown approaches in melanoma cells showed that upon SPRY2 knock-down by siRNA, ERK signaling is increased, but only in wild-type BRAF cells. The data suggested that SPRY2 is an inhibitor of the Ras/ERK pathway, but only when the BRAF mutation is absent. A potential mechanism of SPRY2 inhibition involves direct binding of SPRY2 to BRAF, as demonstrated by co-immunoprecipitation experiments. This interaction is disrupted by the presence of mutations in the kinase domain of BRAF, providing a potential explanation for the absence of SPRY2-dependent inhibition in the presence of BRAF mutations. The results of the functional analysis suggested that SPRY2 may play a role as a tumor suppressor being down-regulated in melanoma of wild-type BRAF status, and it is therefore of interest to understand its expression regulation. In this study, we constructed a computational model, which was based upon information provided by the functional analysis of SPRY2 mentioned above. The model was then used to explore SPRY2 expression regulation, by studying its responsiveness to an ERK-induced feedback loop. The computational model proposed that the Elk1-phosphatase PP2B modulates the ERK-induced regulation of SPRY2 expression, and predicted down-regulation of PP2B in a melanoma cell line. The model-driven hypotheses were then confirmed with biological experiments. The findings suggest that the behavior of the Ras/ERK pathway depends on alterations such as the BRAF mutation, differential SPRY2 levels and PP2B. Each of these pathway components may vary across different melanoma tumors, giving rise to complex pathway behaviors that can be evaluated by high-throughput profiling and computational approaches