Huifang Guo

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

IntroductionProtein 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.ResultsFresh 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.ConclusionsWith 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 PI3K/AKT Pathway and Renal Cell Carcinoma

The phosphatidylinositol 3 kinase (PI3K)/AKT pathway is genetically targeted in more pathway components and in more tumor types than any other growth factor signaling pathway, and thus is frequently activated as a cancer driver. More importantly, the PI3K/AKT pathway is composed of multiple bifurcating and converging kinase cascades, providing many potential targets for cancer therapy. Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer that is notoriously resistant to traditional chemotherapies or radiotherapies. The PI3K/AKT pathway is modestly mutated but highly activated in RCC, representing a promising drug target. Indeed, PI3K pathway inhibitors of the rapalog family are approved for use in RCC. Recent large-scale integrated analyses of a large number of patients have provided a molecular basis for RCC, reiterating the critical role of the PI3K/AKT pathway in this cancer. In this review, we summarize the genetic alterations of the PI3K/AKT pathway in RCC as indicated in the latest large-scale genome sequencing data, as well as treatments for RCC that target the aberrant activated PI3K/AKT pathway.

Site-specific activation of AKT protects cells from death induced by glucose deprivation

The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose-deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGF receptor or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1 and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but not AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling.

Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.

Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium

The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer–dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development. DOI: http://dx.doi.org/10.7554/eLife.06866.001

Glycogen metabolism provides nutritional support to renal cancer cells under conditions of stress and may serve as a marker of response to antiangiogenic therapy with bevacizumab

Purpose: The clear cell type of renal cell carcinoma (ccRCC) derives its histologic appearance from accumulation of glycogen, but the functional significance of this phenomenon is yet unexplored. In physiological conditions, liver and muscle are the main tissues capable of producing and storing glycogen, which is mobilized to produce energy through glycolysis under stress conditions or limited nutrient supply. Tumors frequently experience fluctuations in nutrient availability, uptake and metabolic stress. The purpose of this study is to test the hypothesis that renal tumors express active glycogen synthase (GYS1) to produce glycogen, which may provide with an alternative energy source to support growth and survival under conditions of energetic stress. Agents blocking glycogen utilization (glycogen phosphorylase inhibitors) may contribute to beneficial therapeutic strategies for renal cell carcinoma. In addition, glycogen levels may determine response or resistance to therapeutic strategies that alter nutrient availability (antiangiogenic therapy). Methods: Cultured 786-0 renal cancer cells, clear-cell renal tumors and normal renal cortex samples were subjected to protein extraction or homogenization with sterile water. To detect the levels of GYS1, protein lysates were tested using reverse-phase protein arrays and Western blotting. Lysates in water were used to detect glycogen with the glucoamylase chromogenic method. We tested the functional significance of GYS1 by knocking down its levels with GYS1-specific siRNA, and measured cell numbers and proliferation under normal culture conditions or under metabolic stress mediated by the glycolysis inhibitor 2-deoxyglucose (2DG) or an inhibitor of oxidative phosphorylation (metformin). In order to determine whether GYS1 correlates with responses to antiangiogenic therapy, we tested phospho and total GYS1 expression in renal tumors from bevacizumab-treated patients who participated in a previously published study (Tsavachidou-Fenner et al., Annals of Oncol, 2010). Results: ccRCC tissues and renal cancer cell lines contained glycogen at significantly higher levels than the normal renal cortex. They also expressed higher levels of GYS1. We show that the activation status of GYS1 is controlled by the PI3K/AKT/GSK3α/β cascade via phosphorylation at serine 640, which is considered to be an inactivating event. siRNA-mediated knockdown of GYS1 expression abrogated glycogen synthesis in 786-0 cells. It also suppressed cell growth under conditions of energetic stress induced by 2DG. Blocking glycogen utilization with a glycogen phosphorylase inhibitor had a similar growth suppressing effect on 786-0 cells in vitro , in the presence of metabolic stress mediated by 2DG or metformin. Tumors from patients responding to bevacizumab expressed the inactive form of GYS1 (GYS1-pSer640). In particular, renal tumors from patients with long progression-free survival exhibited higher levels of phospho-GYS1 (p value = 0.00075), consistent with the notion that tumors with lower glycogen production may respond more favorably to anti-angiogenic therapy. Conclusions: Clear-cell renal carcinomas and renal cancer cells produce large amounts of glycogen as a consequence of increased GYS1 levels. Increased glycogen serves as an alternative energy source, enabling cell growth under conditions of metabolic stress. Consequently, glycogen may be responsible for bypassing limitations in nutrient supply imposed by angiogenic therapies, as evidenced by the increased levels of inactive GYS1 in bevacizumab responders. The glycogen pathway may prove a valuable therapeutic target as well as a marker of response to therapies. This talk is also presented as Poster B18.

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and “time to result” are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed “batch effects” which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting.

AKT isoform-specific expression and activation across cancer lineages

BackgroundAberrant AKT activation is prevalent across human cancer lineages, providing an important therapeutic target. AKT comprises three isoforms that mediate critical non-redundant, even opposing functions in cancer pathophysiology. Therefore, targeting specific AKT isoforms in particular cancers may be more effective than pan-AKT inhibition while avoiding disadvantages of pan-AKT inhibition. Currently, AKT isoform-specific expression and activation in cancer are not clearly characterized.MethodsWe systematically characterized AKT isoform-specific expression and activation in 211 cancer cell lines derived from different lineages and genetic backgrounds using a reverse-phase protein array platform.ResultsWe found that phosphorylation, but not expression, of AKT1 and AKT2 was coordinated in most but not all cells. Different cancer lineages displayed differential AKT1 and AKT2 expression and phosphorylation. A PIK3CA hotspot mutation H1047R but not E545K was associated with selective activation of AKT2 but not AKT1.ConclusionsOur study identified and validated AKT isoform-specific expression and phosphorylation in certain cell lines and demonstrated that genetic changes can affect AKT isoform-specific activation. These results provide a more precise understanding of AKT isoform-specific signaling and, in addition, facilitate AKT isoform targeting for personalized cancer therapies.

Abstract C199: Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules.

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated due to the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. While basal AKT2 phosphorylation was sensitive to inhibition of PI3K, basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C199. Citation Format: Huifang Guo, Meng Gao, Yiling Lu, Jiyong Liang, Philip L. Lorenzi, Shanshan Bai, David H. Hawke, Jie Li, Turgut Dogruluk, Kenneth L. Scott, Eric Jonasch, Gordon B. Mills, Zhiyong Ding. Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C199.

Abstract LB-35: Site specific activation of AKT protects cells from death induced by glucose deprivation .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKT phosphorylation at Thr308 and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKT Thr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose-deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKT at Thr308 but not Ser473. Our results reveal a novel mechanism of AKT activation under prolonged metabolic stress that protects cells from metabolic stress. The selective activation of AKT phosphorylation at Thr308 that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. Citation Format: Meng Gao, Jiyong Liang, Yiling Lu, Huifang Guo, Peter German, Shanshan Bai, Eric Jonasch, Xingsheng Yang, Gordon B. Mills, Zhiyong Ding. Site specific activation of AKT protects cells from death induced by glucose deprivation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-35. doi:10.1158/1538-7445.AM2013-LB-35