Increased PDGFR-beta and VEGFR-2 protein levels are associated with resistance to platinum-based chemotherapy and adverse outcome of ovarian cancer patients.

Despite frequent initial response rates of epithelial ovarian cancer to platinum-based chemotherapy the majority of patients develop drug resistance. Our aim was to evaluate differential expression of signaling-pathway proteins in platinum-sensitive versus platinum-resistant primary epithelial ovarian cancer specimens to identify predictive biomarkers for treatment response. 192 patients were studied comprising of independent training (n = 89) and validation (n = 103) cohorts. Full-length proteins were extracted from paraffin-embedded samples including multiple regions per tumor to account for intratumoral heterogeneity. Quantitative reverse-phase-protein-arrays were used to analyze protein and phospho-protein levels of 41 signaling molecules including growth-factor receptors, AKT and MAPK signaling pathways as well as angiogenesis and cell-adhesion. Platinum-resistant ovarian cancers (56/192) demonstrated significantly higher intratumoral levels of the angiogenesis-associated growth-factor receptors PDGFR-beta and VEGFR2 compared to platinum-sensitive tumors. In addition, patients with high PDGFR-beta expression had significantly shorter overall and progression-free survival (HR 3.6 and 2.4; p < 0.001). The prognostic value of PDGFR-beta and VEGFR2 was confirmed in publicly available microarray-datasets. High intratumoral levels of the angiogenesis-related growth-factor receptors PDGFR-beta and VEGFR2 might serve as novel predictive biomarkers to identify primary resistance to platinum-based chemotherapy. Those ovarian cancer patients might particularly benefit from additional anti-vascular therapy including anti-VEGF antibody or receptor tyrosine-kinase-inhibitor therapy.

Quantitative and integrated proteome and microRNA analysis of endothelial replicative senescence.

Age-related changes in vascular functioning are a harbinger of cardiovascular disease but the biological mechanisms during the progression of endothelial senescence have not been studied. We investigated alterations in the proteome and miRNA profiles in the course of replicative senescence using primary human umbilical vein endothelial cells as an in vitro vascular model. Quantitative proteomic profiling from early growth stage to senescence was performed by isotope-coded protein label coupled to LC-ESI-MS/MS analysis. Some proteins consistently changed their expression during the senescence whereas others appeared as deregulated only during the late senescence. The latter was accompanied by alterations in morphology of senescent endothelial cells. MicroRNA expression profiling revealed transient changes in the level of miR-16-5p, miR-28-3p and miR-886-5p in the early senescence, decrease in the level of miR-106b-3p at the late stage, and continuous changes in the expression of miR-181a-5p and miR-376a-3p during the whole senescence process. Integrating data on proteomic and microRNA changes indicated potential crosstalk between specific proteins and non-coding RNAs in the regulation of metabolism, cell cycle progression and cytoskeletal organization in the endothelial senescence. The knowledge of molecular targets that change during the senescence can ultimately contribute to a better understanding and prevention of age-related vascular diseases.

Variation in cell signaling protein expression may introduce sampling bias in primary epithelial ovarian cancer.

Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5-9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17-53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12-48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several distinct locations to avoid sampling bias.

Characterization of signalling pathways by reverse phase protein arrays.

Reverse phase protein array (RPPA) is a very suitable technique to analyze large numbers of proteins in small samples like for example tumor biopsies. Beside their small size another major hindrance for the analysis of proteins from biopsies is the extraction of proteins from formalin-fixed and paraffin-embedded (FFPE) tissues. Here we describe a protocol, allowing quantitative extraction of large numbers of proteins from FFPE tissues and their subsequent analysis by RPPA. To elucidate the role of epidermal growth factor receptor (EGFR) signalling in ovarian cancer, we analyzed 23 primary tumors and corresponding metastases for the expression of 25 proteins involved in EGFR signalling with special emphasis on epithelial-mesenchymal transition (EMT). We found a significant correlation of Snail with EGFR((Tyr1086)) and p38 MAPK((Thr180/Tyr182)) in primary ovarian carcinoma and with EGFR((Tyr1086)) in their corresponding metastases. Additionally, we showed that high expression levels of the E-cadherin repressor Snail in primary tumors combined with high expression levels of the pp38 MAPK((Thr180/Tyr182)) in metastasis lead to an increased risk for death in ovarian carcinoma patients.

The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation.

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas.

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

Common protein biomarkers assessed by reverse phase protein arrays show considerable intratumoral heterogeneity in breast cancer tissues.

Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2-5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22-43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range 18-38%) or between different tumor zones (CV 24%, range 17-38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18-34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29-98%) and lymph node metastases (CV 65%, range 40-146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.

Intratumoral heterogeneity of microRNA expression in breast cancer.

Profiling studies have identified specific microRNA (miRNA) signatures in malignant tumors including breast cancer. Our aim was to assess intratumoral heterogeneity in miRNA expression levels within primary breast cancers and between axillary lymph node metastases from the same patient. Specimens of 16 primary breast cancers were sampled in 8-10 distinct locations including the peripheral, intermediate, and central tumor zones, as well as two to five axillary lymph node metastases (n = 9). Total RNA was extracted from 132 paraffin-embedded samples, and the expression of miR-10b, miR-210, miR-31, and miR-335 was assessed as well as the reproducibility of RNA extraction and miRNA analysis by quantitative RT-PCR. Considerable intratumoral heterogeneity existed for all four miRNAs within primary breast cancers (CV 40%). No significant differences within (CV 34%) or between different tumor zones (CV 33%) were found. A similar variation in miRNA expression was observed between corresponding lymph node metastases (mean CV 40%). In comparison, the variation among different patients showed a CV of 80% for primary tumors and 103% for lymph node metastases. Both miRNA extraction procedures and quantitative RT-PCR showed high reproducibility (CV ≤ 2%). Thus, the intratumoral heterogeneity of miRNA expression in breast cancers can lead to significant sampling bias. Assessment of breast cancer miRNA profiles may require sampling at several different tumor locations and of several tumor-involved lymph nodes when deriving miRNA expression profiles of metastases.

uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases.

The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-ß, ILK, pGSK3-ß, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-ß, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-ß). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.

Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues.

AIMS: Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation. METHODS AND RESULTS: In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays. CONCLUSION: This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.

Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HER3 and uPAR.

In the last few years, new approaches and developments in patient-tailored cancer therapies have raised the need to select, more precisely, those patients who will respond to personalized treatments. Therefore, the most efficient way for optimal therapy and patient selection is to provide a tumour-specific protein network portrait prior to treatment. The aim of our study was to monitor protein networks in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissues, with special emphasis on epidermal growth factor receptor 2 (HER2)-mediated signalling pathways, to identify and validate new disease markers. For this purpose we used a recently developed technology to extract full-length proteins from FFPE tissues and analysed 23 molecules involved in HER2-related signalling by reverse phase protein microarray (RPPA) in a series of 106 FFPE breast cancer tissue samples. We found a significant correlation of HER2 with human epidermal growth factor receptor 3 (HER3/erbB3), epidermal growth factor receptor 1 (EGFR/HER1/erbB1) and urokinase plasminogen receptor (uPAR) in routinely used FFPE breast cancer tissues. Thus, targeting HER2, EGFR, HER3 and uPAR together may offer a more efficient treatment option for patients with breast cancer.

Discovery of new molecular subtypes in oesophageal adenocarcinoma.

A large number of patients suffering from oesophageal adenocarcinomas do not respond to conventional chemotherapy; therefore, it is necessary to identify new predictive biomarkers and patient signatures to improve patient outcomes and therapy selections. We analysed 87 formalin-fixed and paraffin-embedded (FFPE) oesophageal adenocarcinoma tissue samples with a reverse phase protein array (RPPA) to examine the expression of 17 cancer-related signalling molecules. Protein expression levels were analysed by unsupervised hierarchical clustering and correlated with clinicopathological parameters and overall patient survival. Proteomic analyses revealed a new, very promising molecular subtype of oesophageal adenocarcinoma patients characterised by low levels of the HSP27 family proteins and high expression of those of the HER family with positive lymph nodes, distant metastases and short overall survival. After confirmation in other independent studies, our results could be the foundation for the development of a Her2-targeted treatment option for this new patient subgroup of oesophageal adenocarcinoma.

Use of formalin-fixed and paraffin-embedded tissues for diagnosis and therapy in routine clinical settings.

Formalin-fixed and paraffin-embedded (FFPE) tissues are used routinely everyday in hospitals world-wide for histopathological diagnosis of diseases like cancer. Due to formalin-induced cross-linking of proteins, FFPE tissues present a particular challenge for proteomic analysis. Nevertheless, there has been recent progress for extraction-based protein analysis in these tissues. Novel tools developed in the last few years are urgently needed because precise protein biomarker quantification in clinical FFPE tissues will be crucial for treatment decisions and to assess success or failure of current and future personalized molecular therapies. Furthermore, they will help to conceive why only a subset of patients responds to individualized treatments. Reverse phase protein array (RPPA) is a very promising new technology for quick and simultaneous analysis of many patient samples allowing relative and absolute protein quantifications. In this chapter, we show how protein extraction from FFPE tissues might facilitate the implementation of RPPA for therapy decisions and discuss challenges for application of RPPA in clinical trials and routine settings.

Producing reverse phase protein microarrays from formalin-fixed tissues.

In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray.

Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis.

The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.

Molecular profiling of signalling pathways in formalin-fixed and paraffin-embedded cancer tissues.

In most hospitals word-wide, histopathological cancer diagnosis is currently based on formalin-fixed and paraffin-embedded (FFPE) tissues. In the last few years new approaches and developments in patient-tailored cancer therapy have raised the need to select more precisely those patients, who will respond to personalised treatments. The most efficient way for optimal therapy and patient selection is probably to provide a tumour-specific protein network portrait prior to treatment. The discovery and characterisation of deregulated signalling molecules (e.g. human epidermal growth factor receptor 2, mitogen-activated protein kinases) are very promising candidates for the identification of new suitable therapy targets and for the selection of those patients who will receive the greatest benefit from individualised treatments. The reverse phase protein array (RPPA) is a promising new technology that allows quick, precise and simultaneous analysis of many components of a network. Importantly it requires only limited amounts of routine clinical material (e.g. FFPE biopsies) and can be used for absolute protein measurements. We and other research groups have described successful protein extraction from routine FFPE tissues. In this manuscript we show how these recent developments might facilitate the implementation of RPPA in clinical trials and routine settings.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains.

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of virus-cell fusion confirming the correlation between post-translational modification of tubulin and virus-cell fusion. These results thus identify tubulin and its post-translational modification as a new cellular target for interference with HIV-cell fusion.