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This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF ␤-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF ␣-and ␤-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF ␤-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF ␤-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF ␤-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF ␤-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF ␣-and ␤-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF ␤-receptor expression significantly correlates with less favorable clinicopatho-

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor ␤ (PDGFR␤) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFR␤ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR␤ in porcine aortic endothelial cells transfected with the ␤-receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR␤. We furthermore visualized tyrosine phosphorylated PDGFR␤ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology. Molecular & Cellular Proteomics 6:1500 -1509, 2007.

BackgroundThe identification of new prognostic markers for prostate cancer is highly warranted, since it is difficult to identify patients requiring curative treatment. Data from both experimental models and clinical samples have identified important functions of PDGFRβ on pericytes and fibroblasts in the tumor stroma.Methodology/Principal FindingsIn this study the prognostic significance of PDGFRβ in prostate cancer stroma, and in matched non-malignant tissue, was evaluated with immunohistochemistry. PDGFRβ expression was analyzed in normal and tumor stroma from more than 300 prostate cancer patients. High PDGFRβ expression in tumor stroma was associated with large tumor size, advanced stage, high Gleason score and high vessel density. Perivascular PDGFRβ staining in tumors was also correlated with high Gleason score. Correlations were also observed between PDGFRβ status in tumor stroma and non-malignant stroma. Similarly, high PDGFRβ expression in adjacent non-malignant tissue stroma correlated with large tumor size, advanced stage, high Gleason score and proliferation in non-malignant epithelium. Interestingly, high levels of PDGFRβ in the stroma of tumor and non-malignant tissue were associated with shorter cancer specific survival in prostate cancer patients.Conclusions/SignificanceThe study revealed a number of novel associations between stromal PDGFRβ expression in prostate tumors and several important clinical characteristics, including survival.

Background A better definition of biomarkers and biological processes related to local recurrence and disease progression is highly warranted for ductal breast carcinoma in situ (DCIS). Stromal–epithelial interactions are likely of major importance for the biological, clinical, and pathological distinctions between high- and low-risk DCIS cases. Methods Stromal platelet derived growth factor receptor (PDGFR) was immunohistochemically assessed in two DCIS patient cohorts (n = 458 and n = 80). Cox proportional hazards models were used to calculate the hazard ratios of recurrence. The molecular mechanisms regulating stromal PDGFR expression were investigated in experimental in vitro co-culture systems of DCIS cells and fibroblasts and analyzed using immunoblot and quantitative real-time PCR. Knock-out of JAG1 in DCIS cells and NOTCH2 in fibroblasts was obtained through CRISPR/Cas9. Experimental data were validated by mammary fat pad injection of DCIS and DCIS-JAG1 knock-out cells (10 mice per group). All statistical tests were two-sided. Results PDGFRα(low)/PDGFRβ(high) fibroblasts were associated with increased risk for recurrence in DCIS (univariate hazard ratio = 1.59, 95% confidence interval [CI] = 1.02 to 2.46; P = .04 Wald test; multivariable hazard ratio = 1.78, 95% CI = 1.07 to 2.97; P = .03). Tissue culture and mouse model studies indicated that this fibroblast phenotype is induced by DCIS cells in a cell contact-dependent manner. Epithelial Jagged1 and fibroblast Notch2 were identified through loss-of-function studies as key juxtacrine signaling components driving the formation of the poor prognosis-associated fibroblast phenotype. Conclusions A PDGFRα(low)/PDGFRβ(high) fibroblast subset was identified as a marker for high-risk DCIS. The Jagged-1/Notch2/PDGFR stroma–epithelial pathway was described as a novel signaling mechanism regulating this poor prognosis-associated fibroblast subset. In general terms, the study highlights epithelial–stromal crosstalk in DCIS and contributes to ongoing efforts to define clinically relevant fibroblast subsets and their etiology.

PDGF receptors (PDGFRs) exert cell type-specific effects in many different tumor types. They are emerging as key regulators of mesenchymal cells of the tumor microenvironment, and of many common malignancies, such as cancer of the breast, colon and prostate. In some tumor types PDGFRs are genetically activated and are thus directly involved in stimulation of malignant cell growth. Recent studies have uncovered clinically relevant variations in stromal PDGFR expression. High stromal PDGFRβ expression or activation is associated with poor prognosis in breast and prostate cancer. Indications of prognostic significance of stromal PDGFRβ expression in various GI tract tumor types also exist. The prognostic significance of PDGFRα and β in malignant cells of common epithelial tumor types should be further studied. Collectively data suggest that continued characterization of PDGFR expression in human tumors should present opportunities for improved accuracy in prognosis and also allow novel biomarker-based clinical studies exploring the efficacy of PDGFR-directed tumor therapies.

Cancer-associated fibroblasts (CAF) attract increasing attention as potential cancer drug targets due to their ability to stimulate, for example, tumor growth, invasion, angiogenesis, and metastasis. However, the molecular mechanisms causing the tumor-promoting properties of CAFs remain poorly understood. Forkhead box F1 (FoxF1) is a mesenchymal target of hedgehog signaling, known to regulate mesenchymalepithelial interactions during lung development. Studies with FoxF1 gain-and loss-of-function fibroblasts revealed that FoxF1 regulates the contractility of fibroblasts, their production of hepatocyte growth factor and fibroblast growth factor-2, and their stimulation of lung cancer cell migration. FoxF1 status of fibroblasts was also shown to control the ability of fibroblasts to stimulate xenografted tumor growth. FoxF1 was expressed in CAFs of human lung cancer and associated with activation of hedgehog signaling. These observations suggest that hedgehog-dependent FoxF1 is a clinically relevant lung CAF-inducing factor, and support experimentally the general concept that CAF properties can be induced by activation of developmentally important transcription factors. Cancer Res; 70(7); 2644-54. ©2010 AACR.

Dasatinib exhibits antitumor effects in vitro that could be increased by the combination with another RTK inhibitor targeting c-Met.