Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL. We conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments. The study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression. Application of a combined proteomic strategy recently presented as “Pitchfork” enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.

中文翻译：

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通过全面的蛋白质组学表征鉴定治疗 1 类人类嗜铬细胞瘤和副神经节瘤的潜在分子靶点

嗜铬细胞瘤和副神经节瘤（PPGL）是罕见的神经内分泌肿瘤。有助于敏感 PPGL 成像的新药物靶点和蛋白质可以改善 PPGL 患者（即患有复发或转移性疾病的患者）的治疗和生活质量。使用组合蛋白质组学策略，我们在肿瘤细胞表面上调的整合膜蛋白（IMP）和 PPGL 中的非膜药物酶中寻找此类临床相关靶点。我们对 22 个特征明确的人类 PPGL 样本和来自肾上腺髓质的正常嗜铬组织进行了详细的蛋白质组学分析。肿瘤裂解物的标准定量蛋白质组分析主要提供非膜蛋白的信息，并伴随着特定的膜蛋白质组目标方法，即使用凝集素亲和力进行糖肽富集、通过酰肼化学捕获糖肽以及膜嵌入疏水性的富集跨膜片段。该研究发现，与对照嗜铬组织相比，PPGL 中 67 种细胞表面整合膜蛋白的表达强烈上调。我们根据已记录的蛋白质在癌细胞生长或进展中的直接作用对蛋白质进行优先排序。使用特异性抗体证实了七个最有希望的药物靶标（CD146、CD171、ANO1、CD39、ATP8A1、ACE 和 SLC7A1）的表达增加。我们的实验策略还提供了可溶性蛋白质的表达数据。在 PPGL 中上调的可成药非膜酶中，我们鉴定了三个潜在的药物靶点（SHMT2、ARG2 和自分泌运动因子）并验证了它们的表达上调。最近提出的“Pitchfork”组合蛋白质组学策略的应用能够对膜和非膜蛋白质组进行定量分析，并鉴定出人类 PPGL 中的 10 个潜在药物靶点。7 种位于细胞表面的膜蛋白和 3 种非膜药物酶蛋白被鉴定并证实在 PPGL 中显着上调。所有这些蛋白质先前已被证明在几种人类癌症中表达上调，并在癌症进展中发挥直接作用。这些蛋白质的显着上调及其定位以及在肿瘤进展中确立的直接作用使这些分子有希望作为敏感 PPGL 成像的药物靶点或蛋白质。