Cellular or Extracellular Targeting of Lysyl Oxidase Propeptide for Oral Cancer

描述（由申请人提供）：口腔癌的发病率正在上升，现在是第8种最常见的癌症。预后很差;至少50%的患者在诊断后五年内死亡。赖氨酰氧化酶（LOX）是一种重要的细胞外基质蛋白和酶。LOX被合成为50 kDa的前酶原（Pro-LOX），其被分泌，然后在细胞外被前胶原C-蛋白酶加工以形成活性30 kDa赖氨酰氧化酶和18 kDa赖氨酰氧化酶前肽（LOX-PP）。最初归因于LOX酶的肿瘤抑制活性反而取决于LOX-PP。LOX-PP是一种天然存在的细胞外基质蛋白，可能具有有限的毒性，同时具有重要的抗癌活性。LOX-PP的不同区域在其抑制RAS依赖性癌症途径中结合不同的靶点。一些靶点是细胞内的（例如c-RAF和微管蛋白），而其他靶点是细胞外的（例如FGFR 1）。目前尚不清楚LOX-PP的哪些位置和靶点对其肿瘤抑制活性最重要。关于功能位置（细胞内或细胞外）的知识对于基于rLOX-PP的治疗剂的配制是重要的。所提出的研究将确定rLOX-PP的哪个位置在其体外抑制口腔癌表型（Aim 1）和体内小鼠原位口腔肿瘤生长（Aim 2）的能力中起作用。将特别关注HSP 70癌细胞标志物，其是rLOX-PP结合伴侣，并且在细胞外和细胞内发生，并参与RAS依赖性信号转导和细胞转化。将产生融合蛋白衍生物，其被设计为通过与IgG 4的Fc结构域融合而保持在细胞外，或被设计为通过与细胞穿透肽序列或棕榈酸融合而增加细胞摄取，并在体外测试抗癌活性和细胞外/细胞内定位。预期这些rLOX-PP衍生物具有增加的体内稳定性。将最具活性的衍生物全身施用至小鼠，其中使用UMSCC 2（侵袭性）和CAL 27（较低侵袭性）口腔癌细胞系植入舌原位肿瘤，并测定肿瘤生长和肿瘤标志物的表达以及活性RAS活性效应物。预计将确定至少一种具有增加的稳定性和抗口腔肿瘤生长有效性的rLOX-PP衍生物。将特别评价rLOX-PP的HSP 70结合结构域的细胞外与细胞内相互作用的重要性。为进一步完善抗肿瘤分子的设计提供了重要的信息。

DESCRIPTION (provided by applicant): Oral cancer incidence is rising and is the now the 8th most common form of cancer. Prognosis is poor; at least 50% of patients die within five years of diagnosis. Lysyl oxidase (LOX) is a critically important extracellular matrix protein and enzyme. LOX is synthesized as a 50 kDa pre-proenzyme (Pro-LOX) that is secreted, and then processed extracellularly by procollagen C-proteinases to form active 30 kDa lysyl oxidase enzyme, and the 18 kDa lysyl oxidase propeptide (LOX-PP). The tumor suppressor activity originally attributed to LOX enzyme depends instead on LOX-PP. LOX-PP is a naturally occurring extracellular matrix protein, and likely has limited toxicity while having important anti-cancer activities. Different regions of LOX-PP bind to different targets in its inhibition of RAS-dependen cancer pathways. Some targets are intracellular (c-RAF and tubulin for example), while others are extracellular (FGFR1, for example). It is currently unknown which location and targets of LOX-PP are most important for its tumor suppressor activity. Knowledge regarding the functional location (intracellular or extracellular) is important for formulation of therapeutics based on rLOX-PP. The proposed research will establish which location of rLOX-PP is functional in its ability to inhibit oral cancer phenotype in vitro (Aim 1), and orthotopic oral tuor growth in mice in vivo (Aim 2). Particular attention will be paid to the HSP70 cancer cell marker which is a rLOX-PP binding partner and occurs both extracellularly and intracellularly and participates in RAS-dependent signal transduction and cell transformation. Fusion protein derivatives which are designed to either remain extracellular by fusion with the Fc-domain of IgG4, or are designed for increased cell uptake by fusion with cell penetrating peptide sequences, or palmitic acid, will be created and tested for anti-cancer activities and extracellular/intracellular location in vitro. These rLOX-PP derivatives are expected to have increased in vivo stability. The most active derivative will be systemically administered to mice i which tongue orthotopic tumors have been implanted using UMSCC2 (aggressive) and CAL 27 (less aggressive) oral cancer cell lines and tumor growth and expression of tumor markers and active RAS activity effectors will be determined. It is expected that at least one rLOX-PP derivative with both increased stability and anti-oral tumor growth effectiveness will be identifie. The importance of extracellular compared to intracellular interactions of the HSP70-binding domain of rLOX-PP in particular will be evaluated. Important information will be gained to further refine the design of anti-tumor molecules.