Cellular or Extracellular Targeting of Lysyl Oxidase Propeptide for Oral Cancer

描述(申请人提供)：口腔癌发病率正在上升，目前是第八种最常见的癌症。预后很差；至少50%的患者在确诊后五年内死亡。赖氨酰氧化酶(LOX)是一种重要的细胞外基质蛋白和酶。Lox50 kDa前酶原(Pro-LOX)被合成，经前胶原C-蛋白酶胞外处理后，形成30 kDa赖氨酸氧化酶活性酶和18 kDa赖氨酸氧化酶前肽(LOX-PP)。最初归因于LOX酶的肿瘤抑制活性依赖于LOX-PP。LOX-PP是一种天然存在的细胞外基质蛋白，在具有重要的抗癌活性的同时，可能具有有限的毒性。LOX-PP的不同区域在其抑制RAS依赖的癌症通路中结合到不同的靶点。有些靶点在细胞内(例如c-raf和微管蛋白)，而另一些靶点在细胞外(例如FGFR1)。目前尚不清楚LOX-PP的哪些部位和靶点对其肿瘤抑制活性最重要。关于功能定位(细胞内或细胞外)的知识对于基于rLOX-PP的治疗方案的制定非常重要。这项拟议的研究将确定rLOX-PP的哪个位置在体外抑制口腔癌表型的能力(目标1)，以及在体内抑制小鼠原位口腔肿瘤生长的能力(目标2)。HSP70肿瘤细胞标记物是rLOX-PP的结合伙伴，存在于细胞外和细胞内，参与RAS依赖的信号转导和细胞转化。通过与IgG4的Fc结构域融合而保持在细胞外的融合蛋白衍生物，或通过与细胞穿透性多肽序列或棕榈酸融合而增加细胞摄取的融合蛋白衍生物将被创造出来，并在体外进行抗癌活性和细胞外/细胞内定位的测试。这些rLOX-PP衍生物有望提高体内稳定性。最有效的衍生物将系统地应用于使用UMSCC2(侵袭性)和CAL 27(侵袭性较弱)口腔癌细胞系植入舌部原位肿瘤的小鼠I，并将测定肿瘤生长和肿瘤标记物和活性RAS活性效应物的表达。预计将至少鉴定一种同时具有增强稳定性和抗口腔肿瘤生长效果的rLOX-PP衍生物。与rLOX-PP的HSP70结合区域的细胞内相互作用相比，细胞外的相互作用的重要性将被特别评估。将获得重要信息，以进一步完善抗肿瘤分子的设计。

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.