Early Diagnosis of Oral Carcinoma Using Mutant PP32R1

描述（由申请人提供）：大多数口腔白斑病变表现为角化上皮过度增生（良性）、发育不良或鳞状细胞癌。病变必须进行活组织检查和显微镜检查才能诊断。但是，形态学本身并不能回答哪些非侵入性病变会发展为癌症的问题。由于不确定病变是否会发展为癌症，由于手术并发症，最好的治疗选择是完全切除。因此，拟议研究要回答的问题是，“给定的口腔白斑病变有可能发展成癌症吗？”该研究将通过检测现有活检标本中pp32r1 （GenBank AF008216）的基因组序列来回答这个问题。在烟草相关的口腔白斑病变中pp32r1基因的初步测序调查中，首席研究员在口腔鳞状细胞癌中发现了一个突变的、生长加速的pp32r1基因。该项目的第一个具体目标是在福尔马林固定石蜡包埋的口腔白斑标本中扩增和测序pp32r1基因。基本假设是这些病变内的某些肿瘤细胞含有编码残基136-172内非保守氨基酸取代的突变。这些突变早在恶性细胞克隆扩大并形成临床癌症之前就确定了它们。另一个问题涉及突变的、与癌症相关的PP32R1蛋白在加速细胞生长时的离散分子活动。pp32r1基因是一个基因家族的成员，该基因家族编码具有无数活性的蛋白质。所有家族成员都含有富含亮氨酸的n端重复结构域，形成蛋白质相互作用的适配器位点。而且，所有家族成员都含有极酸性的c -末端，这些c -末端通过离子力形成蛋白质-蛋白质相互作用的替代适配器位点。另一个共同的物理特征是其他家族成员，特别是PP32，经常在自然界中与其他细胞内蛋白结合。据认为，与野生型PP32R1s和PP32家族的其他成员相比，突变型、癌相关、生长加速的PP32R1s与细胞内蛋白的结合存在差异。由此产生的异常大分子复合物以加速生长的方式改变细胞内稳态。该项目的第二个具体目标是PP32R1结合蛋白的共免疫沉淀，通过二维凝胶电泳分离结合伙伴，并通过基质辅助激光解吸/电离飞行时间质谱法进行鉴定。细胞内蛋白配体的鉴定将为突变体PP32R1S提供一个特定的功能背景。

DESCRIPTION (provided by applicant): Most oral leukoplakic lesions represent hyperkeratotic epithelium (benign), dysplasia, or squamous cell carcinoma. The lesions must be biopsied and examined microscopically for diagnosis. But, morphology alone does not answer the question about which non-invasive lesions will progress to cancer. The uncertainty of whether a given lesion will progress to cancer or not often compromises the use of the best therapeutic option which is complete excision because of the complications of surgery. So, the question to be answered by the proposed research is, "Will a given leukoplakic oral lesion likely progress to cancer?" The research will begin to answer the question by examining the genomic sequences of pp32r1 (GenBank AF008216) present within existing biopsy specimens. In a pilot sequencing survey of the pp32r1 gene in tobacco-associated, oral, leukoplakic lesions, the principal investigator found a mutant, growth-accelerating pp32r1 gene in an oral squamous cell carcinoma. The first specific aim of the project is to amplify and sequence the pp32r1 gene within formalin-fixed, paraffin embedded specimens of oral leukoplakia. The basic hypothesis is that certain neoplastic cells within those lesions contain mutations encoding non-conservative amino acid substitutions within residues 136-172. These mutations define malignant cells long before they clonally enlarge and form a clinical cancer. Another question concerns the discrete molecular activities of mutant, carcinoma-associated PP32R1 proteins as they accelerate cellular growth. The pp32r1 gene is a member of a family of genes that encode proteins with myriad activities. All the family members contain N-terminal domains with leucine-rich repeats that form adapter sites for protein-protein interactions. And, all the family members contain extremely acidic C-terminals that form alternative adapter sites for protein-protein interactions by ionic forces. Another common physical characteristic is that other family members, particularly PP32, often appear in nature bound to other intracellular proteins. It is thought that mutant, carcinoma-associated, growth-accelerating PP32R1s differentially bind intracellular proteins in comparison to wild-type PP32R1s and other members of the PP32 family. The resultant abnormal macromolecular complexes change cellular homeostasis in ways that accelerate growth. The second specific aim of the project is co-immunoprecipitation of PP32R1 binding proteins, separation of the binding partners by 2-dimensional (2D) gel electrophoresis, and identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Identification of the intracellular protein ligands will provide a specific functional context for mutant PP32R1S.