Early Diagnosis of Oral Carcinoma Using Mutant PP32R1

描述(申请人提供)：大多数口腔白斑病变表现为角化过度(良性)、不典型增生或鳞状细胞癌。必须对病变进行活组织检查和显微镜检查才能诊断。但是，仅靠形态学并不能回答哪些非侵袭性病变会进展为癌症的问题。由于手术的并发症，给定的病变是否会进展为癌症的不确定性通常会影响最佳治疗方案的使用，即完全切除。因此，这项拟议中的研究要回答的问题是，“给定的白斑口腔病变是否有可能进展为癌症？”这项研究将通过检查现有活检标本中存在的pp32r1(GenBank AF008216)的基因组序列来开始回答这个问题。在对烟草相关的口腔白斑病变中的pp32r1基因进行的试点测序调查中，首席调查者在口腔鳞状细胞癌中发现了一个突变的、促进生长的pp32r1基因。该项目的第一个具体目标是在福尔马林固定的、石蜡包埋的口腔白斑标本中扩增pp32r1基因并进行测序。基本假设是，这些病变内的某些肿瘤细胞含有编码136-172残基内非保守氨基酸替代的突变。这些突变在恶性细胞克隆性扩大并形成临床癌症之前很久就定义了它们。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性。Pp32r1基因是编码具有多种活性的蛋白质的基因家族的成员。所有的家族成员都含有N-末端结构域，带有富含亮氨酸的重复序列，形成蛋白质-蛋白质相互作用的适配位点。而且，所有的家族成员都含有极端酸性的C-末端，通过离子作用力形成蛋白质-蛋白质相互作用的替代适配位点。另一个共同的生理特征是，其他家族成员，特别是PP32，在自然界中经常与其他细胞内蛋白结合。人们认为，与野生型PP32R1和PP32家族的其他成员相比，突变的、与癌症相关的、促进生长的PP32R1与细胞内蛋白有不同的结合。由此产生的异常大分子复合体以加速生长的方式改变了细胞的动态平衡。该项目的第二个具体目标是免疫共沉淀PP32R1结合蛋白，通过二维(2D)凝胶电泳分离结合伙伴，并通过基质辅助激光解吸/电离飞行时间质谱仪进行鉴定。细胞内蛋白配体的鉴定将为突变体PP32R1S提供特定的功能背景。

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.