Mechanisms of PTEN-Long Function in Cancer

描述(申请人提供)：癌症的发生是肿瘤抑制基因失活和癌基因激活的结果。PTEN是人类肿瘤中最常失活的抑癌基因之一。PTEN基因大小约为100000个碱基，位于染色体10q23上。PTEN基因从单个启动子转录而来，由9个外显子拼接而成，根据其3‘非翻译区的长度，其长度从2-6kb不等。所有转录本都包含一个403个氨基酸的开放阅读框架，编码PTEN蛋白，该蛋白由一个磷酸酶、C2和尾部结构域组成。PTEN蛋白编码一种脂磷酸酶，可将3‘磷酸从三磷酸肌醇(PIP3)的肌醇环上移除。PTEN可通过错义突变、纯合缺失、转录沉默和酶抑制等多种改变在癌症中失活。最近对PTEN转录本基因5‘端的研究表明，CTG起始密码子能够在与经典PTEN蛋白相同的阅读框架中翻译576个氨基酸的开放阅读框架，从而包含一个氨基酸末端173个氨基酸的交替翻译结构域，紧随其后的是磷酸酶、C2和尾部结构域。不同动物物种的比较表明，交替翻译的结构域在脊椎动物中高度保守。全长cDNA在细胞中的表达导致了75 kDa和56 kDa蛋白条带的表达，这取决于它们各自表达的完整起始密码子。对173个氨基酸交替翻译结构域的检测表明，它具有分泌信号序列和细胞穿透多精氨酸序列。我们命名为PTEN-Long的75 kDa形式的PTEN可以从细胞中分泌出来，并显示出细胞穿透活性。一旦进入细胞内，PTEN-Long就能够抑制AKT信号并抑制肿瘤生长。此外，PTEN-长红色荧光蛋白融合蛋白能够进入细胞。基于这些初步数据，我们建议更好地了解肿瘤突变如何影响PTEN-Long分泌、细胞结合、摄取和PIP3信号，并检测其肿瘤抑制和融合货物递送能力。

DESCRIPTION (provided by applicant): Cancers develop as a result of inactivation of tumor suppressor genes and activation of oncogenes. PTEN is one of the most frequently inactivated tumor suppressor genes in human cancer. The PTEN gene is approximately 100,000 bp in size and is located on chromosome 10q23. PTEN mRNA is transcribed from a single promoter, spliced together from 9 exons, and varies from 2-6 kb in length based upon the length of its 3' untranslated region. The transcripts all contain a 403 amino acid open reading frame that encodes the PTEN protein, which is composed of a phosphatase, C2 and tail domains. The PTEN protein encodes a lipid phosphatase that removes the 3' phosphate from the inositol ring of phosphoinositol-3, 4, 5-trisphosphate (PIP3). PTEN can be inactivated in cancers by a variety of alterations that include missense mutation, homozygous deletion, transcript silencing, and enzyme inhibition. Recent examination of the 5' end of the PTEN transcript cDNA indicated the potential for a CTG initiation codon capable of translating a 576 amino acid open reading frame in the same reading frame as classical PTEN protein, such that it would contain an amino-terminal 173 amino acid alternatively translated domain followed by a phosphatase, C2 and tail domain. Comparison of different animal species indicated that the alternatively translated domain was highly conserved among vertebrates. Expression of the full length cDNA in cells led to the expression of 75 kDa and 56 kDa protein bands, which were dependent upon intact initiation codons for their respective expression. Examination of the 173 amino acid alternatively translated domain revealed that it had a secretion signal sequence and a cell penetration polyarginine sequence. The 75 kDa form of PTEN, which we have named PTEN-Long, could be secreted from cells and displayed cell penetrating activity. Once inside the cell, PTEN-Long was able to inhibit signaling to AKT and regress tumor growth. Moreover, a PTEN-Long Red Fluorescent Protein fusion protein was able to enter cells. Based upon these preliminary data, we propose to better understand how tumor mutations affect PTEN-Long secretion, cell binding, uptake and PIP3 signaling and examine its tumor suppressive and fused cargo delivery capacities.