Mechanisms of PTEN-Long Function in Cancer

描述（由申请人提供）：癌症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基因的大小约为100,000 bp，位于染色体10q23上。PTEN mRNA由单个启动子转录，由9个外显子拼接在一起，根据其3'非翻译区的长度，长度在2-6 kb之间变化。转录本都包含一个403个氨基酸的开放阅读框，编码PTEN蛋白，该蛋白由磷酸酶、C2和尾部结构域组成。PTEN蛋白编码一种脂质磷酸酶，该酶可从磷酸肌醇- 3,4,5 -三磷酸（PIP3）的肌醇环上去除3'磷酸。PTEN在癌症中可以通过多种改变失活，包括错义突变、纯合缺失、转录物沉默和酶抑制。最近对PTEN转录物cDNA 5'端的检测表明，CTG起始密码子能够翻译与经典PTEN蛋白相同的576个氨基酸的开放阅读框，因此它将包含一个氨基末端173个氨基酸的交替翻译结构域，随后是一个磷酸酶，C2和尾部结构域。不同物种的比较表明，交替翻译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75 kDa和56 kDa蛋白带的表达，这两个蛋白带的表达依赖于完整的起始密码子。对173个氨基酸交替翻译结构域的检测显示，它具有分泌信号序列和细胞穿透聚精氨酸序列。我们将PTEN命名为PTEN- long，它可以从细胞中分泌并具有穿透细胞的活性。一旦进入细胞，PTEN-Long就能够抑制AKT的信号传导并减缓肿瘤的生长。此外，PTEN-Long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信号传导，并研究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.