Tumor Progression of Nuclear EGFR in Breast Cancer

描述（由申请人提供）：在上一个资助期内，我们已成功完成所有三个建议目标。以核HER2 （ErbB2）为模型，我们将研究扩展到核EGFR，并取得了很大进展。目前的竞争性更新建议是进一步研究细胞核EGFR的新功能和EGFR从细胞表面转运到细胞核的分子机制。我们之前报道过EGFR复合物识别并结合细胞周期蛋白D1转录的AT-rich序列（ATRS）。随后，我们证明了EGFR与细胞核中的STAT3相互作用，EGFR/STAT3复合物识别iNOS启动子的ATRS和STAT结合序列，并共同调节iNOS基因的转录。细胞周期蛋白D1和iNOS都促进肿瘤进展，这提高了核EGFR参与肿瘤进展的可能性。为了支持这一点，我们和其他人已经表明核EGFR与乳腺癌、卵巢癌、食道癌和口腔癌的不良临床结果相关。我们发现细胞核EGFR与PCNA（一种众所周知的核特异性抗原）相互作用，并使染色质结合的PCNA在tyr211残基上磷酸化，从而稳定活性PCNA并刺激DNA复制。此外，我们在细胞核中发现了几种有趣的与EGFR相互作用的蛋白质，包括RHA和SWI/SNF复合物。RHA是一种识别与ATRS序列相同的DNA序列的DNA结合蛋白，可能是没有DNA结合结构域的核EGFR的潜在DNA结合伙伴。SWI/SNF是一种参与染色质重塑的蛋白质复合体。这些新发现的EGFR相互作用蛋白已被证明在肿瘤进展中发挥作用。最后，扩展我们的观察，即细胞内吞作用和核孔复合体（NPC）参与了EGFR和HER2从细胞表面的核转运，我们有初步的结果表明，细胞表面EGFR向细胞核的转运涉及逆行转运机制。因此，我们假设核EGFR通过肿瘤促进基因的转录上调和与染色质重塑相关的关键蛋白复合物的相互作用，有助于肿瘤的进展。这项提议的长期目标是了解核EGFR的新功能及其在肿瘤进展中的作用。具体目标1：细胞核EGFR的转录调控和肿瘤进展；特异性目标2：核EGFR和染色质重塑复合物SWI/SNF在乳腺肿瘤发展中的作用特异性目标3:EGFR从细胞表面转运到细胞核的分子机制。由于EGFR长期以来被认为是一种细胞表面受体，其核功能几十年来一直被忽视。随着越来越多的证据表明细胞核中存在受体酪氨酸激酶，并逐渐发现其核功能和核EGFR在多种人类癌症中的预后价值，目前的建议代表了一个及时的项目，解决了关键但“几乎被忽视”的问题。

DESCRIPTION (provided by applicant): In the last funding period, we have successfully completed all three proposed aims. Using nuclear HER2 (ErbB2) as a model, we extended our investigation to nuclear EGFR and much progress has been made. The current competitive renewal proposal is to further investigate the novel functions of nuclear EGFR and molecular mechanism of EGFR trafficking from the cell surface to the nucleus. We previously reported that the EGFR complex recognizes and binds to an AT-rich sequence (ATRS) of cyclin D1 transcription. Later, we demonstrated that EGFR interacts with STAT3 in the nucleus and the EGFR/STAT3 complex recognizes the ATRS and STAT binding sequence of the iNOS promoter and co-regulates the transcription of the iNOS gene. Both cyclin D1 and iNOS contribute to tumor progression raising the possibility that nuclear EGFR may be involved in tumor progression. In support of this, we and others have shown that nuclear EGFR correlates with poor clinical outcomes in breast, ovarian, esophagus, and oral cancers. We showed that nuclear EGFR interacts with PCNA, a well-known nucleus-specific antigen and phosphorylates the chromatin-bound PCNA at Tyr-211 residue resulting in stabilization of active PCNA and stimulation of DNA replication. In addition, we identified several interesting proteins interacting with EGFR in the nucleus including RHA and the SWI/SNF complex. RHA is a DNA-binding protein that recognizes the DNA sequence identical to the ATRS sequence and may be a potential candidate to be a DNA-binding partner of nuclear EGFR, which does not have a DNA-binding domain. SWI/SNF is a protein complex involved in chromatin remodeling. These newly identified EGFR interacting proteins have been shown to play a role in tumor progression. Finally, extending our observation that endocytosis and the nuclear pore complex (NPC) are involved in the nuclear translocation from cell surface for both EGFR and HER2 we have preliminary results to suggest that a retrograde transport mechanism is involved in the trafficking of cell surface EGFR to the nucleus. Thus, we hypothesize that nuclear EGFR contributes to tumor progression through transcriptional upregulation of tumor promoting genes and interacting with critical protein complexes involved in chromatin remodeling. The long-term goal of this proposal is to understand the novel functions of nuclear EGFR and their roles in the tumor progression. Three specific Aims are proposed: Specific Aim 1: Transcriptional regulation and tumor progression of nuclear EGFR; Specific Aim 2: Nuclear EGFR and chromatin remodeling complex SWI/SNF in breast tumor development; Specific Aim 3: Molecular mechanism of EGFR trafficking from cell surface to nucleus. As EGFR has long been considered a cell surface receptor, its nuclear functions have been overlooked for decades. With increasing evidence of receptor tyrosine kinases in the nucleus and gradual discovery of their nuclear functions and prognostic value of nuclear EGFR in multiple human cancers, the current proposal represents a timely project that addresses the critical but "almost neglected" issues. PUBLIC HEALTH RELEVANCE: The long-term goal of this proposal is to understand the novel functions of nuclear EGFR and their roles in the tumor progression. With increasing evidence of receptor tyrosine kinases in the nucleus and gradual discovery of their nuclear functions including prognostic value of nuclear EGFR in multiple human cancers, the current proposal represents a timely project that addresses the critical but "almost forgotten" issues.