The roles of TRAF2 and RIP1 in breast cancer cell survival

乳腺癌（BC）是最常见的恶性肿瘤，也是第二大癌症原因 西方女性的死亡率。与大多数其他实体瘤的情况一样，转移和耐药性是肿瘤发生的主要原因。 主要死因。在约80%的BC病例中，由于PI 3 K-AKT通路的异常激活， 编码途径组分的基因的改变，例如Ras、Her 2、PTEN、PIK 3C和AKT。这 途径调节多种细胞过程以促进BC发育、生长、转移和药物治疗。 阻力因此，目前全世界正在进行100多项临床试验，以评估治疗效果。 PI 3 K和AKT抑制剂在BC中的疗效;然而，初始数据显示， 无效或经常导致产生耐药性和疾病复发。因此， 迫切需要另外的靶点和治疗组合。我们之前绘制了TRAF 2 TRAF 2 Ser-11磷酸化增强NF-κB活化，促进NF-κB活化。 癌细胞在细胞应激条件下的存活。最近，我们发现抑制BC中的AKT， 细胞导致TBK 1对TRAF 2的磷酸化增加，并且抑制AKT和TBK 1 在体外协同诱导BC细胞系凋亡并显著抑制异种移植BC肿瘤生长 in vivo. TBK 1及其同源物IKKε是丝氨酸/苏氨酸激酶，在65-70%的BC中过表达， 主要通过TRAF 2在Ser-11的直接磷酸化在BC细胞存活中起关键作用。在癌细胞中， TRAF 2组成型募集有效的E3连接酶cIAP 1和cIAP 2（cIAP）到RIP 1，以催化其非经典的 泛素化，这不仅是必要的NF-κB活化，而且还为抑制RIP 1依赖的 凋亡和坏死性凋亡。通过一系列的功能分析，我们鉴定了一种肽（Tp-14）， TRAF 2与RIP 1相互作用并与AKT抑制协同诱导BC细胞凋亡， 过表达RIP 1。生物信息学分析显示TRAF 2和RIP 1在侵袭性BC中过表达， 且与预后不良显著相关。因此，我们假设联合抑制AKT和 TRAF 2磷酸化或与RIP 1的相互作用协同诱导过表达TRAF 2的BC细胞凋亡。 TRAF 2和RIP 1。为了验证这一假设，我们提出了以下具体目标。目标一确定 PI 3 K-AKT和TBK 1-TRAF 2通路之间的信号传导串扰机制，并鉴定生物标志物 预测BC细胞对AKT和TBK 1联合抑制的反应。目标二评估以下药物的治疗效果 在BC异种移植模型中的组合AKT和TBK 1抑制。目标三确定TRAF 2的阻断 与RIP 1的相互作用影响体外BC细胞存活和体内异种移植肿瘤生长。我们的提议将使 BC细胞对AKT抑制的抵抗机制的新发现，并确定了 联合抑制AKT和TRAF 2磷酸化或与RIP 1相互作用用于BC治疗。

Breast cancer (BC) is the most frequently diagnosed malignancy and the second leading cause of cancer mortality in Western women. As is the case for most other solid tumors, metastasis and drug resistance are the main causes of death. In ~80% of BC cases, the PI3K-AKT pathway is aberrantly activated, due to the alterations in genes encoding the pathway components, such as Ras, Her2, PTEN, PIK3C and AKT. This pathway regulates multiple cellular processes to promote BC development, growth, metastasis, and drug resistance. Consequently, over 100 clinical trials are currently underway worldwide to evaluate the therapeutic efficacy of PI3K and AKT inhibitors in BC; however, initial data revealed that inhibition of this pathway is either not effective or often results in development of resistance and relapse of the disease. Thus, identification of additional targets and therapeutic combinations are urgently needed. We previously mapped TRAF2 phosphorylation sites and reported that TRAF2 Ser-11 phosphorylation enhances NF-κB activation to promote cancer cell survival under conditions of cellular stresses. Recently, we discovered that inhibition of AKT in BC cells leads to increased phosphorylation of TRAF2 by TBK1, and that inhibition of both AKT and TBK1 synergistically induces apoptosis in BC cell lines in vitro and significantly suppresses xenograft BC tumor growth in vivo. TBK1 and its close homologue IKKε are serine/threonine kinases overexpressed in 65-70% of BC and play critical roles in BC cell survival mainly by direct phosphorylation of TRAF2 at Ser-11. In cancer cells, TRAF2 constitutively recruits potent E3 ligases cIAP1 and cIAP2 (cIAPs) to RIP1 to catalyze its noncanonical ubiquitination, which is not only essential for NF-κB activation but also for the suppression of RIP1-dependent apoptosis and necroptosis. Through a series of functional assays, we identified a peptide (Tp-14) that blocks TRAF2 interaction with RIP1 and synergizes with AKT inhibition to induce apoptosis in BC cells that overexpress RIP1. Bioinformatic analyses revealed that TRAF2 and RIP1 are overexpressed in invasive BC, and significantly correlate with poor prognosis. Thus, we hypothesize that combined inhibition of AKT and TRAF2 phosphorylation or interaction with RIP1 synergistically induce apoptosis in BC cells that overexpress TRAF2 and RIP1. To test this hypothesis, we propose the following specific aims. Aim-1. Determine the mechanisms of signaling crosstalks between the PI3K-AKT and TBK1-TRAF2 pathways and identify biomarkers that predict BC cell response to combined AKT and TBK1 inhibition. Aim-2. Evaluate the therapeutic efficacy of combined AKT and TBK1 inhibition in BC xenograft models. Aim-3. Determine how blockade of TRAF2 interaction with RIP1 affects BC cell survival in vitro and xenograft tumor growth in vivo. Our proposals will shed new lights on the mechanisms underlying BC cell resistance to AKT inhibition and identify biomarkers for combined inhibition of AKT and TRAF2 phosphorylation or interaction with RIP1 for BC therapy.