Enhancing Direct Tumor Cell Cytotoxicity By Manipulating Growth Factor Signaling

本申请的广泛、长期目标是通过抑制信号转导途径的相关组分来优化丝氨酸蛋白酶光动力疗法（PDT），从而改善癌症胸膜和腹膜扩散患者的结果。表皮生长因子受体（EGFR）信号通路的激活被认为限制了PDT作为治疗方法的有效性。初步数据表明，EGFR的抑制将增加PDT的治疗指数，增加肿瘤细胞杀伤而不伴随正常组织毒性的增加。此外，虽然PDT被认为主要通过凋亡机制杀死细胞，但通过抑制EGFR信号传导增加癌细胞死亡可能通过刺激坏死/非凋亡细胞死亡途径发生。因此，本项目将集中于定义和表征抑制生长因子信号传导对BPD介导的卵巢癌和肺癌细胞PDT后的信号传导、细胞毒性和细胞死亡机制的影响。为了最大限度地提高这些发现的外部有效性、普遍性和临床适用性，将使用一组卵巢癌（QvCa）和非小细胞肺癌（NSCLC）细胞系（包括OVCAR-5和H460）以及OVCAR-5和H460肿瘤异种移植物进行实验。我们将使用化学抑制剂和siRNA来抑制EGFR及其受体后信号传导伙伴，以研究抑制EGFR信号传导增强BPD介导的PDT细胞毒性的分子机制。我们还将检查PDT和EGFR通路抑制的测序和定时，以最大限度地增强BPD介导的PDT细胞毒性。在这些研究中将分析的具体终点是：1）EGFR和相关信号传导蛋白的水平和活化2）EGFR的核质分布及其与核STAT的关联3）细胞和肿瘤的克隆形成存活。最后，我们将确定调节生长因子信号传导对BPD介导的PDT后细胞死亡机制的影响，以进一步表征这种新的治疗方法，并进一步确定增强细胞毒性的领域。在这些研究中将分析的特定终点是：1）具有凋亡与坏死细胞死亡表型的细胞百分比; 2）细胞死亡相关蛋白的表达和活化; 3）对p53的影响。Bax和巴克蛋白表达对克隆形成细胞存活率和具有凋亡与坏死细胞死亡表型的细胞百分比的影响这些研究应该帮助我们理解PDT +生长因子抑制组合作为恶性肿瘤患者的治疗方法的潜在临床应用。鉴于腹腔内卵巢癌或胸腔内NSCLC扩散患者的预后极差，这种治疗方法的成功临床应用可能会为这些人群带来新的临床护理标准。

The broad, long-term goal of this application is to optimize serosal photodynamic therapy (PDT) through the inhibition of relevant components of the signal transduction pathways thereby improving the outcome of patients with pleural and peritoneal spread of cancers. Activation of the epidermal growth factor receptor (EGFR) signaling pathway is thought to limit the effectiveness of PDT as a therapeutic approach. Preliminary data suggest that inhibition of EGFR will increase the therapeutic index of PDT, increasing tumor cell kill without a concomitant increase in normal tissue toxicity. Moreover, while PDT is thought to kill cells primarily through apoptotic mechanisms, augmentation of cancer cell death through inhibition of EGFR signaling may occur through stimulation of necrotic/non-apoptotic cell death pathways. Therefore, this project will focus on defining and characterizing the impact of inhibiting growth factor signaling on the signaling, cytotoxicity and mechanisms of cell death following BPD-mediated PDT of ovarian and lung cancer cells. To maximize the external validity, generalizability and clinical applicability of these findings, experiments will be performed using a panel of ovarian cancer (QvCa) and non-small cell lung cancer (NSCLC) cell lines that includes OVCAR-5 and H460 and also using OVCAR-5 and H460 tumor xenografts. We will use chemical inhibitors and siRNA to inhibit EGFR and its post-receptor signaling partners to investigate the molecular mechanism by which the inhibition of EGFR signaling enhances BPD-mediated PDT cytotoxicity. We will also examine the sequencing and timing PDT and EGFR pathway inhibition in order to maximize the enhancement of BPD-mediated PDT cytotoxicity. The specific endpoints that will be analyzed in these studies are: 1) Levels and activation of EGFR and related signaling proteins 2} The nucleo-cytoplasmic distribution of EGFR and its association with nuclear STAT3: 3) The clonogenic survival of cells and tumors. Lastly, we will determine the effect of modulating growth factor signaling on the mechanism of cell death following BPD-mediated PDT in order to further characterize this novel treatment approach and to further identify areas for enhancement of cytotoxicity. The specific endpoints that will be analyzed in these studies are: 1) The percent of cells with apoptotic vs necrotic cell death pheotypes; 2) The expression and activation of cell death-related proteins: 3) The effect on p53. Bax and Bak protein expression on clonogenic cell survival and the percent of cells with apoptotic vs necrotic ceil death phenotypes These studies should help us to understand the potential clinical application of combination PDT + growth factor inhibition as a therapeutic approach for patients with serosal malignancies. Given the extremely poor prognosis for patients with spread of either ovarian cancer within the abdominal cavity or NSCLC within the chest cavity, successful clinical application of this treatment approach could lead to a new standard of clinical care in these populations.