Ceramide synthase as a therapy target in prostate cancer

描述（由申请人提供）：本项目的目标是开发能够克服人前列腺肿瘤细胞辐射抗性的辐射反应调节剂。虽然放射能够永久根除局部的人前列腺肿瘤，但由于残留的耐放射性克隆原，近30%接受潜在治愈性放射治疗的患者在照射肿瘤部位复发。本研究旨在探讨人前列腺癌细胞株LNCaP和CRW 22 Rv 1对辐射诱导凋亡的抵抗机制。初步研究表明，佛波醇酯12，O-十四烷酰基佛波醇-13-乙酸酯（TPA）激活蛋白激酶C（PKC）亚型可诱导这些肿瘤细胞产生中度凋亡反应，并使它们对辐射敏感，诱导凋亡。我们还表明，这种凋亡反应是由神经酰胺合成酶（CS）和从头合成神经酰胺介导的。我们以前报道过CS活性受共济失调毛细血管扩张突变（ATM）基因的负调控。我们与该应用相关的初步研究表明，在LNCaP和CRW 22 Rvl细胞中，TPA降低ATM水平。我们观察到类似的减少ATM蛋白水平和放射增敏使用二酰基甘油（DAG）-内酯，HK 654，或反义寡核苷酸ATM（AS-ATM-ODNs）。在这个项目中，我们建议确定PKC α和PKC 5参与TPA通过激活ATM-CS途径诱导的细胞凋亡。我们还将研究CS参与线粒体凋亡的机制，线粒体内神经酰胺的产生，以及神经酰胺，PKC 6和/或Bax之间的相互作用。最后，我们提出研究ATM-CS途径在裸鼠中原位生长的LNCaP和CWR 22 Rvl肿瘤中的体内作用。 我们将研究对TPA的反应（辐射对单剂量辐射以及分次辐射的反应），并将这些动物中的肿瘤生长与组织读数相关联。提出的具体目标是互动的，并解决TPA（前列腺癌细胞和前列腺动物模型中辐射诱导的细胞死亡）的新的和迄今未知的机制。我们的研究计划提出了一个基于信号的方法，调查新的假设，辐射抗性的机制，这可能提供一个基础，在耐药的人前列腺肿瘤克隆的调制的辐射反应，在人前列腺癌的辐射管理的临床应用潜力。

DESCRIPTION (provided by applicant): The goal of this project is to develop modulators of radiation response able to overcome radiation resistance in human prostate tumor cells. Although radiation is able of permanently eradicate localized human prostate tumors, nearly 30% of patients treated with potentially curative radiotherapy relapse at the sites of the irradiated tumor due to residual radiation-resistant clonogens. In this application we propose to explore the mechanisms of resistance to radiation-induced apoptosis for the highly resistant, well-characterized human prostate cancer cell lines-LNCaP and CRW22Rv 1, Our preliminary data Show that activation of protein kinase C (PKC) isoforms by the phorbol ester 12,O-tetradecanoyl phorbol-13-acetate, (TPA), induces a moderate apoptotic response in these tumor cells, and sensitizes them towards radiation, induced apoptosis. We also showed that this apoptotic response is mediated by the enzyme ceramide synthase (CS) and de novo synthesis of ceramide. CS activity was previously reported by us to be negatively regulated by the Ataxia Telangiectasia-Mutated (ATM) gene. Our preliminary studies related to this application showed that in LNCaP and CRW22Rvl cells, TPA reduces ATM levels. We observed a similar reduction in ATM protein levels and radiosensitization using a diacylgycerol (DAG)-lactone, HK654, or antisense oligonucleotides to ATM (AS-ATM-ODNs). In this project, we propose to define the involvement of PKCa and PKC5 in TPAinduced apoptosis via activation of the ATM-CS pathway. We will also study the mechanisms of CS involvement in mitochondrial apoptosis, ceramide generation within the mitochondria, and the interaction between ceramide, PKC6 and/or Bax. Finally, we propose to study the role of the ATM-CS pathway in vivo in LNCaP and CWR22Rvl tumors growing orthotopically in nude mice. We will study the response to TPA ( radiation in response to both single-dose radiation as well as to fractionated radiation and will correlate tumor growth in these animals with tissue readouts. The proposed Specific Aims are interactive and address new and heretofore unknown mechanisms of TPA(radiation-induced cell death in prostate cancer cells and prostate animal models. Our research plan presents a signaling-based approach, to investigate new I hypotheses, about mechanisms of radiation resistance, which could provide a basis for modulation of the l radiation response in resistant human prostate tumor clones, with potential for clinical applications in the radiation management of human prostate cancer.