The Role of Carboxypeptidase E in Cancer

羧肽酶 E 在癌症中的作用

• 批准号: 9550379
• 负责人: Yoke Peng Loh
• 金额: $ 42.82万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9550379
• 关键词: BCL2 gene; Binding; Biological Assay; Biological Markers; Cancer Patient; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Cellular Neurobiology; Collaborations; Colorectal Cancer; Complex; Culture Media; Cytoplasm; Disseminated Malignant Neoplasm; Ectopic Expression; Environment; Enzymes; Future; Gene Targeting; Genes; HDAC1 gene; HDAC4 gene; Human; Hypoxia; IL8 gene; Lead; MAPK3 gene; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Measures; Mediating; Messenger RNA; Metabolic stress; Methods; N-terminal; Neoplasm Metastasis; Neuroendocrine Tumors; Northern Blotting; Nuclear Protein; Nutrient; Ovarian; Pathway interactions; Patients; Phenotype; Pheochromocytoma; Phosphorylation; Play; Primary Neoplasm; Primary carcinoma of the liver cells; Protein Isoforms; Proteins; RNA Splicing; Rattus; Recombinants; Regulation; Reporting; Resistance; Role; Secretory Vesicles; Serum; Signal Transduction; Sorting - Cell Movement; Starvation; Stress; TCF Transcription Factor; TNF gene; Tumor Cell Line; Universities; WNT Signaling Pathway; Work; beta catenin; cancer cell; cancer type; carboxypeptidase H; cell growth; cell type; cytotoxic; cytotoxicity; exosome; experimental study; extracellular; fibrosarcoma; gain of function; hepatocellular carcinoma cell line; improved; inhibitor/antagonist; knock-down; loss of function; metastatic process; migration; mortality; mouse model; neoplastic cell; neutralizing antibody; outcome forecast; overexpression; pancreatic cancer cells; pancreatic cell line; predictive marker; prohormone; protein complex; resilience; targeted cancer therapy; therapeutic target; treatment strategy; tumor; tumor growth; tumor progression; tumorigenesis

We have now cloned the mRNA encoding a N-terminal truncated splice isoform of the prohormone processing enzyme, carboxypeptidase E with Mol. Wt. 40kD, (CPE-deltaN) from hepatocellular carcinoma (HCC) cell line. Northern blot analysis shows this mRNA (1.83 kb in size) is present in metastatic hepatocellular carcinoma, ovarian and pancreatic cell lines. 40kD CPE-deltaN lacks the N-terminus and hence the secretory pathway sorting signal found in wild-type CPE. Wild-type CPE is localized in secretory granules; however, CPE-deltaN is translocated from the cytoplasm to the nucleus of metastatic cancer cells where it could bind to a nuclear protein complex to promote the expression of metastatic genes in a histone deacetylase (HDAC1/2)-dependent manner. In Panc-1 pancreatic cancer cells, knockdown of all forms of endogenous CPE inhibited cell growth and invasion; in contrast, ectopic expression of 40kD N-terminal truncated CPE isoform significantly promoted proliferation, colony formation, invasion and migration, whereas overexpression of WT-CPE had only a small effect. These results suggest that the 40kD CPE- deltaN isoform plays a role in cell growth and metastasis. In collaboration with Dr. Arbersfeld (Tel Aviv University), we have shown that 40kD CPE-deltaN can activate the canonical Wnt pathway in HEK293 cells, resulting in increased beta-catenin expression. Beta-catenin functions with T-cell factor/lymphoid enhancer factor in the nucleus to activate expression of Wnt target genes, some of which are known to promote cancer cell proliferation. It is well known that such a mechanism involving the wnt pathway could lead to colorectal cancer progression. We have also examined the extracellular role and mechanism of action of CPE in tumor cell growth and survival from several cancer types using purified recombinant CPE. We showed that rat pheochromocytoma cells, a neuroendocrine tumor cell line (PC12) secretes CPE and addition of an anti-CPE neutralizing antibody in the cell medium resulted in increased cytotoxic effects and poor survival of the cells under metabolic stress (nutrient starvation and hypoxia). This loss of function experiment demonstrates that CPE is involved in maintaining the durability and resilience of neuroendocrine tumors under this type of stress. In gain of function experiments, we found that HCC cells, that do not synthesize much CPE, and showed significantly less cytotoxicity under these metabolic stress conditions when purified recombinant CPE protein was added to the culture medium. This effect was also observed when CPE was treated with 5microM GEMSA, a specific and potent inhibitor of CPE, indicating that the extracellular role of CPE in imparting resistance to the cells during metabolic stress is independent of its enzymatic activity. We found that treatment of HCC cells under metabolic stress, with CPE, resulted in increased phosphorylation of ERK1/2 and an increase in the expression of the survival gene BCL-2, at the mRNA and protein levels. Thus CPE is a tumor pro-survival factor during metabolic stress, acting through ERK-signaling. In addition, CPE treatment caused an increase of phospho-GSK3beta; (Ser9) and active-beta-catenin, suggesting the involvement of the canonical Wnt signaling pathway. Several other genes (TNF, NF-kappa,beta, I-kappa,beta,alpha, and IL-8), which could support tumor cell survival were also up-regulated in the CPE-treated HCC cells under metabolic stress. We demonstrated the ability of extracellular CPE to inhibit migration and invasion of a very aggressive fibrosarcoma cell line, HT1080, suggesting that CPE has anti-metastatic effects in these cells. However, this anti-metastatic effect was not observed in HCC cells. The mechanism underlying the inhibition of migration or invasion by CPE is less clear. Since the Wnt pathway components can mediate cancer cell invasion, one can speculate that the negative regulation of the Wnt pathway by CPE that we reported previously could be responsible for the inhibition of migration and invasion observed with CPE treatment. Our studies also indicated that CPE can drive tumor cell survival through ERK-BCL-2 signaling, as well as activate the wnt pathway during metabolic stress. Thus CPE can have different effects in different cancer cell types. Hence, the level of expression of CPE, the tumor environment and contributions from other pathways, all dictate the final phenotype of the tumor (Murthy et al, Cancer Lett. 2013). Recently, we have developed an assay that can quantitatively measure CPE/CPE-deltaN mRNA copy numbers in exosomes prepared from cell culture media and serum from normal and cancer patients. Our results on HCC, pancreatic, ovarian and cervical cancer cell lines show significantly elevated levels of CPE/CPEdeltaN mRNA in high versus low metastatic cells of these different cancer types. Moreover, the size and exosome numbers were no different for high and low metastatic cells. Work is now in progress to evaluate the use of this assay for serum exosomes to distinguish between patients with cancer versus normal controls.

现在，我们已经将编码的mRNA克隆，该mRNA编码了激素加工酶，羧肽酶E的N末端截短的剪接同工型。 wt。 40KD，（CPE-DELTAN）来自肝细胞癌（HCC）细胞系。北印迹分析表明，这种mRNA（大小1.83 kb）存在于转移性肝细胞癌，卵巢和胰腺细胞系中。 40KD CPE-DELTAN缺乏N端，因此在野生型CPE中发现的分泌途径分类信号。野生型CPE位于分泌颗粒中；然而，CPE-deltan从细胞质转移到转移性癌细胞的细胞核，在那里它可以与核蛋白复合物结合，以促进组蛋白脱乙酰基酶（HDAC1/2）依赖性方式的转移基因的表达。在PANC-1胰腺癌细胞中，所有形式的内源性CPE敲低抑制了细胞的生长和侵袭。相反，40KD N末端截短的CPE同工型的异位表达显着促进了增殖，菌落形成，侵袭和迁移，而WT-CPE的过表达仅具有较小的作用。这些结果表明，40KD CPE-Deltan同工型在细胞生长和转移中起作用。 与Arbersfeld博士（特拉维夫大学）合作，我们表明40KD CPE-DELTAN可以激活HEK293细胞中的规范WNT途径，从而导致β-catenin表达增加。 β-catenin在细胞核中具有T细胞因子/淋巴样因子的功能，以激活Wnt靶基因的表达，其中一些已知会促进癌细胞增殖。众所周知，涉及WNT途径的这种机制可能导致结直肠癌的进展。 我们还使用纯化的重组CPE研究了CPE在几种癌症类型的肿瘤细胞生长中的细胞外作用和作用机理。我们表明，大鼠嗜铬细胞瘤细胞，一种神经内分泌肿瘤细胞系（PC12）分泌CPE，并在细胞培养基中添加抗CPE中和抗体的抗CPE中和抗体导致细胞毒性的增加和细胞不良的生存率（营养恒星和低氧疾病）。这种功能丧失实验表明，CPE参与了这种压力下神经内分泌肿瘤的耐用性和弹性。在功能实验的增益中，我们发现，当将纯化的重组CPE蛋白添加到培养基中时，在这些代谢应力条件下，在这些代谢应激条件下显示的HCC细胞不会显着降低细胞毒性。当CPE用5microm Gemsa处理（一种特异性且有效的CPE抑制剂）时，还观察到了这种效果，表明CPE在代谢应激期间对细胞施加抗性的细胞外作用与其酶活性无关。我们发现，在代谢应激下对HCC细胞的治疗（用CPE）在mRNA和蛋白质水平下，ERK1/2的磷酸化增加，生存基因Bcl-2的表达增加。因此，CPE是代谢应激期间肿瘤促卵巢因子，它通过ERK信号作用。此外，CPE治疗导致磷酸化GSK3BETA的增加。 （Ser9）和活性-Beta-catenin，表明规范Wnt信号通路的参与。在代谢应激下，在CPE处理的HCC细胞中也上调了其他几个基因（TNF，NF-KAPPA，Beta，I-Kappa，I-Kappa，I-Kappa，beta，alpha和IL-8）。我们证明了细胞外CPE抑制非常侵略性纤维肉瘤细胞系HT1080的迁移和侵袭的能力，这表明CPE在这些细胞中具有抗转移性作用。但是，在HCC细胞中未观察到这种抗转移性作用。 CPE抑制迁移或侵袭的基础机制尚不清楚。由于Wnt途径成分可以介导癌细胞侵袭，因此可以推测我们先前报告的CPE对Wnt途径的负调控可能是抑制迁移和通过CPE治疗观察到的迁移和侵袭。我们的研究还表明，CPE可以通过ERK-BCL-2信号传导驱动肿瘤细胞的存活，并在代谢应激期间激活WNT途径。因此，CPE在不同的癌细胞类型中可能具有不同的作用。因此，CPE的表达水平，肿瘤环境和其他途径的贡献都决定了肿瘤的最终表型（Murthy等人，Cancer CancerLett。2013）。 最近，我们开发了一种可以定量测量由细胞培养基和正常患者和癌症患者血清制备的外泌体中的CPE/CPE-DELTAN mRNA拷贝数。我们在HCC，胰腺，卵巢和宫颈癌细胞系上的结果显示，在这些不同癌症类型的高转移细胞和低转移细胞中，CPE/CPEDELTAN mRNA水平显着升高。此外，对于高转移细胞和低转移性细胞，大小和外泌体数量没有什么不同。 现在正在进行评估该测定法对血清外泌体的使用，以区分癌症与正常对照的患者。