The Role of Carboxypeptidase E in Cancer

羧肽酶 E 在癌症中的作用

• 批准号: 10001293
• 负责人: Yoke Peng Loh
• 金额: $ 22.76万
• 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/10001293
• 关键词: Automobile Driving; BCL2 gene; Biological Assay; Biological Markers; CXCL12 gene; CXCR4 gene; Cancer Cell Growth; Cancer cell line; Cell Communication; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Cellular Neurobiology; Collaborations; Complex; Culture Media; Cytoplasm; DNA; Data; Down-Regulation; E protein; Ectopic Expression; Environment; Future; Genes; Glioma; Growth; Human; Hypoxia; IL8 gene; IL8RB gene; Lead; Length; Lung; MAPK3 gene; MMP3 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; MicroRNAs; N-terminal; Names; Neoplasm Metastasis; Neuroendocrine Tumors; Northern Blotting; Nutrient; Ovarian; Pathway interactions; Patients; Phenotype; Pheochromocytoma; Phosphorylation; Play; Primary Neoplasm; Primary carcinoma of the liver cells; Process; Protein Isoforms; Proteins; RNA; RNA Splicing; Rattus; Recombinants; Regulation; Reporting; Resected; Resistance; Role; Serum; Signal Transduction; Sorting - Cell Movement; Starvation; Stress; TCF Transcription Factor; TNF gene; Translating; Tumor Cell Line; Universities; WNT Signaling Pathway; Western Blotting; base; beta catenin; cancer cell; cancer type; carboxypeptidase H; cell growth; cell type; colorectal cancer progression; cytotoxic; cytotoxicity; exosome; experimental study; extracellular; extracellular vesicles; fibrosarcoma; gain of function; glioma cell line; improved; inhibitor/antagonist; knock-down; loss of function; metastatic process; migration; mortality; mouse model; nanosized; neoplastic cell; neutralizing antibody; novel; novel therapeutic intervention; outcome forecast; overexpression; pancreatic cancer cells; pancreatic cell line; predictive marker; resilience; small hairpin RNA; targeted cancer therapy; therapeutic target; treatment strategy; tumor; tumor growth; tumor progression; tumorigenesis

Northern blot analysis revealed two carboxypeptidase E (CPE) mRNAs (2.4 and 1.7 kb in size) present in human metastatic hepatocellular carcinoma (HCC), ovarian, glioma, lung and pancreatic cell lines. We have cloned both these mRNAs from HCC97H cells. The 2.4 kb mRNA represents CPE-WT and the 1.7kb is a novel mRNA encoding a 40kD N-terminal truncated splice isoform of CPE, we named 40kD CPE-deltaN. Western blot analysis of these cancer cell lines shows expression of the 40kD CPE-deltaN which lacks the N-terminal secretory pathway sorting signal, and is translocated from the cytoplasm to the nucleus. However wild-type CPE (WT-CPE) mRNA is poorly translated into protein in these cancer lines except in glioma cells, and it is mainly secreted into the medium. In HCC97H and Panc-1 pancreatic cancer cells, siCPE-RNA knockdown of all forms of endogenous CPE inhibited cell proliferation, migration and invasion. Ectopic expression of 40kD N-terminal truncated CPE-deltaN in HCC and Panc-1 cells promoted proliferation, colony formation and invasion, whereas overexpression of WT-CPE promoted proliferation but not invasion. We have examined the mechanism underlying the promotion of invasion in HCC and Panc-1 cells by 40kD CPE-deltaN and found that several metastasis-related genes such as CXCL12, CXCR2, CXCR4 and MMP3 were up-regulated when CPE-deltaN was overexpressed in these cells. These findings suggest that the 40kD CPE- deltaN plays an important role in cancer cell growth and metastasis through up-regulating expression of metastasis-related genes (10.18632/genesandcancer,193. (2019)). 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-WT 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, 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 pro-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. Such an effect has been reported for CPE-WT secreted from glioma cell lines. However, this anti-metastatic effect was not observed in HCC cells which synthesize and secrete only low levels of CPE-WT. The mechanism underlying the inhibition of migration or invasion by CPE is unclear. 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-WT can drive tumor cell survival through ERK-BCL-2 signaling, as well as activate the wnt pathway during metabolic stress. However, it does not appear to be involved in invasion, and in glioma and HT1080 cells even have anti-invasion effects, unlike CPE-deltaN. Thus CPE-WT can have different effects in different cancer cell types. Hence, the level of expression of CPE and CPE-deltaN, the tumor environment and contributions from other pathways, all dictate the final phenotype of the tumor. Exosomes represent a class of nano-sized extracellular vesicles, which are widely associated with cancer progression and metastasis. They carry biomolecules (proteins, DNA, mRNA and miRNA) unique to their cell of origin and deliver them to recipient target cells, thereby mediating cell-cell communication. Recently, we have developed an assay that can quantitatively measure CPE/CPE-deltaN mRNA copy numbers in exosomes prepared from cell culture media. 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, although the size and exosome numbers were no different for high and low metastatic cells. Content analysis of exosomes derived from HCC high-metastatic (HCC97H) cells revealed presence of full length CPE-WT protein by Western Blot and CPE-WT mRNA using semi-quantitative PCR. We showed that exosomes released from HCC97H cells were able to enhance invasion and proliferation of HCC cells with poor metastatic ability (HCC97L). However, when CPE expression was suppressed in the HCC97H cells before exosome isolation, the exosomes had no effect on proliferation and invasion. These data show the ability of exosomes to confer metastasis in cancer cells and the role of exosomal CPE in driving the process. Since our previous studies have shown that down-regulation of CPE expression by CPE shRNA can reverse tumor growth and metastasis in a HCC mouse model, our future studies will focus on using exosomes as a vehicle for delivering CPE shRNA into cancers cells to inhibit growth and metastasis. This could potentially be developed as a new therapeutic approach to treating cancer.

Northern印迹分析显示，人类转移性肝细胞癌（HCC），卵巢，胶质瘤，肺和胰腺细胞系中存在两个羧肽酶E（CPE）mRNA（2.4和1.7 kb）。我们已经从HCC97H细胞中克隆了这两个mRNA。 2.4 kb mRNA代表CPE-WT，1.7KB是一个新颖的mRNA，编码了CPE的40KD N末端截断的剪接同工型，我们命名为40KD CPE-DELTAN。这些癌细胞系的蛋白质印迹分析显示了缺乏N末端分泌途径分类信号的40KD CPE-DELTAN的表达，并从细胞质转移到细胞核。然而，除神经胶质瘤细胞外，在这些癌细胞系中，野生型CPE（WT-CPE）mRNA被不良转化为蛋白质，并且主要分泌到培养基中。在HCC97H和PANC-1胰腺癌细胞中，所有形式的内源性CPE的SICPE-RNA敲低抑制了细胞的增殖，迁移和侵袭。 HCC和PANC-1细胞中40KD N末端截短的CPE-DELTAN的异位表达促进了增殖，菌落形成和侵袭，而WT-CPE的过表达促进了增殖，但不是侵袭。我们已经检查了40KD CPE-DELTAN促进HCC和PANC-1细胞促进侵袭的基础机制，并发现当CPE-Deltan在这些细胞中过度表达时，在CPE-DELTAN过度表达时，在CXCL12，CXCR2，CXCR2，CXCR2，CXCR2，CXCR4和MMP3等几个基因。这些发现表明，通过上调与转移相关基因的表达，40KD CPE-Deltan在癌细胞生长和转移中起着重要作用（10.18632/Genesandcancer，193。（2019）。 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-WT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，没有合成太多CPE的HCC细胞在这些代谢应力条件下显示出明显较小的细胞毒性。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治疗导致ERK1/2的磷酸化增加，并在mRNA和蛋白质水平下促生寿险基因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-BETA-ALPHA和IL-8），这些基因也可以支持肿瘤细胞存活。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。据报道了从神经胶质瘤细胞系分泌的CPE-WT的效果。但是，在合成和分泌低水平的CPE-WT的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-WT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。但是，与CPE-DELTAN不同，它似乎没有参与侵袭，在神经胶质瘤和HT1080细胞中甚至具有抗侵入作用。因此，CPE-WT在不同的癌细胞类型中可能具有不同的影响。因此，CPE和CPE-DELTAN的表达水平，肿瘤的环境和其他途径的贡献都决定了肿瘤的最终表型。 外泌体代表一类纳米大小的细胞外囊泡，这些囊泡与癌症的进展和转移广泛相关。它们携带生物分子（蛋白质，DNA，mRNA和miRNA），其原始细胞独有，并将其传递到受体靶细胞中，从而介导细胞 - 细胞通信。最近，我们开发了一种可以定量测量从细胞培养基制备的外泌体中的CPE/CPE-DELTAN mRNA拷贝数。我们在HCC，胰腺，卵巢癌和宫颈癌细胞系上的结果显示，这些不同癌症类型的高转移性细胞和低转移性细胞中CPE/CPEDELTAN mRNA水平显着升高，尽管对于高和低转移性细胞，大小和外泌体数量并没有什么不同。 源自HCC高量转移（HCC97H）细胞的外泌体的含量分析显示，使用半定量PCR，通过蛋白质印迹和CPE-WT mRNA存在全长CPE-WT蛋白。我们表明，从HCC97H细胞释放的外泌体能够增强转移能力不良（HCC97L）的HCC细胞的浸润和增殖。但是，当外泌体分离前的HCC97H细胞中CPE表达被抑制时，外泌体对增殖和侵袭没有影响。这些数据表明，外泌体赋予癌细胞转移的能力以及外泌体CPE在推动过程中的作用。由于我们先前的研究表明，在HCC小鼠模型中，CPE SHRNA对CPE表达的下调可以反向肿瘤的生长和转移，因此我们未来的研究将集中于使用外泌体作为将CPE SHRNA输送到癌细胞中的载体，以抑制生长和转移。这可能会作为治疗癌症的一种新的治疗方法发展。

项目成果

Carboxypeptidase E promotes cancer cell survival, but inhibits migration and invasion.

• DOI: 10.1016/j.canlet.2013.08.011
• 发表时间: 2013-12-01
• 期刊: CANCER LETTERS
• 影响因子: 9.7
• 作者: Murthy, Saravana R. K.;Dupart, Evan;Al-Sweel, Najla;Chen, Alexander;Cawley, Niamh X.;Loh, Y. Peng
• 通讯作者: Loh, Y. Peng