No abstract

Metastasis is a major cause of mortality in cancer patients. However, the mechanisms governing the metastatic process remain elusive, and few accurate biomarkers exist for predicting whether metastasis will occur, something that would be invaluable for guiding therapy. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. mRNA encoding CPE-ΔN was found to be elevated in human metastatic colon, breast, and hepatocellular carcinoma (HCC) cell lines. In HCC cells, cytosolic CPE-ΔN was translocated to the nucleus and interacted with histone deacetylase 1/2 to upregulate expression of the gene encoding neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9) -which has been shown to promote melanoma metastasis. Nedd9 upregulation resulted in enhanced in vitro proliferation and invasion. Quantification of mRNA encoding CPE-ΔN in HCC patient samples predicted intrahepatic metastasis with high sensitivity and specificity, independent of cancer stage. Similarly, high CPE-ΔN mRNA copy numbers in resected pheochromocytomas/ paragangliomas (PHEOs/PGLs), rare neuroendocrine tumors, accurately predicted future metastasis or recurrence. Thus, CPE-ΔN induces tumor metastasis and should be investigated as a potentially powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients. IntroductionCancer mortality often results from metastatic disease and is not the direct effect of the primary tumor. With advances in cancer treatment, control of the primary tumor can be managed by multimodal therapy; however, metastatic disease is frequently refractory to the same therapeutic approaches. Thus, identification of biomarkers that could accurately predict future metastasis from the state of the primary tumor would be invaluable for guiding therapy.Currently, determination of the likelihood of developing metastatic disease is based on morphological and histological criteria such as the size of the primary tumor, local invasion, tumor differentiation together with vascular and capsular invasion, and the presence of cancer cells in lymph nodes. Although these criteria put many patients in a very high-risk category for metastatic disease, a significant number of them do not develop metastatic spread. On the other hand, patients with favorable conventional prognostic factors may still develop metastasis.Numerous mechanisms have been described that modulate metastatic potential, including those both endogenous and exogenous to the tumor cells. Such alterations may recruit genes

Major depressive disorder is often linked to stress. Whereas short-term stress is without effect in mice, prolonged stress leads to depressive-like behavior, indicating that an allostatic mechanism exists in this difference. Here we demonstrate that mice after short-term (1h/day for 7days) chronic restraint stress (CRS), do not display depressive-like behavior. Analysis of the hippocampus of these mice showed increased levels of neurotrophic factor-α1(NF-α1) (also known as carboxypeptidase E, CPE), concomitant with enhanced fibroblast growth factor 2 (FGF2) expression, and an increase in neurogenesis in the dentate gyrus. In contrast, after prolonged (6h/day for 21days) CRS, mice show decreased hippocampal NF-α1 and FGF2 levels and depressive-like responses. In NF-α1-knock out mice, hippocampal FGF2 levels and neurogenesis are reduced. These mice exhibit depressive-like behavior which is reversed by FGF2 administration. Indeed, studies in cultured hippocampal neurons reveal that NF-α1 treatment directly up-regulates FGF2 expression through ERK-Sp1 signaling. Thus, during short-term CRS, hippocampal NF-α1 expression is up-regulated and it plays a key role in preventing the onset of depressive-like behavior through enhanced FGF2-mediated neurogenesis. To evaluate the therapeutic potential of this pathway, we examined, rosiglitazone, a PPARγ agonist, which has been shown to have antidepressant activity in rodents and humans. Rosiglitazone up-regulates FGF2 expression in a NF-α1-dependent manner in hippocampal neurons. Mice fed rosiglitazone show increased hippocampal NF-α1 levels and neurogenesis compared to controls; thereby indicating the antidepressant action of this drug. Development of drugs that activate the NF-α1/FGF2/neurogenesis pathway can offer a new approach to depression therapy.

Carboxypeptidase E (CPE), a prohormone processing enzyme is highly expressed and secreted from (neuro)endocrine tumors and gliomas, and has been implicated in cancer progression by promoting tumor growth. Our study demonstrates that secreted or exogenously applied CPE promotes survival of pheochromocytoma (PC12) and hepatocellular carcinoma (MHCC97H) cells under nutrient starvation and hypoxic conditions, but had no effect on their proliferation. CPE also reduced migration and invasion of fibrosarcoma (HT1080) cells. We show that CPE treatment mediates survival of MHCC97H cells during metabolic stress by up-regulating the expression of anti-apoptotic protein BCL-2, and other pro-survival genes, via activation of the ERK1/2 pathway.

Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE(-/-) mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of neural stem cell/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing β-catenin. NF-α1 induced differentiation of neural stem cells to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-KO mice showed ~49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating anti-proliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment.

SummaryExpression of carboxypeptidase E, a prohormone processing enzyme in different cancer types was analyzed from data in the GEO profile database, and experimentally in pheochromocytomas. Microarray data from the GEO profile database indicated that significantly elevated levels of CPE mRNA was found in many non-endocrine cancers: cervical, colon rectal, renal cancers, Ewing sarcomas (bone cancer) and various types of astrocytomas and oligodendrogliomas, but expression of CPE mRNA was virtually absent in their respective counterpart normal tissues. Moreover there was a good correlation of high CPE mRNA expression with metastasis in cervical cancer and anaplastic oligodendrogliomas and neoplastic astrocytomas. Elevated CPE mRNA expression was found in neuroendocrine tumors in lung and pituitary adenomas, although the significance is unclear since endocrine and neuroendocrine cells normally express CPE. However studies of a neuroendocrine tumor, pheochromocytoma, revealed expression of not only wild type CPE, but a variant which was correlated with tumor behavior. Extremely high CPE mRNA copy numbers of the variant were found in very large or invasive tumors, both of which usually indicate poor prognosis. Thus, all the data suggest that CPE is involved in tumorogenesis and that it may play a role in promoting tumor growth and invasion. CPE could potentially serve as a diagnostic and prognostic biomarker for different cancer types.

Patients with Alzheimer's disease (AD), a common dementia among the aging population, often also suffer from depression. This comorbidity is poorly understood. Although most forms of AD are not genetically inherited, we have identified a new human mutation in the carboxypeptidase E (CPE)/neurotrophic factor-α1 (NF-α1) gene from an AD patient that caused memory deficit and depressive-like behavior in transgenic mice. This mutation consists of three adenosine inserts, introducing nine amino acids, including two glutamines into the mutant protein, herein called CPE-QQ. Expression of CPE-QQ in Neuro2a cells demonstrated that it was not secreted, but accumulated in the endoplasmic reticulum and was subsequently degraded by proteasomes. Expression of CPE-QQ in rat hippocampal neurons resulted in cell death, through increased ER stress and decreased expression of pro-survival protein, BCL-2. Transgenic mice expressing CPE-QQ did not show any difference in the processing enzyme activity of CPE compared with wild-type mice. However, the transgenic mice exhibited poor memory, depressive-like behavior, severely decreased dendrites in the hippocampal CA3 region and medial prefrontal cortex indicative of neurodegeneration, hyperphosphorylation of tau at Ser396, and diminished neurogenesis in the dentate gyrus at 50 weeks old. All these pathologies are associated with AD and the latter with depression and were observed in 50-week-old mice. Interestingly, the younger CPE-QQ mice (11 weeks old) did not show deficits in dendrite outgrowth and neurogenesis. This study has uncovered a human CPE/NF-α1 gene mutation that could lead to comorbidity of dementia and depression, emphasizing the importance of this gene in cognitive function.