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. Author manuscript; available in PMC: 2011 Mar 15.
Published in final edited form as: Cell Mol Neurobiol. 2010 Nov;30(8):1377–1381. doi: 10.1007/s10571-010-9592-y

Carboxypeptidase E: Elevated expression correlated with tumor growth and metastasis in pheochromocytomas and other cancers

Saravana RK Murthy 1, Karel Pacak 2, Y Peng Loh 1,3
PMCID: PMC3057539  NIHMSID: NIHMS230055  PMID: 21061162

Summary

Expression 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.

Keywords: Carboxypeptidase E, pheochromocytomas, neuroendocrine tumors, non-endocrine cancers, biomarker, metastasis

Introduction

Carboxypeptidase E (CPE) is a prohormone processing exopeptidase and it also functions as a prohormone sorting receptor for the regulated secretory pathway (RSP) in endocrine and neuroendocrine cells (Fricker et al., 1983, 1999; Cool et al., 1997, 1998.) Precursors of peptide hormones and neuropeptides are synthesized at the rough endoplasmic reticulum (RER) (neuro)endocrine cells. These precursors are then processed sequentially in the secretory granules, first by prohormone convertases (PC1 and PC2), by cleaving either in between or on the carboxyl side of paired basic residues to yield basic residue-extended peptides (Jean et al., 1993; Johanning et al., 1998; Rouille et al., 1995). A subset of soluble CPE molecules, in the secretory granules, then cleaves the basic residues from these intermediates to generate mature peptide hormones and neuropeptides (Hook et al., 1984). Another subset of CPE is the membrane form, which anchors to cholesterol/sphingolipid-rich microdomains known as lipid rafts or detergent-resistant membranes at the trans-Golgi network (TGN), and functions as a receptor for sorting prohormones away from other non-RSP proteins at the TGN, and into secretory granules in the RSP of pituitary (Cool et al., 1997; Shen et al., 1997; Dhanvantari et al., 2000, 2002; Zhang et al., 2003), and pancreatic ß-cells (Dhanvantari et al., 2003), as well as in neuronal cell lines (Normant et al., 1998). Additionally, the C-terminal tail of CPE drives bi-directional transport of BDNF vesicles to maintain vesicle homeostasis and secretion in hippocampal neurons by binding to microtubule motors via dynactin (Lou et al., 2005; Park et al., 2008). Thus CPE plays many cell biological roles in the endocrine and nervous system. Hence mutations of the CPE gene or lack of CPE in the CPE-knock out mice lead to many pathophysiological conditions, such as diabetes, infertility, obesity, low bone mineral density and deficits in learning and memory (Chen et al., 2001; Coleman et al., 1990; Naggert et al., 1995; Srinivasan et al., 2004; Cawley et al., 2003, 2004, 2010; Jacob et al., 2003).

CPE in non-endocrine cancers

While CPE has always been known to be expressed in endocrine cells and peptidergic neurons, it has now been found in epithelia-derived cancer cells as well, for example in cancer cells in the liver, which does not normally express CPE (Ge et al., 2005), although the function in tumor cells is not known. The first evidence for the expression of CPE in cancerous cells was documented by Grimwood et al., 1989 in human hepatoma (Hep G2) cells, although this form of CPE was slightly larger in size than that found in endocrine cells. Tumor cells of non-endocrine tissues such as breast cancer cells have also have been shown to express CPE (Du et al., 2001; Tang et al., 2009). CPE is normally not expressed in cervical tissues, but during cancerous state it is highly expressed (Fig. 1) (Bachtiary et al., 2006). Given that 70% of the cases of cervical cancer are correlated with human papillomavirus (HPV) infection (Walboomers et al., 1999) it would be interesting to determine if CPE expression is triggered by viral antigens in this pathological condition. CPE expression in colon is minimal when compared to other tissues, but during early onset colorectal cancer (CRC) and during lymph node metastasis, CPE mRNA expression is substantially elevated (Fig. 2 and 3) (Provenzani et al., 2006; Hong et al., 2007). CPE expression in normal kidney is comparatively lower than colon. Interestingly CPE expression is significantly elevated in neoplastic clear cell sarcoma of the kidney compared to the normal controls. Surprisingly, CPE expression is down regulated in Wilms' tumors (Fig. 4) (Cutcliffe et al., 2005). Additionally, microarray studies by Lenburg et al., 2003 have also convincingly shown that CPE mRNA expression is significantly elevated in renal clear cell tumor which has high metastatic potential, compared to adjacent normal tissue isolated from the same surgical samples (Fig. 5). Bone marrow has no detectable CPE expression, but in Ewing sarcomas, malignant round-cell tumors in bone, CPE expression is exceptionally high (Fig. 6), although less than rhabdomyosarcomas, malignant tumors derived from skeletal muscle. In brain, neuronal cells in particular express large amounts of CPE, with the highest concentrations found in hippocampus, amygdala and cortex. Astrocytes and glial cells also show notable expression of CPE. In pathological state, human gliomas have been shown to have elevated levels of CPE expression compared to normal glial cells in several studies. Studies by Bredel el at, 2005 in 50 human gliomas of various histogenesis, using cDNA microarrays (Fig. 7), and studies by Liu et al., 2007 in 12 primary brain tumor biopsies (Fig. 8) have demonstrated significant elevation in CPE expression compared to normal brain. It is important to note that oligodendroglioma and anaplastic oligodendroglioma (Fig. 7) and a mixed tumor (LGAG in Fig. 8) which is known to be malignant, have elevated levels of CPE in both these studies, Astrocytic tumors also shows elevated CPE expression compared to gliomas. Astrocytomas are CNS neoplasms, predominantly of astrocyte origin. Different subtypes of astrocytoma brain tumors showed differential expression of CPE (Fig. 9).

CPE in neuroendocrine cancers

Neuroendocrine tumors are of special interest because of the abundance of CPE expression in these tumors. CPE is required for biosynthesis of neuropeptides which serve as autocrine growth factors in these tumors (Rozengurt E, 2002). In a drug-resistant form of small-cell carcinoma of the lung (SCCL) cell line (NCI H82), expression of both 55 and 49kDa forms of CPE have been reported (North et al., 1998). Quantitative immunostaining of CPE has shown that elevated CPE expression is a statistically significant predictor of good prognosis in pulmonary neuroendocrine tumors such as bronchial carcinoids, small cell lung cancers, and large cell neuroendocrine carcinomas (He et al., 2004). However, this is in contrast to recent studies, which suggest that significantly elevated levels of CPE expression in lung cancer patients indicate poor prognosis (Krajnik et al., 2010; Mousa et al., 2010). In other endocrine tumors, significantly high levels of CPE expression were detected in some pituitary adenomas compared to normal pituitary. An oligonucleotide array analysis carried out on pituitary adenomas showed that CPE is differentially expressed (Morris et al., 2005, Fans et al., 2002) (Fig. 10). Interestingly, CPE mRNA expression in GH-secreting pituitary adenomas were significantly higher than normal pituitary, but in ACTH- secreting pituitary adenomas, it was lower. In contrast, another study specific to nonfunctioning pituitary macroadenoma showed no CPE immunorectivity (Tatsumi et al., 2003).

Pheochromocytomas/paragangliomas (PHEO/PGL)

PHEO/PGL are rare typical neuroendocrine tumors. There are different categories of PHEOs/PGLs; familial including mutation of succinate dehydrogenase subunites B, C, and D (SDHB/C/D), multiple endocrine neoplasia type 2 (MEN2), neurofibromatosis type 1 (NF1), and von Hippel-Lindau (VHL) syndrome that all together account for about 25% and the rest are sporadic (Neumann HP et al. 2002). The incidence of metastatic PHEO/PGL is currently estimated to be 3–36% or even higher, depending on genetic background and primary tumor localization (Pacak K et al., 2007). Currently, there are no reliable markers for diagnosing metastatic PHEO/PGL (Lenders et al., 2005). To understand the role of CPE in these tumors, we recently investigated CPE expression in these tumors by quantitative real time PCR (qRTPCR). Seven resected sporadic tumors from National Institutes of Health were analysed and these patients were followed up at the NIH. The patients were chosen based on frozen tumor and pathological availability. Interestingly during screening for an optimal amplicon of CPE for PCR, we came across a CPE variant smaller than the predicted amplicon, while amplifying at the 5′ UTR of CPE mRNA. We further designed specific primers and analyzed the sporadic PHEO/PGL samples and normal adrenals for this CPE variant. Wild type CPE was highly expressed in the PHEO samples and in normal adrenals. To our surprise, the new short CPE variant was not detected in normal adrenals, but was present only in the PHEO samples. Moreover, the mRNA copy numbers of this variant correlated with the state of the tumor with regards to tumor growth and invasiveness (Table 1). For example, patient S71 with a very large benign tumor (8 cm in diameter) had a ∼ 30 times higher CPE mRNA copy number than other patients (S39, S48, S49, S67) with benign tumors of < 5 cm and were disease free for at least 3 years following surgical resection of the primary tumor. Moreover, patient S76 who had a high CPE mRNA copy number also had a very aggressive tumor showing capsular and vascular invasion, although the tumor was diagnosed as benign based on pathology and biochemistry. Patient S45 who had a tumor with a high CPE mRNA copy number had metastatic papillary thyroid cancer two years prior to showing first symptoms of PHEO and was diagnosed with the disease 3 years later. These data suggest that highly elevated CPE variant mRNA levels in the primary PHEO tumor could be an indicator of poor prognosis.

Table 1. CPE expression in various cancers.

Serial # Cancer Type Sample type State Number of samples CPE expression fold change& ± SEM p value Geo profile ID
a Cervical cancer Biopsies Metastatic 33 7.4 0.426 <0.003-22 GDS2416/CPE
b Colorectal cancer Biopsies Metastatic 24 9.5 0.623 <0.0128 GDS2609/CPE
c Colorectal cancer Primary cells Metastatic 6 7 1.020 <0.0007 GDS1780/CPE
dΔ Clear cell sarcoma Biopsies Metastatic 14 2.49 0.260 <0.0001 GDS1282/CPE
eΔ Wilms' tumor Biopsies Metastatic 15 -0.5 0.260 <0.001-4 GDS1282/CPE
f$ Renal Clear Cell Carcinoma Biopsies Metastatic 8 16.3 1.894 <0.0007 GDS505/CPE
g Oligodendroglioma Biopsies Benign 8 1.75 0.726 <0.0097 GDS1813/CPE
h Anaplastic Oligodendroglioma Biopsies Metastatic 6 1.59 0.698 <0.0112 GDS1813/CPE
i Glioblastoma Biopsies Metastatic 30 1.48 0.425 <0.0029 GDS1813/CPE
j Astrocytic Tumor Biopsies Metastatic 5 1.65 0.325 <0.0070 GDS1813/CPE
k Non-functioning pituitary adenoma Biopsies Benign 5 -0.33 Pooled samples GDS1253/CPE
l ACTH-secreting pituitary adenoma Biopsies Benign 5 -0.5 Pooled samples GDS1253/CPE
m GH-secreting pituitary adenoma Biopsies Benign 5 1.75 Pooled samples GDS1253/CPE
n PRL-secreting pituitary adenoma Biopsies Benign 5 1 Pooled samples GDS1253/CPE
o Ewing's sarcoma* Biopsies Metastatic 11 8.5 0.650 <0.031 GDS971/CPE
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*

CPE expression fold changes in Ewing sarcomas vs rhabdomyosarcomas.

&

CPE expression fold changes in metastatic or begnin tumors versus normal tissues.

Δ

CPE expression fold changes compared to normal human fetal kidney.

$

CPE expression fold changes compared to adjacent normal human kidney.

Conclusions

In this study we have mined various microarray data from GEO profile database to analyze CPE mRNA expression in various non-endocrine and (neuro)endocrine cancers. As well, we have presented our data on the expression of a CPE variant in PHEOs/PGLs. Our analyses revealed that many non-endocrine cancers: cervical, colon, and kidney cancers and Ewing sarcomas show significant levels of expression of CPE mRNA which is virtually absent in their respective counterpart normal tissues. Moreover, the levels were even more elevated in metastatic and malignant tumors compared to the primary tumor. For example, CPE mRNA expression in lymph node metastasis (SW620) colorectal cancer patients' isogenic cell lines were much higher compared to those derived from the primary tumor (SW480). In addition, anaplastic oligodendroglioma which is known to be malignant, express much higher levels of CPE mRNA than normal brain. Likewise, low and high grade astrocytoma brain tumors which are neoplastic express much higher levels of CPE mRNA compared to benign glioma cells. Among the endocrine tumors examined, pituitary adenomas secreting growth hormone exhibited the highest level of CPE compared to normal pituitary. This was rather surprising since CPE was not required to process pro-growth hormone. In our studies on PHEOs, we found that a CPE variant mRNA was expressed that correlated with tumor size and invasiveness, although patient sample size was very small. A PHEO which was very large and one which was very invasive, although both diagnosed as benign, had CPE variant mRNA copy numbers more than 10 times greater than benign small tumors found in 4 patients, although they all were diagnosed as benign. In addition, another patient (S45) that had highly elevated CPE variant mRNA copy number in the resected PHEO, previously had metastatic papillary thyroid cancer. Our results suggest that the CPE variant could be a prognostic biomarker for PHEOs. Thus our extensive analysis of microarray data from the Geo profile database and studies on PHEOs have yielded correlative findings that strongly suggest that CPE and/or its variant is involved in tumorigenesis. The data also suggest that CPE may promote tumor growth and metastasis. Finally, CPE can potentially be a diagnostic and prognostic biomarker for malignancy in different cancer types.

Table 2. Determination of CPE variant copy number in Sporadic PHEO/PGL tumors.

Table 2 shows the expression of CPE variant mRNA copy number in sporadic pheochromocytoma/paraganglioma (PHEO/PGL) samples. Total RNA was extracted from frozen PHEOs/PGLs after homogenization in TRlzol reagent and 0.2μg was then converted to cDNA. A standard curve was set up to determine the CPE mRNA copy numbers by qRTPCR using CPE plasmid construct of eight different concentrations. Copy numbers were calculated using a standard formula. QRTPCR was done on PHEOs/PGLs samples and the crossing point for each sample was plotted on the standard curve to determine the mRNA copy number.

Sample ID Diagnosis* Genotype Copy Numbers
S39 Benign SPORADIC 175,080
S45 BenignΔ SPORADIC 6,181,873
S48 Renign SPORADIC 184,761
S49 Benign SPORADIC 181,713
S67 Benign SPORADIC 135,279
S71 Benign* SPORADIC 6,451,057
S76 Benign& SPORADIC 7,228,701
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*

At the time of surgery

#

Large tumor (8cms)

Δ

Thyroid Cancer

&

Capsular and vascular invasion

Acknowledgments

We thank Dr.Tamara Prodanov (NICHD) for helping us in providing pheochromocytomas patients' clinical information. This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development and National Cancer Institute, National Institutes of Health, USA.

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