Carboxypeptidase E and its splice variants: Key regulators of growth and metastasis in multiple cancer types

Abstract

Mechanisms driving tumor growth and metastasis are complex, and involve the recruitment of many genes working in concert with each other. The tumor is characterized by the expression of specific sets of genes depending on its environment. Here we review the role of the carboxypeptidase E (CPE) gene which has been shown to be important in driving growth, survival and metastasis in many cancer types. CPE was first discovered as a prohormone processing enzyme, enriched in endocrine tumors, and later found to be expressed and secreted from many epithelial-derived tumors and cancer cell lines. Numerous studies have shown that besides wild-type CPE, a N-terminal truncated splice variant form of CPE (CPE-ΔN) has been cloned and found to be highly expressed in malignant tumors and cell lines derived from prostate, breast, liver and lung cancers and gliomas. The mechanisms of action of CPE and the splice variant in promoting tumor growth and metastasis in different cancer types are discussed. Mechanistically, secreted CPE activates the Erk/wnt pathways, while CPE-ΔN interacts with HDACs in a protein complex in the nucleus, to recruit various cell cycle genes and metastatic genes, respectively. Clinical studies suggest that CPE and CPE-ΔN mRNA and protein are potential diagnostic and prognostic biomarkers for multiple cancer types, assayed using solid tumors and secreted serum exosomes. CPE has been shown to be a therapeutic target for multiple cancer types. CPE/CPE-ΔN siRNA transported via exosomes and taken up by recipient high metastatic cancer cells, suppressed growth and proliferation of these cells. Thus future studies, delivering CPE/CPE-ΔN siRNA, perhaps via exosomes, to the tumor could be a novel treatment approach to suppress tumor growth and metastasis.

Keywords: Exosomes; Glioblastoma; Hepatocellular carcinoma; Lung adenocarcinoma; Osteosarcoma.

Fig. 1.. Schematic representation of CPE mRNA and protein:

A. CPE mRNA structure of wild type CPE, showing the position of the three putative start codons and the stop codon. UTR-untranslated regions. In cancer cells (HCC) and clinical tumors, the WT-CPE and CPE variants were found to use an alternate transcription start site at 133 nt. The 1.7kb CPE-ΔN variant has been cloned in HCC cells, and the corresponding deletions (yellow box) in the first exon and 589 nt deletions in the 3’-UTR, in comparison to WT-CPE are shown. The 1.7kb transcript encodes a 40kD CPE-ΔN variant was detected in HCC patient samples and various cancer cell lines. Numbers refer to nt postions in homo sapiens CPE NM001873.2 sequence. nt- nucleotide. B. Structure of full-length CPE, mature CPE and 40kD CPE-ΔN protein. 40kD CPE-ΔN variant lacks 112 amino acids at the N’ end. Numbers refer to amino acid positions deduced from homo sapiens CPE NM001873.2. SP- signal protein, P- propeptide, TM- transmembrane protein.

Fig. 2.. CPE-ΔN differentially regulates tumor associated genes.

A. Bar graphs show genes that are increased ≥2 fold in MHCC97L-CPE-ΔN vs MHCC97L cells. RNA from MHCC97L cells (low-metastatic HCC cells) transfected with CPE-ΔN plasmid or un-transfected MHCC97L cells were subjected to genome-wide gene expression analysis. All genes that were increased at least two-fold were identified and plotted in a bar graph. Thirty four genes were up-regulated in CPE-ΔN transfected cells versus un-transfected control cells. B. Bar graphs show genes that are decreased ≥2 fold in MHCC97L-CPE-ΔN vs MHCC97L cells. All genes that were decreased at least two-fold were identified and plotted in a bar graph. Twenty two genes were down-regulated in CPE-ΔN transfected cells versus un-transfected control cells. C. RNA from MHCC97L cells transfected with CPE-ΔN plasmid, or MHCC97H (high-metastatic) and un-transfected MHCC97L cells were subjected to genome-wide gene expression level analysis. Venn diagram shows the number of genes up-regulated ≥2 fold in CPE-ΔN transfected cells (right green circle) and MHCC97H (left green circle) versus un-transfected MHCC97L cells. Six genes, listed, were found to overlap in these two conditions. D. RNA from MHCC97L cells transfected with CPE-ΔN plasmid, or MHCC97H (high metastatic) and un-transfected MHCC97L cells were subjected to genome-wide gene expression level analysis. Venn diagram shows the number of genes down-regulated ≥2 fold in CPE-ΔN transfected cells (right red circle) and MHCC97H (left red circle) versus un-transfected MHCC97L cells. Twenty genes, listed, were found to overlap in these two conditions.

Fig. 3.. CPE inhibits glioma cell migration.

Soluble CPE possibly binds to a putative receptor (blue) to activate the mTOR pathway, causing phosphorylation of the downstream target, ribosomal protein S6 (RSP6), which inturn inhibits RAC1, and RAC1 induced cell migration. Lactate, produced by the metabolic switch from glycolysis to tricarboxylic acid cycle (TCA) also suppresses cell motility [35]. In addition, it is proposed that CPE regulates expression of SLUG, downstream of ERK1/2 and MMP-2, to modulate glioma cell migration [36]. Modified from Ilina I.E et al, 2017 [35].

Fig. 4.. Proposed mechanism for CPE-WT regulation of tumorigenesis.

CPE present in the extracellular space binds to a putative receptor (HTR1E), to activate oncogenic signaling pathways such as ERK1/2, NF-κB or Wnt3a, to regulate tumor growth and metastasis, by inducing the expression of target genes involved in cell cycle regulation and survival/ anti-apoptosis.

Fig. 5.. 5-HTR1E expression in cancer cells.

Western blot showing the 45kDa 5-HTR1E band in different cancer cells as indicated. Cells were lysed in RIPA lysis buffer and 30 μg lysate was loaded in each well of a 4–12% SDS-PAGE gel. Western blot membrane was incubated with 5-HTR1E, S31 rabbit ab (Cat. no. ab154813, dilution-1:2500) overnight at 4°C followed by one hour incubation with anti-rabbit secondary ab IRDye® 800CW (LI-COR- 925–32211). Blots were visualized on a LI-COR Odyssey system. Previous studies show that Panc-1, BxPC3 and ASPC1 [23], LN 18, LNT229 [34], HCC97H [17], U118 and SHSY5Y (our unpublished data) cells also express CPE.

Fig. 6.. Proposed mechanism of action of CPE-ΔN in tumorigenesis.

A. Data abstracted from Yeast-two-Hybrid analysis indicated that CPE interacts with HDAC1 and HDAC2 [48]. Bioinformatic analysis comparing HDAC interacting domain in other proteins identified a HDAC interacting domain in CPE, and CPE-ΔN (sequence in blue). B. Schematic of proposed mechanism involving CPE-ΔN interaction with HDACs in a protein complex on a promoter to up-regulate metastasis-related and anti-apoptotic gene, or down-regulate tumor suppressor gene expression.