An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers
Terence K. Lee, Saravana R.K. Murthy, Niamh X. Cawley, Savita Dhanvantari, Stephen M. Hewitt, Hong Lou, Tracy Lau, Stephanie Ma, Thanh Huynh, Robert A. Wesley, Irene O. Ng, Karel Pacak, Ronnie T. Poon, Y. Peng Loh
Terence K. Lee, Saravana R.K. Murthy, Niamh X. Cawley, Savita Dhanvantari, Stephen M. Hewitt, Hong Lou, Tracy Lau, Stephanie Ma, Thanh Huynh, Robert A. Wesley, Irene O. Ng, Karel Pacak, Ronnie T. Poon, Y. Peng Loh
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Research Article Oncology

An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers

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.

Authors

Terence K. Lee, Saravana R.K. Murthy, Niamh X. Cawley, Savita Dhanvantari, Stephen M. Hewitt, Hong Lou, Tracy Lau, Stephanie Ma, Thanh Huynh, Robert A. Wesley, Irene O. Ng, Karel Pacak, Ronnie T. Poon, Y. Peng Loh

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Figure 6

CPE-ΔN interacts with HDAC1/2 to upregulate NEDD9 expression.

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CPE-ΔN interacts with HDAC1/2 to upregulate NEDD9 expression. (A) Immuno...
(A) Immunofluorescence confocal microscopy of MHCCLM3 cells transfected with si-Scr (top row) or CPE-ΔN siRNA (bottom row). Cells were immunostained with CPE monoclonal antibody and TRITC-conjugated goat anti-mouse IgG secondary antibody (red). The slide was counterstained with fluorescein phalloidin for F-actin (green) and DAPI for nuclei (blue). Note CPE-ΔN in the nucleus of MHCCLM3 si-Scr cells. Original magnification, ×600. (B) MHCC97L cells were transduced with an adenovirus expressing CPE-ΔN. Twenty-four hours after transduction, a cell lysate was prepared and immunoprecipitated with CPE or HDAC1 monoclonal antibodies as indicated. The immunoprecipitated proteins were analyzed by Western blot for both CPE and HDAC1 and HDAC2. Nonspecific monoclonal antibodies (IgG) or omission of antibodies (None) were used as negative controls as indicated. HDAC1 and HDAC2 (two forms) were detected in the lanes (CPE) where CPE-ΔN was immunoprecipitated. CPE-ΔN was detected in the lane where HDAC1 was immunoprecipitated, indicating interaction of CPE-ΔN with HDAC1- and HDAC2-containing complex. Asterisks indicate IgG heavy and light chains. (C) Effect of HDAC inhibitor depsipeptide on NEDD9 levels in MHCC97L cells stably expressing CPE-ΔN. Cells were treated with depsipeptide at various concentrations (0–2 ng/ml) for 24 hours. Cells were harvested and fractionated into cytosolic and nuclear fractions and analyzed by Western blot. A representative Western blot shows expression of NEDD9 in cytoplasm (c) and CPE-ΔN in nucleus (n) at different concentrations of depsipeptide, as indicated. The graph shows the quantification of NEDD9 and CPE-ΔN bands from Western blots of 3 different experiments. Values are the mean ± SEM.