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. 2015;14(12):1771-8.
doi: 10.1080/15384101.2015.1026518.

Human ACAP2 is a homolog of C. elegans CNT-1 that promotes apoptosis in cancer cells

Kelly D Sullivan  1 , Akihisa Nakagawa, Ding Xue, Joaquín M Espinosa
Affiliations

Human ACAP2 is a homolog of C. elegans CNT-1 that promotes apoptosis in cancer cells

Kelly D Sullivan et al. Cell Cycle. 2015.

Abstract

Activation of caspases is an integral part of the apoptotic cell death program. Collectively, these proteases target hundreds of substrates, leading to the hypothesis that apoptosis is "death by a thousand cuts". Recent work, however, has demonstrated that caspase cleavage of only a subset of these substrates directs apoptosis in the cell. One such example is C. elegans CNT-1, which is cleaved by CED-3 to generate a truncated form, tCNT-1, that acquires a potent phosphoinositide-binding activity and translocates to the plasma membrane where it inactivates AKT survival signaling. We report here that ACAP2, a homolog of C. elegans CNT-1, has a pro-apoptotic function and an identical phosphoinositide-binding pattern to that of tCNT-1, despite not being an apparent target of caspase cleavage. We show that knockdown of ACAP2 blocks apoptosis in cancer cells in response to the chemotherapeutic antimetabolite 5-fluorouracil and that ACAP2 expression is down-regulated in some esophageal cancers, leukemias and lymphomas. These results suggest that ACAP2 is a functional homolog of C. elegans CNT-1 and its inactivation or downregulation in human cells may contribute to cancer development.

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Figures

Figure 1.
Figure 1.
ACAP2 is a mammalian homolog of CNT-1. (A) Sequence alignment of 2 C. elegans CNT-1 isoforms (CNT-1a and CNT-1b) with human ACAP2 and ACAP3. Residues that are identical in all 4 proteins are shaded in yellow and residues that are identical in 3 of the 4 proteins are shaded in blue. The PH domain is underlined. The black arrowhead indicates the conserved lysine residue critical for lipid binding. The red arrow indicates the CED-3 cleavage site in CNT-1. (B) Schematic alignment of ACAP2 with CNT-1a with conservation information.
Figure 2.
Figure 2.
ACAP2 has an identical lipid-binding pattern to that of CNT-1. (A) Neither ACAP2 nor ACAP3 is cleaved by caspase-3 in vitro. PARP, ACAP2 and ACAP3 were synthesized and labeled with S35-Methionine(*) in rabbit reticulocyte lysate, incubated with or without 1 unit of purified caspase-3 for 2 hours, and then resolved by 15% SDS-PAGE. (B) ACAP2 and ACAP3 are not cleaved during apoptosis. HCT116 cells were treated with DMSO or 375 μM 5FU for 24 hours and cell lysates subjected to immunoblotting. ACAP2 and ACAP3 are not cleaved during apoptosis, whereas Caspase-3, Caspase-8, and PARP are. Nucleolin serves as a loading control. (C) ACAP2, but not ACAP3, displays a similar phosphoinositide binding activity to that of tCNT1a. ACAP2, ACAP3, and GST-tCNT-1a were synthesized and labeled with S35-MET(*) as in A and quantified as described in Materials and Methods. 40 nM of each protein was then added to the membrane strips.
Figure 3.
Figure 3.
Knockdown of human ACAP2 reduces 5FU-induced apoptosis in cancer cells. (A, C) HCT116 cells stably expressing shRNAs targeting ACAP2, ACAP3, or a non-targeting control (Ctrl) were treated with 375 μM 5FU for 24 hours prior to analysis of phosphatidylserine externalization (Annexin V) via flow cytometry. Where indicated, cells were pretreated with 2 μM Z-VAD-FMK for 1 hour. (B) A549 cells stably expressing shRNAs targeting ACAP2 or a non-targeting control (Ctrl) were treated and analyzed as in A. All Data shown represent at least 3 independent experiments +/− SEM. P-values were calculated using Student's t-test (unpaired, 2-tailed). (D) Immunoblots showing degree of ACAP2 and ACAP3 knockdown for 2 independent shRNAs.
Figure 4.
Figure 4.
For figure caption, see page 1776.Figure 4 (See previous page) Oncomine analysis of ACAP2 and ACAP3 expression levels in cancer versus normal cells. (Left) A summary of comparisons of ACAP2 and ACAP3 expression levels in different cancer vs. normal tissue datasets. The bottom rows in each case indicate the total number of unique analyses and the number of unique analyses that show significant overexpression (red) or underexpression (blue) of the target gene in the cancer samples relative to the normal tissue samples. Color intensity indicates the percentile rank of genes (key at bottom) displaying significant overexpression or underexpression. (Right) Box and whisker plots of representative studies profiling decreased expression of ACAP2 in esophageal cancer, leukemia and lymphoma. Details include study name, P-value, t-test score and fold change. Tissue or cancer type is indicated below the plots with the number of analyzed samples indicated in parentheses.
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