Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Nov;10(33):e2305096.
doi: 10.1002/advs.202305096. Epub 2023 Oct 16.

A Cell-Penetrant Peptide Disrupting the Transcription Factor CP2c Complexes Induces Cancer-Specific Synthetic Lethality

Seung Han Son  1 , Min Young Kim  1 , Sungwoo Choi  1 , Ji Sook Kim  1   2 , Yong Sang Lee  1 , Sangwon Lee  1 , Yeon Ju Lee  1 , Jin Youn Lee  1 , Seol Eui Lee  1 , Young Su Lim  1 , Dae Hyun Ha  1 , Eonju Oh  3 , Young-Bin Won  1 , Chang-Jun Ji  1 , Mi Ae Park  1 , Boram Kim  4 , Kyu Tae Byun  4 , Min Sung Chung  5 , Jaemin Jeong  5 , Dongho Choi  5 , Eun Jung Baek  6 , Eung-Ho Cho  7 , Sang-Bum Kim  7 , A Reum Je  8 , Hee-Seok Kweon  8 , Hyun Sook Park  9 , Dongsun Park  10 , June Sung Bae  11 , Se Jin Jang  11   12   13 , Chae-Ok Yun  3 , Ji Hyung Chae  1 , Jin-Won Lee  1 , Su-Jae Lee  1 , Chan Gil Kim  4 , Ho Chul Kang  1 , Vladimir N Uversky  14 , Chul Geun Kim  1   15
Affiliations

A Cell-Penetrant Peptide Disrupting the Transcription Factor CP2c Complexes Induces Cancer-Specific Synthetic Lethality

Seung Han Son et al. Adv Sci (Weinh). 2023 Nov.

Abstract

Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.

Keywords: CP2c complex disruption; a cell-penetrant peptide; pan-anticancer drug; synthetic lethality; transcription factor CP2c.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Efficient induction of cell death by cell‐penetrant Pep #5‐2 (ACP52C) in most of cancer cells. A) Schematic diagram illustrating the functional domains of the CP2c protein and the binding region of Pep #5. The lower left box displays amino acid sequences and the N‐ and C‐terminal modifications of Pep #5‐2, a truncated form of Pep #5, and its derivatives. TAD, transactivation domain; DBD, DNA binding domain; TD, tetramerization domain; DD, dimerization domain; PPI, protein‐protein interaction; dnCP2c, dominant negative form of human CP2c (Q234L and K236E). B) GI50 values of ACP52C in various human cancer and non‐cancer cells. Duplicated data are expressed as means ± SD. C) Cell photographs (left) and quantification of cell survival (right) demonstrate the cancer cell‐killing effect of ACP52C. Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; **p < 0.01. Scale bar represents 50 µm. D) ACP52C exhibits anticancer effects on mammosphere‐cultured MCF7 cells. Scale bar represents 50 µm. Additional data can be found in Figure S6 (Supporting Information). E) Representative photographs showing gross cell morphology (left) and subcellular structures observed through electron microscopy (right) in MDA‐MB‐231 cells treated with 2 µm ACP52C for 48 h. Scale bar represents 20 µm. F) Immunofluorescent microscopic images reveal apoptotic cell death in ACP52C‐treated MDA‐MB‐231 cells. Scale bar represents 10 µm.
Figure 2
Figure 2
ACP52C induces cancer cell synthetic lethality through G2/M arrest, genomic instability, and apoptosis. A,B) Cell cycle distribution in synchronized cell populations treated with ACP52C over time. Experimental schematics (upper panels) and the cell cycle distribution in cells (lower panels) demonstrate the effect of ACP52C on cell cycle progression in synchronized cells at the G1/S boundary through thymidine double block (A) or at the G2/M boundary via thymidine/nocodazole treatment (B). Duplicated data are expressed as means ± SD. ANOVA test; *p < 0.05; **p < 0.01; ***p < 0.001. Refer to Figure S7B,C (Supporting Information) for the original data. C) Schematic depicting the emergence of polyploidy and the subG1‐fraction of cells in synchronized cell populations following ACP52C treatment over time. D) WBs display the expression of cell cycle‐related markers over time or at varying concentrations of ACP52C treatment in MCF7 cells (left), along with a proposed pathway leading to G2/M arrest (right). E) WBs illustrate the expression of apoptosis‐related markers over time in mock‐treated and 2 µm ACP52C‐treated cells (left), accompanied by a proposed pathway leading to apoptosis (right). Additional data can be found in Figure S7D (Supporting Information).
Figure 3
Figure 3
ACP52C dissociates CP2c complexes and induces apoptosis in a CP2c transcriptional activity‐independent manner. A,B) Colony‐forming ability tests of ACP52C in HepG2 (A) and MDA‐MB‐231 (B) cells with varying expression levels of CP2c. Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; *p < 0.05; **p < 0.01. C) Schematic illustrating the effect of ACP52C on cell growth and transcription inhibition through the dissociation of CP2c complexes. D–H) Pep #5‐2 disrupts CP2c TF complexes. D,E) DNA‐IP assays demonstrate that Pep #5‐2 inhibits the DNA binding activity of the homotetrameric [C4] and heteromeric CBP ([C2B2P2]2, 4) complexes to the wildtype Hba‐a2 promoter probe, similar to Pep #5. Data are means ± SD of two independent biological replicates. Two‐tailed unpaired Student's t‐test; **p < 0.01; *p < 0.05. F) DSP XL‐WB shows efficient disruption of the endogenous nuclear and cytosolic CP2c complexes by Pep #5‐2C. Band intensities were quantified from two independent biological replicates. Two‐tailed unpaired Student's t‐test; **p < 0.01. G) Pep #5‐2C induces instability of CP2c complex proteins in the nucleus (top) and cytosol (bottom), inhibiting CP2c complex formation. New protein synthesis was inhibited by cycloheximide (CHX) treatment. H) Schematic depicting how Pep #5‐2 disrupts CP2c TF complexes. Refer to Figure S10 (Supporting Information) for additional data. I,J) Growth inhibition occurs at lower concentration of ACP52C in cancer cells compared to transcriptional inhibition of CP2c targets. Cell viability and relative mRNA levels of CP2c transcriptional targets (FN1, OCT4, and SNAI2) were assessed in HepG2 (I) and MDA‐MB‐231 (J) cells at 36 h after ACP52C treatment through MTT assays and RT‐qPCR, respectively. GI50 and TI50 (50% transcription inhibition) values were estimated from the cell growth and transcription inhibition profiles, respectively. Duplicated data are expressed as means ± SD. K) Schematic depicting the induction kinetics of cell growth inhibition and transcription inhibition in a specific cell type following ACP52C treatment, where cell growth inhibition positively correlates with the concentrations of total cellular free CP2c isoforms, while transcription inhibition inversely correlates with the functional nuclear CP2c TF complexes.
Figure 4
Figure 4
ACP52C induces cancer‐specific apoptosis via the CP2c/YY1/MDM2 p90/p53 pathway. A–E) ACP52C induces cancer‐specific apoptosis through the CP2c/YY1/MDM2 p90/p53 pathway. A) WBs and B) RT‐qPCR depict the expression profiles of the p53‐ and CP2c‐centered markers upon ACP52C treatment over time or concentration. Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; *p < 0.05; ns, non‐significant. C) Hypothesis of apoptosis induction by ACP52C‐mediated dissociation of CP2c complexes through the CP2c/YY1/MDM2 p90/p53 pathway. D) WBs display the modulation of proteins in MDA‐MB‐231 cells with Tet‐inducible KD or OE of each CP2c, YY1, MDM2 p90, or p53. E) ACP52C‐mediated GI50 values in cells with KD or OE of each factor in MDA‐MB‐231 and HepG2 cells. Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; *p < 0.05; **p < 0.01; ns, non‐significant. Refer to Figure S14 (Supporting Information) for additional data, including the original WBs in HepG2 cells. F–I) YY1/MDM2 p90/p53 axis‐mediated cell death induction is also effective in hMSCs. F,G) WB show marker protein expression in hMSCs 48 h after conditional CP2c OE (F) or YY1 KD (G). H,I) Representative photographs of cells during conditional CP2c OE (H) or YY1 KD (I). Scale bar represents 50 µm.
Figure 5
Figure 5
ACP52C induces cancer‐specific apoptosis through TDP2 squelching‐associated catastrophic DNA damage responses. A–D) DNA strand break‐associated DNA damage response (DDR) mechanisms are linked to ACP52C responses in cancer cells. A) Confocal immunofluorescence microscopy shows the activation of γH2AX, a sensitive molecular marker of DNA damage and repair, and DNA synthesis upon ACP52C treatment, suggesting the involvement of DDR pathways. B,C) WBs depict occurrences of DDR in MDA‐MB‐231 (B) and MCF7 (C) cells following ACP52C treatment over time or concentration. Scale bar in (A) represents 5 µm. D) Schematic representation of DDR pathways. E–G) Monomeric CP2cs bind TDP2 to squelch its TOP2 depoisoning function, leading to catastrophic DDR. E) IP assays demonstrate the direct interaction between ectopically overexpressed CP2c and TDP2 in 293T cells. F) Co‐IPs reveal the enhanced interaction between endogenous CP2c and TDP2 in MDA‐MB‐231 cells upon ACP52C treatment. G) ChIP‐qPCRs show the ACP52C‐mediated modulation of DDR factors binding around the representative CP2c target genes, VIM (left) and FN1 (right). Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; *p < 0.05; **p < 0.01. H) Schematic illustration of a catastrophic DDR cascade triggered by ACP52C‐induced TDP2 squelching.
Figure 6
Figure 6
CASP2‐mediated MDM2 p90 cleavage is a factor in ACP52C resistance in some cancer cells, and sensitizing ACP52C resistance with CASP2 inhibition. A–D) ACP52C functions regardless of the p53 mutation status in cancer cells. A) Schematic of the p53 protein showing MDM2 p90‐ and YY1‐binding domains and mutation sites in some cancers (top panel). Comparative domain structures of p53 paralogs, p63 and p73 (middle panel), and differential MDM2 p90 effects on p53, p63, and p73 (bottom panel). CTD, C‐terminal domain; DBD, DNA binding domain; NLS, nuclear localization sequence; OD, oligomerization domain; PR, proline‐rich domain; SAM, sterile alpha motif; TAD, transcriptional activation domain; TID, transcriptional inhibition domain. B) ACP52C‐mediated GI50 values in various cancer cell lines with different p53 mutation statuses. Duplicated data are expressed as means ± SD. C) ACP52C‐mediated GI50 values in HCT116 and MDA‐MB‐231 cells with downregulation of either p53, p63, or p73. Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; *p < 0.05; ns, non‐significant. D) Schematic representation of ACP52C‐mediated anticancer activity induction through MDM2 p90 downregulation, independent of p53 mutation status. E,F) Analysis of marker protein expression levels in resistant cells after ACP52C treatment over time (E) or concentration (F) by WB. G) Schematics of mutual regulation between p53 and MDM2 p90, where the MDM2 p60 form does not induce p53 degradation (top) and immunoblots showing marker protein expression in resistant cells with MDM2 p90 OE (bottom). H) Analysis of MDM2 p90 and p60 expression levels in resistant cells after CASP2 inhibiting peptide (CASP2i; Ac‐VDVAD‐CHO, 10 µm) treatment. I,J) ACP52C resistance can be sensitized by CASP2 inhibition. I) Photographs of A549 and human patient‐derived cultured cancer cells treated with ACP52C (2 µm) and CASP2i (10 µm), alone or in combination for 48 h. Scale bar represents 50 µm. J) GI50 values in representative cancer cell lines with ACP52C (2 µm) alone or in combination with CASP2i (10 µm) (top) and schematic showing the restoration of ACP52 sensitivity by preventing MDM2 p90 degradation via CASP2 inhibitor treatment (bottom). Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test; **p < 0.01; ns, non‐significant.
Figure 7
Figure 7
In vivo efficacies of ACP52C and its derivatives in the mouse tumor models. A–C) Therapeutic effects of ACP52C and ACP52CGK in Hep3B xenograft Balb/c nude mice. Tumor volumes during the treatment phase (A), tumor volumes measured immediately before sacrifice (B), and photographs of tumors and corresponding tumor weights in the sacrificed mice (C). Scale bar represents 10 mm. Data (means ± SE) were analyzed using ANOVA test. *p < 0.05; **p < 0.01. D‐F) Therapeutic effects of ACP52C and ACP52CGK in the MDA‐MB‐231 LM1 xenograft NPG mice. Tumor volumes during the treatment phase (D), tumor volumes measured immediately before sacrifice (E), and photographs of tumors and corresponding tumor weights in the sacrificed mice (F). Scale bar represents 10 mm. Data (means ± SE) were analyzed using ANOVA test. *p < 0.05; **p < 0.01. G,H) Therapeutic effects of ACP52CGK in DEN‐induced orthotopic liver cancer mice. Experimental schemes for tumor induction and the tumor nodule size estimation (G), and photographs of the H&E‐stained liver sections and the estimation of the tumor nodule size in the sacrificed mice (H). Data (means ± SE) were analyzed using ANOVA test. *p < 0.05. I–K) Cellular and in vivo efficacies of ACP52CGK‐α3, a CASP2 inhibiting peptide‐conjugated ACP52CGK. (I) GI50 values of ACP52CGK derivatives (α1 to α4; see Table S3 for sequences, Supporting Information) in the representative cell lines. Duplicated data are expressed as means ± SD. Two‐tailed unpaired Student's t‐test *p < 0.05. J,K) ACP52CGK‐α3 confers a therapeutic effect in the ACP52C‐resistant PC9 CDX Balb/c nude mouse. Anticancer efficacy was assessed by tumor volumes during the treatment phase (J) and tumor weights in the sacrificed mice (K). Data (means ± SE) were analyzed using ANOVA test. *p < 0.05; **p < 0.01. It is worth noting that there were no animal deaths related to drug administration in any of the animal studies, although there was one animal death in some treatment groups.
Figure 8
Figure 8
Schematic diagram depicting cancer‐specific apoptosis induced by ACP52C. This schematic diagram illustrates the multifaceted impact of ACP52C in inducing cancer‐specific apoptosis. In many cancers, elevated expression of the transcription factor CP2c is a distinguishing feature, indicating their reliance on CP2c for growth. ACP52C, a cell‐penetrating peptide, disrupts CP2c complexes, leading to cancer‐specific lethality through two unexpected transcription‐independent DNA damage response pathways triggered by the liberated CP2c monomers. Some cancer cells display resistance to ACP52C due to increased levels of MDM2 p60, a product of CASP2‐mediated cleavage of MDM2 p90. However, this resistance can be overcome by co‐treatment with a CASP2 inhibitor. This insight lays the groundwork for the development of potential pan‐anticancer drugs that have minimal effects on normal cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. a) Hanahan D., Weinberg R. A., Cell 2000, 100, 57; - PubMed
    2. b) Cell 2011, 144, 646. - PubMed
    1. Bucheit A. D., Davies M. A., Biochem. Pharmacol. 2014, 87, 381. - PubMed
    1. Chang L., Ruiz P., Ito T., Sellers W. R., Cancer Cell 2021, 39, 466. - PMC - PubMed
    1. Weinstein I. B., Joe A. K., Nat. Clin. Pract. Oncol. 2006, 3, 448. - PubMed
    1. a) Barnhart K. M., Kim C. G., Banerji S. S., Sheffery M., Mol. Cell. Biol. 1988, 8, 3215; - PMC - PubMed
    2. b) Kim C. G., Barnhart K. M., Sheffery M., Mol. Cell. Biol. 1988, 8, 4270. - PMC - PubMed

Publication types

LinkOut - more resources

-