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. Author manuscript; available in PMC: 2026 Jan 2.
Published in final edited form as: Mol Cancer Res. 2025 Jul 2;23(7):585–596. doi: 10.1158/1541-7786.MCR-24-0702

ANGEL2 modulates wildtype TP53 translation and doxorubicin chemosensitivity in colon cancer

Christopher August Lucchesi 1,2,*, Saisamkalpa Mantrala 1, Darren Tran 1, Neelu Batra 2, Avani Durve 1, Conner Suen 1, Jin Zhang 3, Paramita Ghosh 1,2, Xinbin Chen 3
PMCID: PMC12221813  NIHMSID: NIHMS2066163  PMID: 40052999

Abstract

Multiple lines of correlative evidence support a role for ANGEL2, a novel cancer-relevant RNA-binding protein, in the modulation of chemoresistance and cancer patient survival. However, to date, no study has determined a mechanism by which ANGEL2 modulates cancer progression, nor its role in chemoresistance. Herein, we demonstrate that loss of ANGEL2 leads to a substantial decrease of the key tumor suppressor protein TP53. We show that ANGEL2 directly interacts with EIF4E, the rate limiting protein in cap-dependent translation. This interaction abrogates the ability for the TP53 translation repressor RBM38 to interact with EIF4E thereby enhancing TP53 translation. Loss of ANGEL2 in cancer cell lines resulted in increased 2D and 3D spheroid cell growth, and resistance to doxorubicin and etoposide. With therapeutic potential, treatment with Pep7, a seven amino-acid peptide derived from ANGEL2, rescued wildtype TP53 expression and sensitized cancer cells to doxorubicin. Together, we conclude that ANGEL2 modulates the EIF4E-RBM38 complex to enhance wildtype TP53 translation, and further, the Pep7 peptide may be explored as a therapeutic strategy for cancers which harbor wildtype TP53 expression.

Implications:

Loss of ANGEL2 contributes to decreased wildtype TP53 translation promoting doxorubicin resistance which can be rescued via an ANGEL2-derived peptide.

Introduction

Up to now, over 1,500 RNA-binding proteins (RBP) have been identified, of which more than 450 were found to be differentially expressed in cancers. Accumulating data has demonstrated that RBPs are dysregulated in a multitude of cancers, whereby they enhance oncogenesis by differentially modulating the expression of oncogenes (enhance) and tumor suppressor genes (decrease), such as TP53. For example, RPL26 and ELAVL1 enhance TP53 expression1,2, whereas RNA-binding motif protein 38 (RBM38) decreases TP53 expression3. Functionally, RBM38 inhibits TP53 translation by interacting with eukaryotic translation initiation factor 4E (EIF4E) and TP53 mRNA, consequently preventing EIF4E from binding to the TP53 m7G cap, halting its translation3. Therapeutically, an 8-amino acid peptide (Pep8) derived from a region in RBM38 necessary for its interaction with EIF4E was found to disrupt the RBM38-EIF4E complex, increase wildtype TP53 expression, and inhibit tumor cell growth in vitro and in vivo4.

The TP53 transcription factor is a crucial tumor suppressor and a leading regulator of numerous signaling pathways involved in all aspects of tumor suppression, including, but not limited to, DNA repair, cell cycle arrest, apoptosis, and senescence5. The loss of wildtype TP53 function is a hallmark and a driver of tumor progression. Though many tumors (around 50%) are characterized by loss-of-function mutations in TP53, the prevalence of wildtype TP53 in multiple malignancies including lymphoma (~80%), non-basal cell breast cancer (~60%), and colorectal carcinoma (~30%) (TCGA database) points to other genetic and epigenetic mechanisms that stifle wildtype TP53 expression and function, further contributing to oncogenesis and chemoresistance6,7. For example, elevated levels of MDM2 lead to the degradation of TP53 protein8, overexpression of PPM1D leads to the inactivation of TP53 function9, and aberrant expression of RBM38 inhibits TP53 mRNA translation10. Therefore, targeting non-genetic mechanisms, such as MDM2 and RBM38 that inhibit TP53 protein stability and/or expression is a promising therapeutic tactic for malignancies that carry wildtype TP53.

ANGEL2 (also known as CCR4d) is a novel, cancer relevant RBP, who is a member of the carbon catabolite repression 4 (CCR4) family which consists of CCR4a-CCR4e. Unlike CCR4a and CCR4b who function as deadenylases, ANGEL2 does not harbor deadenylase activity. Uniquely, ANGEL2 was found to contain 2′,3′-cyclic phosphatase activity, supporting its role in tRNA recycling11. Moreover, ANGEL2 was also found to function as a modulator of mRNA stability as it was shown to directly interact with the CDKN1A transcript, a TP53 transcriptional target, thereby increasing its mRNA half-life12. Of clinical significance, loss of ANGEL2 was found to be a driver of diffuse large B-cell lymphomagenesis13, and low ANGEL2 expression across multiple different cancer types correlated with poor overall and disease-free patient survival including in colon and breast cancer (Supp. Fig. 1A)14. Despite this evidence, the mechanistic role that ANGEL2 plays in oncogenesis has yet to be determined. Herein, we demonstrate that ANGEL2 modulates TP53 translation via abrogating the interaction between EIF4E and RBM38, and that loss of ANGEL2 contributes to increased tumor cell growth and resistance to doxorubicin and etoposide. Therapeutically, a 7-amino acid peptide (Pep7) derived from ANGEL2 was found to rescue wildtype TP53 expression, resensitize cancer cells to doxorubicin, and decrease 3D tumor spheroid cell viability in wildtype and ANGEL2-null cancer cells.

Materials and Methods

Human cell lines

RKO (RRID:CVCL_0504), HCT116 (RRID:CVCL_0291), and MCF7 (RRID:CVCL_0031) cells were cultured at 37°C in DMEM (Dulbecco’s Modified Eagle’s medium, Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) in a humidified incubator with 5% CO2. Cell lines RKO (CRL-2577), HCT116 (CCL-247) and MCF7 (HTB-22) were obtained from ATCC between 2016 and 2020 and used below passage 25 or within 2 months after thawing. Cells were tested negative for mycoplasma after thawing and used within two months. Since all cell lines from ATCC have been thoroughly tested and authenticated, we did not authenticate the cell lines used in this study.

Plasmids and cell line generation

Generation of ANGEL2-null cells lines was achieved by using CRISPR-Cas9, pSpCas9(BB)-2A-Puro plasmid (RRID:Addgene_48139) expressing guide RNA’s (ANGEL2 guide #1: 5’- CAC CGC AGG TGT GAT AAA GCG GAA T 3’ and ANGEL2 guide #2 5’- CAC CGT CCA GTG TAA TAC TGG CCG C). The cells were selected with puromycin and each individual clone was confirmed by genotyping and western blot analysis. Generation of RBM38/RBM24-null and TP53-null cells lines was previously described15. TP53 UTR and ORF plasmids were previously described3. GST-RBM38 expression plasmids were generated as previously described 16. ANGEL2 full-length and truncation clones were generated by subcloning into pGEX-GP-1 bacterial vector with the following primers; ANGEL2 full-length (F): ATC GTA GGA TCC GCC ACC ATG GAT TAC AAG GAC GAC GAC GAT AAG GAT TAC AAG GAC GAC GAC GAT AAG GAT TAC AAG GAC GAC GAC GAT AAG ATG GAA GCC TGG CGC TGT GTG AGG A; (R): TAC GAT GCG GCC GC TCA GAG CTC AAG TCT GAA CTT TGC CAA TAA AG; ANGEL2 1–225 (F): Same as Full-length forward primer; (R) TAC GAT GCG GCC GC TCA ATG ATC TTC TTG AAC TTC TTG CAA ACA AAG T. ANGEL2 225–450 (F): ATC GTA GGA TCC GCC ACC ATG GAT TAC AAG GAC GAC GAC GAT AAG GAT TAC AAG GAC GAC GAC GAT AAG GAT TAC AAG GAC GAC GAC GAT AAG CAT TAT GGA GCA GAG ATC AGG CCA A; (R) TAC GAT GCG GCC GC TCA ACT GAA ATG GTG CTG TAA ATT TGA AGA CA. ANGEL2 450–544 (F) TC GTA GGA TCC GCC ACC ATG GAT TAC AAG GAC GAC GAC GAT AAG GAT TAC AAG GAC GAC GAC GAT AAG GAT TAC AAG GAC GAC GAC GAT AAG AGT TTG TCA TCT GTT TAT TCA CAT TAC TTT CCT GA; (R) TAC GAT GCG GCC GC TCA GAG CTC AAG TCT GAA CTT TGC CAA TAA AG. pTXB1-eIF4E plasmid was generated by amplifying EIF4E using His-EIF4E expression plasmid as template. The amplicon was then cloned into pTXB1 via NdeI and SapI. The primers used to amplify EIF4E were forward primer, 5´- GGT GGT CAT ATG GCG ACT GTC GAA CCG GAA ACC -3´, and reverse primer, 5´- GGT GGT TGC TCT TCC GCA AAC AAC AAA CCT ATT TTT AGT GGT GGA G -3´.

Peptide synthesis and delivery

Pep7 peptide (PYAACPA), and control peptide (STLWDTAELWQ) were synthesized by GenScript (Piscataway, NJ) with a N-terminal penetratin fusion cell penetrating peptide ( Pep7-RQIKIWFQNRRMKWKK-PYAACPA and Control- RQIKIWFQNRRMKWKK- STLWDTAELWQ).

Competitive ELISA

Briefly, bacterially purified GST-tagged RBM38 protein was incubated in a 96-well glutathione-coated plate. After washing, equal amounts of purified EIF4E and increasing concentration of purified ANGEL2 protein were added to each well. Finally, each well was extensively washed and incubated with horseradish peroxidase–conjugated EIF4E (Santa Cruz) antibody before being visualized with TMB substrate solution.

Western blotting

Western blot procedures were as previously described 17. Briefly, membranes were blocked in PBST containing 3% milk for 1 hour at 20°C. Primary antibodies in PBST containing 3% milk were incubated at 4°C rocking overnight. The next day membranes were washed 3x with PBST followed by the addition of secondary antibody in PBST containing 3% milk at 20°C for 2 hours. Membranes were then washed 3x with PBS. All western blots figures are representative data of at least 2 independent replicates with approximate molecular weights for each protein indicated on the left side in kDa. Band intensities were calculated using ImageJ 18. Antibodies used where: anti-TP53 (1C12, Cell Signaling, Danvers, MA), anti-actin (Sigma), anti-GAPDH (0411, Santa Cruz, Dallas, TX), anti-GST (B-14, Santa Cruz, Dallas, TX), anti-EIF4E (P-2, Santa Cruz), anti-vinculin (7F9, Santa Cruz), anti-Flag (M2, Millipore Sigma, St. Louis, MO), anti-EGFR (2232, Cell Signaling, Danvers, MA), anti-ANGEL2 pAb generated in house using full-length ANGEL2 protein.

Competitive and non-competitive pull-down assays

For truncated ANGEL2 and EIF4E pull-down assays we expressed full length and each ANGEL2 truncation construct in pGEX-6P1 vector. Plasmids were transformed into BL21 (DE3) competent E. coli. 1-liter culture was grown at 37°C until OD600 = 0.6–0.8 and then induced with a final concentration of 0.1 mM IPTG for 4 hours. Bacteria were spun down and the pellet placed in −80°C overnight. Pellets were lysed, sonicated and centrifuged in 20 mL lysis buffer with 1% Triton-X100, 1 mM DTT and protease inhibitor cocktail. Lysates were then incubated with GST (pGEX) beads rocking at 4°C for 2 hours. The beads were washed 3 times with wash buffer. EIF4E was purified as described below. ANGEL2-GST beads were then resuspended in wash buffer with 0.1% Triton-X100 to make a 50% bead slurry. 100 μl bead slurry was incubated in 650 μl wash buffer and 5 μM purified EIF4E in a 1.5 mL tube. 5% of this sample (100 μl bead slurry, 650 μl wash buffer and 5 μM purified EIF4E) was taken for the input wells. Samples were rocked overnight, washed 3 times with wash buffer and eluted with 60 μl 1x SDS-loading buffer before western blot analysis.

For GST-RBM38 competitive pull-down assays with ANGEL2 and EIF4E, pGEX-6P1-RBM38, or pTXB1-EIF4E, or pTXB1-ANGEL2 plasmids were transformed into BL21 (DE3) competent E. coli. 1-liter culture was grown at 37°C until OD600 = 0.6–0.8 and then induced with a final concentration of 0.1 mM IPTG for 4 hours. Bacteria were spun down and the pellet placed in −80°C overnight. Pellets were lysed, sonicated and centrifuged in 20 mL lysis buffer with 1% Triton-X100, 1 mM DTT and protease inhibitor cocktail. Lysates were then incubated with GST (pGEX) or chitin (pTXB1) beads rocking at 4°C for 2 hours. The beads were washed 3 times with wash buffer. pTXB1-EIF4E and ANGEL2 were eluted by the addition of 50 mM dithiothreitol (DTT) overnight. After brief centrifugation, lysates were carefully removed and buffer exchanged using a PD10 column (Cytiva). pGEX-RBM38 beads were then resuspended in wash buffer with 0.1% Triton-X100 to make a 50% bead slurry. 650 μl wash buffer with 5 μM purified EIF4E (with and without 5 μM ANGEL2) was added to a 1.5mL tube where 5% of this sample was taken for the input wells. After the input was taken out, 100 μl empty bead slurry or with GST-RBM38 was added to each 1.5 mL tube and rocked overnight, washed 3 times with wash buffer and eluted with 60 μl 1x SDS-loading buffer before western blot analysis.

RNA isolation, siRNA ANGEL2 design, RT-PCR, and qRT-PCR

Total RNA was isolated with Quick-RNA Miniprep Kit (Zymo Research). cDNA was synthesized using RevertAid First Strand cDNA Synthesis kit according to the manufacturer’s protocol (Thermo Fisher Scientific). siRNA targeting ANGEL2 were generated by Horizon Discovery with the following sequences: siANGEL2 #1 sense 5’ G.C.A.A.G.A.A.G.U.U.C.A.A.G.A.A.G.A.U.U.U 3’ and antisense 5’ A.U.C.U.U.C.U.U.G.A.A.C.U.U.C.U.U.G.C.U.U 3’; siANGEL2 #2 sense 5’ C.A.G.U.G.A.A.C.C.C.A.G.U.G.G.A.A.U.U.U.U 3’ and antisense 5’ A.A.U.U.C.C.A.C.U.G.G.G.U.U.C.A.C.U.G.U.U 3’. siRNA constructs where transfected with RNAiMax per manufacturers guidelines. For qRT-PCR analysis, 20 μL reactions were set up using 2× qPCR SYBR Green Mix (ABgene) along with 5 μmol/L primers. The reactions were run on a StepOne plus (Invitrogen) using a two-step cycling program: 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 68°C for 30 seconds. A melt curve (57°C–95°C) was generated at the end of each run to verify primer specificity. For TP53 mRNA stability, HCT116 wildtype and ANGEL2-null cells were treated with 5 ug/mL Actinomycin-D for varying timepoints in duplicate. The primers used to amplify human HPRT1 were forward primer 5′-TAT GGC GAC CCG CAG CCC T-3′, reverse primer 5′-CAT CTC GAG CAA GAC GTT CAG-3′. The primers used to amplify TP53 were forward primer 5′- CCT CAG CAT CTT ATC CGA GTG G-3′, and reverse primer 5′- TGG ATG GTG GTA CAG TCA GAG C-3′. The primers used to amplify CDKN1A were forward primer 5′- AGG TGG ACC TGG AGA CTC TCA G-3′, and reverse primer 5′- TCC TCT TGG AGA AGA TCA GCC G-3′. The primers used to amplify MDM2 were forward primer 5′- CCC AAG ACA AAG AAG AGA GTG TGG-3′, and reverse primer 5′- CTG GGC AGG GCT TAT TCC TTT TCT-3′.

RNA immunoprecipitation assay

RNA immunoprecipitation was carried out as previously described19. Cell extracts were prepared with immunoprecipitation buffer [10 mmol/L HEPES, pH 7.0, 100 mmol/L KCl, 5 mmol/L MgCl2, 0.5% Nonidet P-40, and 1 mmol/L Dithiothreitol (DTT)] and then incubated with 1 μg of anti-EIF4E [Anti-EIF4E Antibody (A-10), Santa Cruz Biotechnology], anti-HA [HA-tag (C29F4), Cell Signaling Technology] or an isotype control IgG overnight at 4°C. The RNA–protein immunocomplexes were brought down by magnetic protein A/G beads. RT-PCR analysis was carried out to determine the levels of TP53 and ACTIN (used as a control) transcripts.

Nascent TP53 protein detection with Click-iT chemistry

For MCF7-Tet-on-ANGEL2 assay, cells were plated in duplicate at 2.5×10^6 cells per 10 cm plate with one plate treated with tetracycline (2ug/mL). For wildtype and ANGEL2-null HCT116 cells, cells were plated in quadruplicate at 2.5×10^6 cells per 10 cm plate. The next day the cells were washed three times with methionine-free DMEM and then treated with Click-iT HPG (Thermo Fisher Scientific, C10186) and for HCT116 assay, with Pep7 (20μM) alone or in combination with doxorubicin (3.125 ng/mL) in methionine-free DMEM for 2 hours. The cells were then washed three times with PBS and collected via trypsinization. The pelleted cells were then lysed in 750 μL lysis buffer (25 mmol/L Tris pH 8.0, 100 mmol/L NaCl, and 0.1% Triton X-100) including protease inhibitor cocktail. The homogenized pellets were then subjected to sonication on ice (5 seconds on, 15 seconds off, for 5 pulses). After being lysed, the cells were pelleted and lysates were collected in new tubes. A total of 50 μL from each tube was removed to be used for the input. Next, 15 μL protein A/G magnetic beads were added to each tube followed by 1 μg TP53 (Santa Cruz Biotechnology, catalog no. sc-126) antibody. After rocking overnight at 4°C, the beads were washed five times before being subjected to Click-iT biotin assay (including the input samples) utilizing Click-iT Protein Reaction Buffer Kit (Invitrogen, C10276) according to manufactures protocol. Nascent protein expression was visualized by Western blot analysis using anti-biotin antibody (Santa Cruz Biotechnology, catalog no. sc-57636).

Colony formation

Colony formation assays were performed as previously described20. Briefly, 1,000 cells were plated in 6-well plates. The following day Pep7 and/or doxorubicin were added to the media. Following three-day incubation, the media was replaced with fresh complete DMEM. Cells were grown until colonies were visible. Cells were fixed using (7:1) methanol : acetic acid followed by crystal violet staining.

Two-dimensional cell viability assay

For two-dimensional (2D) cell viability assays, 10,000 cells per well were plated in a triplicate in a 96-well plate. Two hours later, Pep7 or control peptide alone and/or in combination with doxorubicin were added to each well. Twenty-four hours later, cell viability was measured by CellTiter-Glo 3D according to manufacturer’s guidelines (Promega).

Three-dimensional spheroid cultures

3D mini ring culture assays were adapted from Phan and colleagues21. Briefly, single-cell suspensions (15K cells/well) were plated around the rim of the well in 96-well plates in a 4:3 mixture of Matrigel and Mammocult (BD Biosciences CB-40324). After plates were incubated at 37°C with 5% CO2 for 15 minutes to solidify the gel, 100 μL of prewarmed Mammocult containing the indicated peptide/doxorubicin were added to the corresponding well. Seventy-two hours later, 100 μL prewarmed PBS was used to wash the cells three times. Cells were then released from the Matrigel by incubating at 37°C for 45 minutes in 50 mL of 5 mg/mL dispase (Life Technologies #17105–041). Images were taken with a 10X objective and then cell viability was measured by CellTiter-Glo 3D according to manufacturer’s guidelines (Promega).

Statistical analysis

Experimental values are presented as mean ± SEM. Statistical comparisons between experimental groups were analyzed by a two-tailed Student’s t-test. P values < 0.05 were considered statistically significant.

Data availability

The data generated in this study are available within the article and its Supplementary Materials.

Results

ANGEL2 modulates wildtype TP53 protein expression.

Our group previously identified an 8 amino acids peptide (Pep8) derived from RBM38 which was shown to disrupt the RBM38-EIF4E complex, thereby inducing wildtype TP53 mRNA translation22. A subsequent protein BLAST using the NCBI BLAST suite identified ANGEL2 as another human protein that contains a similar domain to RBM38 (RBM38 peptide sequence- YPYAASPA; ANGEL2 peptide sequence PYAACPA)23. Accordingly, we hypothesized that ANGEL2 may also regulate TP53 protein expression. To that end, we overexpressed Flag-tagged ANGEL2 in RKO (colon cancer) and HCT116 (colon cancer) cell lines (both cell lines harbor wildtype TP53). Contrastingly to RBM38 modulation of TP53, ectopic ANGEL2 expression resulted in increased wildtype TP53 protein and mRNA expression (Figs. 1A–D) in both cell lines tested3. Further, we overexpressed ANGEL2 in MCF7 breast cancer cells, which have a very low basal expression of ANGEL224, and demonstrated that TP53 protein and mRNA expression where also enhanced (Supp. Figs. 1B–C). Henceforth, wildtype TP53 will be identified as TP53, unless otherwise stated. As DNA damage induced by chemotherapeutic agents is known to enhance TP53 expression25, we questioned whether increased expression of ANGEL2 would further enhance DNA-damage mediated TP53 expression. In both RKO and HCT116 cell lines, concurrent expression of ANGEL2 with doxorubicin or etoposide (two well studied chemotherapeutic agents) enhanced TP53 expression as compared to non-ANGEL2 expressing controls (Fig. 1E–F). Using CRISPR/Cas9 we developed both RKO and HCT116 ANGEL2 knockout clones. We found that loss of ANGEL2 caused a marked decrease in TP53 protein and mRNA expression (Fig. 1G–J). In addition, both TP53 transcriptional targets, CDKNA1 and MDM2 mRNA were significantly decreased in ANGEL2-null cells (Supp. Figs. 1D–E). We further showed that decreased TP53 protein expression via loss of ANGEL2 was unable to reach the same level as isogenic control cells after treatment with doxorubicin (Fig. 1K–L, compare lane 4 with lanes 5 and 6). Collectively, these data support that ANGEL2, unlike RBM38, positively modulates TP53 protein expression, and in cancer cells with loss of ANGEL2, TP53 expression is attenuated even after treatment with doxorubicin.

Figure 1:

Figure 1:

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Overexpression of ANGEL2 enhances, whereas knockout of ANGEL2 decreases wildtype TP53 protein expression. A-D, Immunoblot (A and C) and qPCR (C and D) for wildtype TP53 in RKO and HCT116 colon cancer cell lines ectopically expressing Flag-ANGEL2. E-F, Immunoblot for wildtype TP53 in RKO and HCT116 colon cancer cell lines ectopically expressing Flag-ANGEL2 and treated with doxorubicin (6.25 ng/mL) or etoposide (200 nM) for 18 hrs. G-J, Immunoblot (G and I) and qPCR (H and J) for wildtype TP53 in wildtype and ANGEL2-KO clones (RKO and HCT116). K-L, Immunoblot for ANGEL2 and wildtype TP53 in wildtype and ANGEL2-KO clones treated with doxorubicin (6.25 ng/mL).

ANGEL2 stabilizes TP53 transcript and enhances its translation.

ANGEL2 has previously been shown to be an RNA-binding protein (RBP), therefore, we performed targeted RIP (RNA immunoprecipitation) assays to evaluate possible interactions between ANGEL2 and TP53 mRNA. MCF7 cells were chosen for ectopic overexpression assays as they have low ANGEL2 expression and where not genetically modified as are the knockout cell lines26. As shown in figure 2A, ANGEL2 was found to interact with TP53 mRNA, and increased ANGEL2 expression was found to enhance TP53 mRNA expression (Fig. 2B). As ANGEL2 expression was shown to regulate TP53 mRNA levels, we performed an mRNA half-life assay in isogenic control and ANGEL2-null cells. We found that loss of ANGEL2 caused a substantial reduction in TP53 mRNA half-life (~4.7 hours) as compared to isogenic control cells (> 12 hours) (Fig. 2C). To confirm knockout of ANGEL2 via CRISPR/Cas9 modulates TP53 mRNA levels, we treated RKO, HCT116 and MCF-7 cells with siRNA to knock down ANGEL2 and found, like in the ANGEL2-null cell lines, TP53 mRNA expression was downregulated in all three cell lines (Supp. Fig. 2A).

Figure 2.

Figure 2.

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ANGEL2 modulates TP53 mRNA stability and translation. A, RNA-ChiP assay for ANGEL2 binding to TP53 mRNA in MCF7 cells with and without ANGEL2 ectopic expression. B, RT-PCR analysis for wildtype TP53 in MCF7 cells transfected with ANGEL2. C, qPCR analysis for TP53 in wildtype and ANGEL2-null HCT116 cells treated with actinomycin D (5 mg/mL) for 8 hours. D, Immunoblot for wildtype TP53 and ANGEL2 (Flag-tag) in TP53-null HCT116 cells co-transfected with wildtype TP53 expression vectors that contain the TP53 coding region (ORF) alone or in combination with its 5’ UTR, 3’ UTR, or both UTR’s and with increasing ANGEL2. E, RIP assay for EIF4E binding to TP53 mRNA in MCF7 cells with and without ANGEL2 ectopic expression. F, Immunoblot for de-novo wildtype TP53 (biotin) in MCF7 cells with and without ANGEL2 ectopic expression.

To explore the role of the TP53 5′ and 3′ UTRs in ANGEL2-mediated expression of TP53 we generated various expression vectors that contain the TP53 coding region (ORF) alone or in combination with its 5′ UTR, 3′ UTR, or both UTR’s (schematic shown in supplemental figure 2B). We co-transfected each TP53 expression vector with increasing concentrations of ANGEL2 in TP53-null HCT116 cells. We showed that ANGEL2 had no effect on TP53 ORF alone (Fig. 2D, first panel), whereas the largest increase in TP53 expression was found with the plasmid containing the TP53 5′ UTR (Fig. 2D, second panel). Further, we found that the plasmid containing the 3′ UTR had a limited ability to increase TP53 expression (Fig. 2D, third panel), supporting that ANGEL2 likely modulates TP53 expression through its interaction with TP53 5′ UTR. Interestingly, ANGEL2 only had a modest, if any, ability to increase TP53 expression in the plasmid containing both the 5′ and 3′ UTR’s (Fig. 2D, fourth panel). To further support that ANGEL2 modulates TP53 expression via interaction with its 5′ UTR, we utilized PRIdictor (Protein-RNA Interaction predictor), a web server capable of predicting mutual binding sites in RNA and protein at the nucleotide- and residue-level27. Likewise, PRIdictor showed a strong probability that ANGEL2 interacts with multiple nucleotides in TP53 5′ UTR, however, was unable to find any binding residues in TP53 ORF or 3′ UTR (Supp. Fig. 2C).

To determine whether ANGEL2 modulates the ability of TP53 mRNA to interact with EIF4E, we performed an RNA immunoprecipitation (RIP) assay. We questioned whether increased expression of ANGEL2 affects the ability of EIF4E, the rate limiting component of cap dependent translation, to interact with TP53 mRNA. Indeed, expression of ANGEL2 enhanced the ability for EIF4E to interact with TP53 mRNA (Fig. 2E). This contrasts with RBM38 which inhibits TP53 mRNA from interacting with EIF4E, shutting down its translation3. To further confirm that ANGEL2 enhances TP53 translation, we performed a de novo protein translation assay. Utilizing Click-IT technology, we determined that overexpression of ANGEL2 enhanced TP53 translation as shown in figure 2F. Taken together, our data supports that ANGEL2 interacts with TP53 mRNA, likely through its 5′ UTR thereby enhancing TP53 translation via increasing TP53 mRNA accessibility to EIF4E.

ANGEL2 directly interacts with EIF4E

As we previously identified that RBM38, and the Pep8 peptide, directly interact with EIF4E, we asked whether ANGEL2 can directly interact with EIF4E as well due to its similar amino acid homology to Pep822. To that end, we generated full-length and three truncated GST-tagged ANGEL2 constructs (Fig 3A). We purified ANGEL2 constructs and the EIF4E proteins expressed in E. coli before setting up a pulldown assay. GST-tagged ANGEL2 proteins were bound to GST beads and washed extensively before being incubated with EIF4E purified protein. Our pulldown assays demonstrated that full-length and ANGEL2 truncation 1–225 interacted the strongest with EIF4E (Fig. 3B). Interestingly, the 225–450 truncation weakly interacted with EIF4E, even though the peptide portion of ANGEL2 corresponding to the RBM38 Pep8 peptide is located between 297–303 (PYAACPA).

Figure 3.

Figure 3.

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ANGEL2 inhibits RBM38 from interacting with EIF4E. A, Graphical representation of the ANGEL2 expression constructs. B, Immunoblots for GST-ANGEL2 constructs pull-down of full-length EIF4E. C, Immunoblots for the competitive pulldown of GST-RBM38 interacting with purified EIF4E with and without the addition of purified ANGEL2 protein. D, Competitive ELISA for GST-tagged RBM38 interacting with EIF4E in the presence of increasing concentrations of purified ANGEL2.

Next, we explored whether ANGEL2 abrogates the interaction between RBM38 and EIF4E. We showed that ANGEL2 suppresses the ability for RBM38 to interact with EIF4E via a competitive pull-down assay and in a dose dependent manner with a competitive ELISA assay (Figs. 3C–D). Collectively, this data supports that ANGEL2 is a novel inhibitor of RBM38 that functions by blocking the interaction between RBM38 and EIF4E, which enhances TP53 mRNA translation.

Loss of ANGEL2 enhances tumor cell growth and chemoresistance.

To further elucidate the role of ANGEL2 in TP53 modulation, we investigated how the loss of ANGEL2 expression in TP53 expressing cancer cells effects cell growth and chemosensitivity. First, a colony formation assay in wildtype and ANGEL2-null RKO colon cancer cells demonstrated that loss of ANGEL2 caused a substantial increase in the ability for RKO cells to form colonies (Fig. 4A). Furthermore, loss of ANGEL2 decreased the sensitivity to the chemotherapeutic doxorubicin in 2D cell culture (Fig. 4B). In addition, ANGEL2-null RKO cells were also resistant to etoposide treatment (Supp. Fig. 3). To rule out any effect caused by CRISPR/Cas9 we also used siRNA in RKO wildtype and RKO TP53-null cells which demonstrated that loss of ANGEL2 caused a significant increase in cell viability in wildtype cells, whereas only a slight increase in the TP53-null cells suggesting that wildtype TP53 plays a role in ANGEL2-mediated growth suppression (Fig. 4C). Conversely, we found that overexpression of ANGEL2 in MCF7 and RKO cell lines decreases 3D tumor spheroid cell viability and sensitizes these cells to doxorubicin (Figs 4D–E). Finally, we showed that loss of ANGEL2 in HCT116 cells results in increased tumor cell viability and decreased sensitivity to doxorubicin (Fig. 4F). Taken together, these data support that ANGEL2 loss enhances tumor growth and contributes to decreased sensitivity to doxorubicin.

Figure 4.

Figure 4.

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Loss of ANGEL2 enhances tumor cell growth and resistance to doxorubicin. A, Colony formation assay for wildtype and ANGEL2-null RKO cells. B, 2D cell viability assay in wildtype and ANGEL2-null RKO cells treated with and without doxorubicin (6.25 ng/mL) for 24 hrs. C, 2D cell viability assay in wildtype and TP53-null RKO cells transfected with scramble siRNA, or siRNA targeted at ANGEL2. D-E, 3D tumor spheroid assay in MCF7 (left) and RKO (right) with and without ectopic ANGEL2 expression and treated with and without doxorubicin (6.25 ng/mL) for 18hrs. F, 3D tumor spheroid assay in wildtype and ANGEL2-null HCT116 cells treated with and without doxorubicin (6.25 ng/mL) for 18hrs. (*, P < 0.05)

ANGEL2-derived Pep7 peptide rescues TP53 expression

To test whether the ANGEL2-dervied peptide Pep7 (which shares high sequence homology with RBM38) can rescue TP53 expression in ANGEL2 deficient cells, we treated wildtype and ANGEL2-null HCT116 and RKO cells with pen-control or pen-Pep7 alone and in combination with doxorubicin. Doxorubicin treatment has been shown to induce TP53 protein accumulation through multiple post-translational modifications28. Further, our group previously demonstrated that RBM38 decreases TP53 expression even after doxorubicin treatment3. Thus, we sought to address if Pep7, which shares high sequence homology with RBM38, cooperates with doxorubicin to enhance TP53 expression. We found that in both cell lines, Pep7 (which is transported via fusion with the cell-penetrating peptide penetratin29) was able to rescue TP53 expression equal to that of wildtype cells (Fig. 5A–B, compare lanes 4 with 5 and 6). Of interest, we found that Pep7 treatment in combination with doxorubicin caused a larger increase TP53 expression in the two ANGEL2-null lines compared to wildtype cells (Fig. 5A–B, compare lanes 7 with 8 and 9). We previously showed that the control peptide (Pen-Control) does not modulate TP53 expression nor tumor cell viability. In confirmation, we treated wildtype and ANGEL2-null HCT116 and RKO cells with mock treatment or with Pen-Control and confirmed that Pen-Control was unable to modulate TP53 expression (Supp. Fig. 4A). Additional, we previously found that peptides derived from RBM38, including Pep7, abrogate the ability for RBM38 to interact with EIF4E, enhancing TP53 translation15. To confirm that Pep7 functions through modulating RBM38 binding we used RBM38/RBM24-null RKO cells. We utilized the dual knockout RBM38/RBM24 cell line as we have previously shown that RBM24 which is predominantly expressed in muscle cells, like RBM38, can modulate TP53 expression, albeit at a much lower extent30. We showed that Pep7 alone or in combination with doxorubicin had no ability to increase TP53 expression in these double knock-out cells (Supp. Fig. 4B). To confirm that Pep7 enhances TP53 translation, we performed a de novo translation assay for TP53. We found that in both wildtype and ANGEL2-null HCT116 cell lines, treatment with Pep7 or doxorubicin alone caused an increase in TP53 translation whereas the largest increase in translation was found when cells were treated concurrently with Pep7 and doxorubicin (Fig. 5C–D). Together, these results demonstrate that the ANGEL2-derived peptide Pep7 can rescue TP53 expression in ANGEL2-null cells by enhancing its translation.

Figure 5.

Figure 5.

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Pep7 enhances wildtype TP53 translation. A-B, Immunoblot for TP53 and vinculin in HCT116 (left) and RKO (right) cells treated with Pen-Control or Pen-Pep7 (20 mM) alone or in combination with doxorubicin (6.25 ng/mL) for 18hrs.C-D, Immunoblot for de-novo wildtype TP53 (biotin) in HCT116 (left) and RKO (right) cells treated with Pen-Control or Pen-Pep7 (20 mM), doxorubicin (6.25 ng/mL), or in combination for 18hrs.

Pep7 sensitizes tumor cells to doxorubicin

As Pep7 alone and in combination with doxorubicin enhances TP53 expression, we next asked whether this leads to a decrease in tumor cell growth. To that end, we performed colony formation assays in wildtype and ANGEL2-null HCT116 cells which were treated with Pep7 and doxorubicin alone and in combination. This assay revealed that both Pep7 and doxorubicin had a similar effect at decreasing colony formation, whereas when treated together there was a more profound effect (Fig. 6A–B). Next, we looked at cell viability in wildtype and ANGEL2-null HCT116 and RKO cell lines grown in 2D treated with Pep7 and doxorubicin alone and in combination. Like with the colony formation assay, we found that both Pep7 and doxorubicin had a similar ability to decrease cell viability, whereas when treated together there was a more pronounced decrease in cell viability (Fig. 6C–D). Finally, we questioned whether the effect of Pep7 alone and in combination with doxorubicin on 2D cultures would be recapitulated in 3D spheroid cultures. Therefore, we treated HCT116 wildtype and ANGEL2-null 3D tumor spheroids with Pep7 and doxorubicin alone and in combination. As in the previous assays, both Pep7 and doxorubicin had similar abilities to decrease cell viability and when combined together had an enhanced ability to decrease tumor spheroid cell viability (Fig. 6E–F). Collectively, these data support that Pep7 alone and in combination with doxorubicin can rescue the loss of ANGEL2 and sensitize tumor cell lines which harbor wildtype TP53 to doxorubicin.

Figure 6.

Figure 6.

Open in a new tab

Pep7 decreases tumor cell growth and sensitizes tumor cells to doxorubicin. A-B, colony formation assay in HCT116 (left) and ANGEL2-null HCT116 (right) cells treated with Pen-Control or Pen-Pep7 (20 mM), doxorubicin (6.25 ng/mL), or in combination. C-D, 2D cell viability assay in wildtype and ANGEL2-null HCT116 (left) and RKO (right) cells treated with Pen-Control or Pen-Pep7 (20 mM), doxorubicin (6.25 ng/mL), or in combination for 24hrs. E-F, 3D tumor spheroid assay in wildtype and ANGEL2-null cells treated with Pen-Control or Pen-Pep7 (20 mM), doxorubicin (6.25 ng/mL), or in combination for 24hrs.

Discussion

Since its discovery in 1979, TP53 has been at the forefront of cancer research with a multitude of signaling pathways discovered which have been shown to regulate, or are regulated by TP5331,32. These regulations are significant in the context of cancer progression as a key hallmark of tumorigeneses is the loss of wildtype TP53 function. This can occur secondary to loss of expression mutations but happens more commonly through loss of function and gain of function mutations33. Of significance, restoration of wildtype TP53 has been determined to be an effective strategy to inhibit tumor growth34–36. Previously our group identified that RBM38, an RNA-binding protein and a TP53 target, inhibits TP53 translation via its interaction with EIF4E3. Furthermore, we developed a peptide derived from RBM38 coined Pep8, which abrogates the ability for RBM38 to interact with EIF4E, increasing TP53 translation, leading to decreased colony/tumor sphere formation and reduced xenograft tumor growth in nude mice4. Pep8, composed of eight amino-acids (YPYAASPA), was shown to have high sequence homology to amino-acids in ANGEL2 (PYAACPA).

Multiple correlative studies have demonstrated a role for ANGEL2 in various malignancies, however ANGEL2 remains widely under studied with only one manuscript demonstrating ANGEL2 ability to regulate CDKN1A mechanistically explaining a potential role for ANGEL2 in cancer37–42. ANGEL2 (also known as CCR4d) is a novel member of the carbon catabolite repression 4 (CCR4) family. Thus far, there have been five Ccr4 homologues identified in human, which include Ccr4a (CNOT6), Ccr4b (CNOT6L), Ccr4c (nocturnin, Ccn4L), Ccr4d (ANGEL2), and Ccr4e (ANGEL1). Between the five, Ccr4a and Ccr4b share the highest homology (74% amino acid identity) and contain leucine-rich repeats facilitating interactions with Caf1a/Caf1b43. These two proteins both facilitate deadenylation contributing to the prevention of cell death and senescence44. Ccr4c is the ortholog of the deadenylase nocturnin which plays a role in circadian function45. ANGEL1 has previously been identified as a EIF4E binding protein which inhibits EIF4G from binding presumably stalling translation46. Unlike Ccr4a and Ccr4b who function as deadenylases, ANGEL2 does not harbor deadenylase activity. Uniquely, ANGEL2 was found to contain 2′,3′-cyclic phosphatase activity, supporting its role in tRNA recycling11. Moreover, ANGEL2 was found to directly interact with CDKN1A transcript increasing its mRNA half-life, suggesting that ANGEL2 may function as an RBP modulating target mRNA stability as well47. Of clinical significance, loss of ANGEL2 was found to be a driver of diffuse large B-cell lymphomagenesis13,47,48, and low ANGEL2 expression across 17 different cancer types correlated with poor overall patient survival14. However, it is still not understood how loss of ANGEL2 occurs in these cancers nor the role ANGEL2 plays in oncogenesis.

For the first time, we demonstrate that ANGEL2 directly interacts with EIF4E, relieving the translational repression caused by RBM38, thus enhancing TP53 translation. Herein, we show that loss of ANGEL2 substantially decreases, whereas ectopic expression of ANGEL2 enhances, TP53 protein expression (Fig. 1). We demonstrate that overexpression and loss of ANGEL2 modulates TP53 across HCT116, RKO and MCF7 cancer cell lines supporting that ANGEL2 is part of a conserved pathway across multiple cancer cell types. Mechanistically, ANGEL2 was found to stabilize the TP53 transcript presumably through its interaction with TP53 5′ UTR and enhancing TP53 translation by stifling the ability for RBM38 to interact with EIF4E. (Figs. 2–3). While it may be argued that the decreased mRNA stability in the ANGEL2-null cells is the sole reason for decreased TP53 translation, we would like to point out that it has been shown by multiple groups that loss of translation of a transcript negatively modulates its mRNA stability49–51. We propose that decreased TP53 translation by loss of ANGEL2 contributes to its mRNA instability, which is compounded by the fact that ANGEL2 directly interacts with its transcript presumably stabilizing it. This is a very interesting mechanism that necessitates further exploration. Of interest, while ANGEL2 was able to modulate the expression of TP53 via its 5′ or 3′ UTR alone, we found that ANGEL2 had no effect on the TP53 construct which contained both 5′ and 3′ UTRs. Previously, it was thought that TP53 3′ UTR restrains TP53 expression as the 3′ UTR has been shown to be modulated by 23 miRNA, 1 lncRNA, and 6 known RNA-binding proteins52. However, recently a paper demonstrated that TP53 3′ UTR modulation of TP53 expression was muted by TP53 coding region repressive effects suggesting that the 3′ UTR has no ability to enhance TP53 expression53. Yet this doesn’t explain why ANGEL2 has no effect on the 5′UTR-ORF-3′UTR TP53 DNA construct. Collectively, our data as well as from other investigators demonstrate that TP53 regulation via its UTR’s is multifaceted and still not fully understood.

Our results support that ANGEL2 interacts with EIF4E via amino acids 1–225, even though the Pep7 peptide motif is in the fragment 225–450 (Fig. 3A–B). While these results were surprising, our approach has limitations. ANGEL2 is a larger protein (544 amino acids) and truncating the protein into 3 domains may have introduced nonnatural folding of these fragments leading to loss of binding. While minimal binding was identified for the 225–450 fragment, our results indicate that ANGEL2 likely forms key interactions with EIF4E beyond the interaction with the Pep7 peptide fragment. This hypothesis is supported by the fact that ANGEL2 1–225 fragment showed robust, while the 225–450 fragment showed limited binding to EIF4E (Fig. 3B). Coupled with the data supporting Pep7 enhances TP53 translation (Fig. 5C–D), which our group has also previously shown that highly homologous peptides interact with EIF4E and dissociates RBM38 as does ANGEL215. Nonetheless, a structure-based approach would be useful to determine the binding interface between ANGEL2 and EIF4E. Previously, it was found that ANGEL1, a family member of ANGEL2, interacts with EIF4E, however, manipulation of ANGEL1 protein levels in vitro had no effect on global translation rates suggesting a more specific regulation43. In the future, it would be beneficial to study the global translation effects of loss of ANGEL2 and if any other translational targets are modulated by loss of ANGEL2 beyond TP53 such as mutant TP53.

Applying synthetic peptides to block cancer-specific protein-protein interactions have particular advantages such as increased potency, target specificity, and enhanced safety54. We previously showed that rationally designed peptides abrogate the EIF4E-RBM38 complex, thereby restoring TP53 expression4,15. Herein, we demonstrate that an ANGEL2-derived peptide, Pep7, rescues TP53 translation in ANGEL2 deficient cell lines. Our data shows that Pep7 treatment, alone and in combination with doxorubicin in both wildtype and ANGEL2-null cell lines was shown to enhance TP53 de novo translation and increase TP53 protein expression (Fig. 5). Pep7 also inhibits colony and 3D tumor spheroid formation in both wildtype and ANGEL2-null tumor cells (Figs. 5–6). Doxorubicin is a widely used first-line agent for a multitude of cancers owning to its effectiveness as a cancer therapeutic agent55. Unfortunately, doxorubicin has substantial systemic toxicities which include severe inflammation of multiple organs and cardiac damage56. Therefore, finding ways to improve doxorubicin efficacy to lower its dosing may be an attractive modality to improve treatment efficacy and improve patient outcomes and quality of life. Of interest, we found that loss of ANGEL2 caused a decrease in sensitivity to etoposide as well (Supp. Fig. 3). As both drugs cause DNA damage, albeit through different modes of action57, we postulate that loss of ANGEL2 may play a role in the DNA damage repair response and/or drug efflux which necessitates further exploration.

Collectively, our findings here support that ANGEL2 may function as a novel tumor suppressor as demonstrated by loss of ANGEL2 which substantially decreases TP53 protein expression via decreased TP53 translation. This leads to enhanced tumor cell growth and acquired resistance to doxorubicin. Therapeutically, utilizing the Pep7 peptide, which is derived from ANGEL2, we can rescue TP53 protein expression and sensitize tumor cells to doxorubicin. However, multiple questions are still yet to be addressed. For example, does ANGEL2 also modulate mutant TP53, and if so, does loss of ANGEL2 in these cells decrease cell growth as many mutant TP53 expressing cancers become addicted to its expression58. Further, as ANGEL2 interacts directly with EIF4E, what are the other potential global consequences to translation because of loss of ANGEL2 expression and potential therapeutic intervention with Pep7.

Supplementary Material

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Acknowledgements

C. Lucchesi has been supported by NCI (K22CA279075).

Footnotes

Competing Interests

Dr. Lucchesi’s work has been funded by the NIH. The authors declare no competing financial interests. The contents reported/presented within do not represent the views of the Department of Veterans Affairs or the United States Government.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2066163-supplement-1.docx (14.1KB, docx)
2
NIHMS2066163-supplement-2.tif (40.6MB, tif)
3
NIHMS2066163-supplement-3.tif (65.6MB, tif)
4
NIHMS2066163-supplement-4.tif (8.4MB, tif)
5
NIHMS2066163-supplement-5.tif (11.3MB, tif)

Data Availability Statement

The data generated in this study are available within the article and its Supplementary Materials.

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