Enhanced G1 arrest and apoptosis via MDM4/MDM2 double knockdown and MEK inhibition in wild‑type <em>TP53</em> colon and gastric cancer cells with aberrant KRAS signaling

Wang,Xiaoxuan; Yamamoto,Yoshiyuki; Imanishi,Mamiko; Zhang,Xiaochen; Sato,Masashi; Sugaya,Akinori; Hirose,Mitsuaki; Endo,Shinji; Natori,Yukikazu; Moriwaki,Toshikazu; Yamato,Kenji; Hyodo,Ichinosuke

doi:10.3892/ol.2021.12819

Enhanced G1 arrest and apoptosis via MDM4/MDM2 double knockdown and MEK inhibition in wild‑type TP53 colon and gastric cancer cells with aberrant KRAS signaling

Authors:
- Xiaoxuan Wang
- Yoshiyuki Yamamoto
- Mamiko Imanishi
- Xiaochen Zhang
- Masashi Sato
- Akinori Sugaya
- Mitsuaki Hirose
- Shinji Endo
- Yukikazu Natori
- Toshikazu Moriwaki
- Kenji Yamato
- Ichinosuke Hyodo
View Affiliations

Affiliations: Department of Gastroenterology, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305‑8575, Japan, Division of Gastroenterology, Ibaraki Prefectural Central Hospital, Kasama, Ibaraki 309‑1793, Japan, BioThinkTank Co. Ltd., Yokohama, Kanagawa 220‑0012, Japan
Published online on: May 25, 2021 https://doi.org/10.3892/ol.2021.12819
Article Number: 558
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Murine double minute homolog 2 (MDM2) is an oncoprotein that induces p53 degradation via ubiquitin‑ligase activity. MDM4 cooperates with MDM2‑mediated p53 degradation, directly inhibiting p53 transcription by binding to its transactivation domain. Our previous study reported that the simultaneous inhibition of MDM2 and MDM4 using nutlin‑3 (an inhibitor of the MDM2‑p53 interaction) and chimeric small interfering RNA with DNA‑substituted seed arms (named chiMDM2 and chiMDM4) more potently activated p53 than the MDM2 or MDM4 inhibitor alone and synergistically augmented antitumor effects in various types of cancer cells with the wild‑type (wt) TP53. Recently, the synergism of MDM2 and mitogen‑activated protein kinase kinase (MEK) inhibitors has been demonstrated in wt TP53 colorectal and non‑small cell lung cancer cells harboring mutant‑type (mt) KRAS. The current study examined whether chiMDM4 augmented the synergistic antitumor effects of MDM2 and MEK inhibition using chiMDM2 or nutlin‑3 and trametinib, respectively. ChiMDM2 and trametinib used in combination demonstrated a synergistic antitumor activity in HCT116 and LoVo colon cancer cells, and SNU‑1 gastric cancer cells harboring wt TP53 and mt KRAS. Furthermore, chiMDM4 synergistically enhanced this combinational effect. Similar results were observed when nutlin‑3 was used instead of chiMDM2. MDM4/MDM2 double knockdown combined with trametinib treatment enhanced G1 arrest and apoptosis induction. This was associated with the accumulation of p53, suppression of phosphorylated‑extracellular signal‑regulated kinase 2, inhibition of retinoblastoma phosphorylation, suppression of E2F1‑activated proteins, and potent activation of pro‑apoptotic proteins, such as Fas and p53 upregulated modulator of apoptosis. The results inidcated that the triple inhibition of MDM4, MDM2 and MEK exerted a potent antitumor effect in wt TP53 colon and gastric cancer cells with mt KRAS. Simultaneous activation of p53 and inhibition of aberrant KRAS signaling may be a rational treatment strategy for gastrointestinal tumors.

View Figures

View References

1	Sionov RV and Haupt Y: The cellular response to p53: The decision between life and death. Oncogene. 18:6145–6157. 1999. View Article : Google Scholar : PubMed/NCBI
2	Vousden KH and Lu X: Live or let die: The cell's response to p53. Nat Rev Cancer. 2:594–604. 2002. View Article : Google Scholar : PubMed/NCBI
3	Levine AJ: The tumor suppressor genes. Annu Rev Biochem. 62:623–651. 1993. View Article : Google Scholar : PubMed/NCBI
4	Moll UM and Petrenko O: The MDM2-p53 interaction. Mol Cancer Res. 1:1001–1008. 2003.PubMed/NCBI
5	Linares LK, Hengstermann A, Ciechanover A, Müller S and Scheffner M: HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53. Proc Natl Acad Sci USA. 100:12009–12014. 2003. View Article : Google Scholar : PubMed/NCBI
6	Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, Bazuine M, van Ham RC, van der Houven van Oordt W, Hateboer G, van der Eb AJ and Jochemsen AG: MDMX: A novel p53-binding protein with some functional properties of MDM2. EMBO J. 15:5349–5357. 1996. View Article : Google Scholar : PubMed/NCBI
7	Wu X, Bayle JH, Olson D and Levine AJ: The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 7:1126–1132. 1993. View Article : Google Scholar : PubMed/NCBI
8	Mahmoodi Chalbatani G, Dana H, Gharagouzloo E, Grijalvo S, Eritja R, Logsdon CD, Memari F, Miri SR, Rad MR and Marmari V: Small interfering RNAs (siRNAs) in cancer therapy: A nano-based approach. Int J Nanomedicine. 14:3111–3128. 2019. View Article : Google Scholar : PubMed/NCBI
9	Imanishi M, Yamamoto Y, Wang X, Sugaya A, Hirose M, Endo S, Natori Y, Yamato K and Hyodo I: Augmented antitumor activity of 5-fluorouracil by double knockdown of MDM4 and MDM2 in colon and gastric cancer cells. Cancer Sci. 110:639–649. 2019. View Article : Google Scholar : PubMed/NCBI
10	Hirose M, Yamato K, Endo S, Saito R, Ueno T, Hirai S, Suzuki H, Abei M, Natori Y and Hyodo I: MDM4 expression as an indicator of TP53 reactivation by combined targeting of MDM2 and MDM4 in cancer cells without TP53 mutation. Oncoscience. 1:830–843. 2014. View Article : Google Scholar : PubMed/NCBI
11	Endo S, Yamato K, Hirai S, Moriwaki T, Fukuda K, Suzuki H, Abei M, Nakagawa I and Hyodo I: Potent in vitro and in vivo antitumor effects of MDM2 inhibitor nutlin-3 in gastric cancer cells. Cancer Sci. 102:605–613. 2011. View Article : Google Scholar : PubMed/NCBI
12	Malmlöf M, Roudier E, Högberg J and Stenius U: MEK-ERK-mediated phosphorylation of Mdm2 at Ser-166 in hepatocytes. Mdm2 is activated in response to inhibited Akt signaling. J Biol Chem. 282:2288–2296. 2007. View Article : Google Scholar
13	Ries S, Biederer C, Woods D, Shifman O, Shirasawa S, Sasazuki T, McMahon M, Oren M and McCormick F: Opposing effects of Ras on p53: Transcriptional activation of mdm2 and induction of p19ARF. Cell. 103:321–330. 2000. View Article : Google Scholar : PubMed/NCBI
14	Hata AN, Rowley S, Archibald HL, Gomez-Caraballo M, Siddiqui FM, Ji F, Jung J, Light M, Lee JS, Debussche L, et al: Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers. Oncogene. 36:6581–6591. 2017. View Article : Google Scholar : PubMed/NCBI
15	Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 22:27–55. 1984. View Article : Google Scholar : PubMed/NCBI
16	Bronkhorst AJ, Wentzel JF, Aucamp J, van Dyk E, du Plessis L and Pretorius PJ: Characterization of the cell-free DNA released by cultured cancer cells. Biochim Biophys Acta. 1863:157–165. 2016. View Article : Google Scholar : PubMed/NCBI
17	Tsujimoto Y and Croce CM: Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA. 83:5214–5218. 1986. View Article : Google Scholar : PubMed/NCBI
18	Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP and Wang X: Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science. 275:1129–1132. 1997. View Article : Google Scholar : PubMed/NCBI
19	Reshi L, Wang HV, Hui CF, Su YC and Hong JR: Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells. Fish Shellfish Immunol. 61:120–129. 2017. View Article : Google Scholar : PubMed/NCBI
20	Rodríguez J, Calvo F, González JM, Casar B, Andrés V and Crespo P: ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma-lamin A complexes. J Cell Biol. 191:967–979. 2010. View Article : Google Scholar
21	Bidère N and Senik A: Caspase-independent apoptotic pathways in T lymphocytes: A minireview. Apoptosis. 6:371–375. 2001. View Article : Google Scholar
22	Wu M, Xu LG, Li X, Zhai Z and Shu HB: AMID, an apoptosis-inducing factor-homologous mitochondrion-associated protein, induces caspase-independent apoptosis. J Biol Chem. 277:25617–25623. 2002. View Article : Google Scholar : PubMed/NCBI
23	Lee TJ, Kim EJ, Kim S, Jung EM, Park JW, Jeong SH, Park SE, Yoo YH and Kwon TK: Caspase-dependent and caspase-independent apoptosis induced by evodiamine in human leukemic U937 cells. Mol Cancer Ther. 5:2398–2407. 2006. View Article : Google Scholar : PubMed/NCBI
24	Mandal R, Raab M, Matthess Y, Becker S, Knecht R and Strebhardt K: pERK 1/2 inhibit caspase-8 induced apoptosis in cancer cells by phosphorylating it in a cell cycle specific manner. Mol Oncol. 8:232–249. 2014. View Article : Google Scholar : PubMed/NCBI
25	Allan LA, Morrice N, Brady S, Magee G, Pathak S and Clarke PR: Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat Cell Biol. 5:647–654. 2003. View Article : Google Scholar : PubMed/NCBI