ROCK signaling is involved in the entosis of both nonepithelial and epithelial tumors, whereas N‑cadherin is involved in the entosis of nonepithelial tumors

Oi,Mizuha; Kushibiki,Raia; Kanehira,Yuki; Nishijima,Yoshimi; Kobayashi,Sayaka; Saio,Masanao

doi:10.3892/etm.2025.12840

ROCK signaling is involved in the entosis of both nonepithelial and epithelial tumors, whereas N‑cadherin is involved in the entosis of nonepithelial tumors

Authors:
- Mizuha Oi
- Raia Kushibiki
- Yuki Kanehira
- Yoshimi Nishijima
- Sayaka Kobayashi
- Masanao Saio
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Affiliations: Laboratory of Histopathology and Cytopathology, Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, Maebashi, Gunma 371‑8514, Japan
Published online on: March 7, 2025 https://doi.org/10.3892/etm.2025.12840
Article Number: 90
Copyright: © Oi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Entosis is a cell‑in‑cell phenomenon wherein cells detaching from the extracellular matrix are internalized by a neighboring cell. The present study examined whether entosis, which is observed in epithelial cells, also occurs in nonepithelial cells. The present study used the MCF‑7 breast cancer cell line as a positive control for entosis and compared this with three representative sarcoma cell lines (RD, HT1080 and ICH‑ERMS‑1). The formation of cell‑in‑cell structures was induced by culturing cells in adherent and nonadherent conditions. Cell lines that formed the cell‑in‑cell structures were cultured in nonadherent conditions with and without Rho‑associated coiled‑coil containing protein kinase (ROCK) inhibition, and the cell‑in‑cell structures were evaluated in slides prepared from cell blocks. It was examined whether ROCK inhibition blocked the formation of cell‑in‑cell structures, and the expression levels of specific cadherins associated with entosis were determined using quantitative PCR. The proportion of cells with cell‑in‑cell structures was significantly higher in nonadherent conditions in both MCF‑7 (P=0.0297) and RD (P=0.0098) cells, whereas few cell‑in‑cell structures were observed in both adherent and nonadherent conditions in HT1080 and ICH‑ERMS‑1 cells. Under nonadherent conditions, ROCK inhibition significantly reduced the proportion of cells with cell‑in‑cell structures in MCF‑7 (P=0.0021) and RD (P=0.0407) cells. Based on quantitative PCR, among the five cadherin genes, the E‑cadherin expression level was the lowest in MCF‑7 cells (ΔCt, 2.6) and the N‑cadherin expression level was lowest in RD cells (ΔCt, 4.8). By contrast, the N‑cadherin expression levels were higher in HT1080 (ΔCt, 11.0) and ICH‑ERMS‑1 (ΔCt, 8.8) cells. These results suggested that the cell‑in‑cell phenomenon observed in RD cells is an entotic process based on its emergence in nonadherent culture conditions and the involvement of ROCK signaling. Entosis observed in RD cells was mediated via N‑cadherin and not E‑cadherin.

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1	Overholtzer M, Mailleux AA, Mouneimne G, Normand G, Schnitt SJ, King RW, Cibas ES and Brugge JS: A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell. 131:966–979. 2007.PubMed/NCBI View Article : Google Scholar
2	Wang M, Niu Z, Qin H, Ruan B, Zheng Y, Ning X, Gu S, Gao L, Chen Z, Wang X, et al: Mechanical ring interfaces between adherens junction and contractile actomyosin to coordinate entotic cell-in-cell formation. Cell Rep. 32(108071)2020.PubMed/NCBI View Article : Google Scholar
3	Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, Yu K, Lan S, Wang Q, Li S, et al: Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. Sci Rep. 5(12223)2015.PubMed/NCBI View Article : Google Scholar
4	Bauer MF, Hildebrand LS, Rosahl MC, Erber R, Schnellhardt S, Büttner-Herold M, Putz F, Ott OJ, Hack CC, Fietkau R and Distel L: Cell-in-cell structures in early breast cancer are prognostically valuable. Cells. 12(81)2022.PubMed/NCBI View Article : Google Scholar
5	Dziuba I, Gawel AM, Tyrna P, Machtyl J, Olszanecka M, Pawlik A, Wójcik C, Bialy LP and Mlynarczuk-Bialy I: Homotypic entosis as a potential novel diagnostic marker in breast cancer. Int J Mol Sci. 24(6819)2023.PubMed/NCBI View Article : Google Scholar
6	Wei Y, Niu Z, Hou X, Liu M, Wang Y, Zhou Y, Wang C, Ma Q, Zhu Y, Gao X, et al: Subtype-based analysis of cell-in-cell structures in non-small cell lung cancer. Am J Cancer Res. 13:1091–1102. 2023.PubMed/NCBI
7	Liu X, Guo R, Li D, Wang Y, Ning J, Yang S and Yang J: Homotypic cell-in-cell structure as a novel prognostic predictor in non-small cell lung cancer and frequently localized at the invasive front. Sci Rep. 14(18952)2024.PubMed/NCBI View Article : Google Scholar
8	Almangush A, Mäkitie AA, Hagström J, Haglund C, Kowalski LP, Nieminen P, Coletta RD, Salo T and Leivo I: Cell-in-cell phenomenon associates with aggressive characteristics and cancer-related mortality in early oral tongue cancer. BMC Cancer. 20(843)2020.PubMed/NCBI View Article : Google Scholar
9	Kim Y, Choi JW, Lee JH and Kim YS: Spindle assembly checkpoint MAD2 and CDC20 overexpressions and cell-in-cell formation in gastric cancer and its precursor lesions. Hum Pathol. 85:174–183. 2019.PubMed/NCBI View Article : Google Scholar
10	Song J, Xu R, Zhang H, Xue X, Ruze R, Chen Y, Yin X, Wang C and Zhao Y: Cell-in-cell-mediated entosis reveals a progressive mechanism in pancreatic cancer. Gastroenterology. 165:1505–1521.e20. 2023.PubMed/NCBI View Article : Google Scholar
11	Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, Gentile M, Luciani F, Parmiani G, Rivoltini L, et al: Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells. Cancer Res. 66:3629–3638. 2006.PubMed/NCBI View Article : Google Scholar
12	Gupta K and Dey P: Cell cannibalism: Diagnostic marker of malignancy. Diagn Cytopathol. 28:86–87. 2003.PubMed/NCBI View Article : Google Scholar
13	Wang S, He M, Li L, Liang Z, Zou Z and Tao A: Cell-in-cell death is not restricted by caspase-3 deficiency in MCF-7 cells. J Breast Cancer. 19:231–241. 2016.PubMed/NCBI View Article : Google Scholar
14	Fais S: Cannibalism: A way to feed on metastatic tumors. Cancer Lett. 258:155–164. 2007.PubMed/NCBI View Article : Google Scholar
15	Humble JG, Jayne WH and Pulvertaft RJ: Biological interaction between lymphocytes and other cells. Br J Haematol. 2:283–294. 1956.PubMed/NCBI View Article : Google Scholar
16	Larsen TE: Emperipolesis of granular leukocytes within megakaryocytes in human hemopoietic bone marrow. Am J Clin Pathol. 53:485–489. 1970.PubMed/NCBI View Article : Google Scholar
17	Sun Q, Cibas ES, Huang H, Hodgson L and Overholtzer M: Induction of entosis by epithelial cadherin expression. Cell Res. 24:1288–1298. 2014.PubMed/NCBI View Article : Google Scholar
18	Sun Q, Luo T, Ren Y, Florey O, Shirasawa S, Sasazuki T, Robinson DN and Overholtzer M: Competition between human cells by entosis. Cell Res. 24:1299–1310. 2014.PubMed/NCBI View Article : Google Scholar
19	Kianfar M, Balcerak A, Chmielarczyk M, Tarnowski L and Grzybowska EA: Cell death by entosis: Triggers, molecular mechanisms and clinical significance. Int J Mol Sci. 23(4985)2022.PubMed/NCBI View Article : Google Scholar
20	Krajcovic M, Johnson NB, Sun Q, Normand G, Hoover N, Yao E, Richardson AL, King RW, Cibas ES, Schnitt SJ, et al: A non-genetic route to aneuploidy in human cancers. Nat Cell Biol. 13:324–330. 2011.PubMed/NCBI View Article : Google Scholar
21	Liang J, Niu Z, Zhang B, Yu X, Zheng Y, Wang C, Ren H, Wang M, Ruan B, Qin H, et al: p53-dependent elimination of aneuploid mitotic offspring by entosis. Cell Death Differ. 28:799–813. 2021.PubMed/NCBI View Article : Google Scholar
22	Wen S, Shang Z, Zhu S, Chang C and Niu Y: Androgen receptor enhances entosis, a non-apoptotic cell death, through modulation of Rho/ROCK pathway in prostate cancer cells. Prostate. 73:1306–1315. 2013.PubMed/NCBI View Article : Google Scholar
23	Zhang X, Niu Z, Qin H, Fan J, Wang M, Zhang B, Zheng Y, Gao L, Chen Z, Tai Y, et al: Subtype-based prognostic analysis of cell-in-cell structures in early breast cancer. Front Oncol. 9(895)2019.PubMed/NCBI View Article : Google Scholar
24	Ishikawa F, Ushida K, Mori K and Shibanuma M: Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling. Cell Death Dis. 6(e1619)2015.PubMed/NCBI View Article : Google Scholar
25	Wang C, Chen A, Ruan B, Niu Z, Su Y, Qin H, Zheng Y, Zhang B, Gao L, Chen Z, et al: PCDH7 inhibits the formation of homotypic cell-in-cell structure. Front Cell Dev Biol. 8(329)2020.PubMed/NCBI View Article : Google Scholar
26	Durgan J, Tseng YY, Hamann JC, Domart MC, Collinson L, Hall A, Overholtzer M and Florey O: Mitosis can drive cell cannibalism through entosis. Elife. 6(e27134)2017.PubMed/NCBI View Article : Google Scholar
27	Mackay HL, Moore D, Hall C, Birkbak NJ, Jamal-Hanjani M, Karim SA, Phatak VM, Piñon L, Morton JP, Swanton C, et al: Genomic instability in mutant p53 cancer cells upon entotic engulfment. Nat Commun. 9(3070)2018.PubMed/NCBI View Article : Google Scholar
28	Bozkurt E, Dussmann H, Salvucci M, Cavanagh BL, Van Schaeybroeck S, Longley DB, Martin SJ and Prehn JHM: TRAIL signaling promotes entosis in colorectal cancer. J Cell Biol. 220(e202010030)2021.PubMed/NCBI View Article : Google Scholar
29	Garanina AS, Kisurina-Evgenieva OP, Erokhina MV, Smirnova EA, Factor VM and Onishchenko GE: Consecutive entosis stages in human substrate-dependent cultured cells. Sci Rep. 7(12555)2017.PubMed/NCBI View Article : Google Scholar
30	Rao X, Huang X, Zhou Z and Lin X: An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 3:71–85. 2013.PubMed/NCBI
31	Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M and Narumiya S: Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 389:990–994. 1997.PubMed/NCBI View Article : Google Scholar
32	Gutjahr MC, Rossy J and Niggli V: Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells. Exp Cell Res. 308:422–438. 2005.PubMed/NCBI View Article : Google Scholar
33	Hatta K, Okada TS and Takeichi M: A monoclonal-antibody disrupting calcium-dependent cell cell-adhesion of brain-tissues-possible role of its target antigen in animal pattern-formation. Proc Natl Acad Sci USA. 82:2789–2793. 1985.PubMed/NCBI View Article : Google Scholar
34	Yoshida-Noro C, Suzuki N and Takeichi M: Molecular nature of the calcium-dependent cell cell-adhesion system in mouse teratocarcinoma and embryonic-cells studied with a monoclonal-antibody. Dev Biol. 101:19–27. 1984.PubMed/NCBI View Article : Google Scholar
35	Nose A and Takeichi M: A novel cadherin cell-adhesion molecule: Its expression patterns associated with implantation and organogenesis of mouse embryos. J Cell Biol. 103 (6 Pt 2):2649–2658. 1986.PubMed/NCBI View Article : Google Scholar
36	Soler AP, Johnson KR, Wheelock MJ and Knudsen KA: Rhabdomyosarcoma-derived cell-lines exhibit aberrant expression of the cell-cell adhesion molecules N-Cam, N-Cadherin, and cadherin-associated proteins. Exp Cell Res. 208:84–93. 1993.PubMed/NCBI View Article : Google Scholar
37	Derycke LD and Bracke ME: N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. Int J Dev Biol. 48:463–476. 2004.PubMed/NCBI View Article : Google Scholar
38	Rapa E, Hill SK, Morten KJ, Potter M and Mitchell C: The over-expression of cell migratory genes in alveolar rhabdomyosarcoma could contribute to metastatic spread. Clin Exp Metastasis. 29:419–429. 2012.PubMed/NCBI View Article : Google Scholar
39	Li M, Nagamori E and Kino-Oka M: Disruption of myoblast alignment by highly motile rhabdomyosarcoma cell in tissue structure. J Biosci Bioeng. 123:259–264. 2017.PubMed/NCBI View Article : Google Scholar
40	Schwegler M, Wirsing AM, Schenker HM, Ott L, Ries JM, Büttner-Herold M, Fietkau R, Putz F and Distel LV: Prognostic value of homotypic cell internalization by nonprofessional phagocytic cancer cells. Biomed Res Int. 2015(359392)2015.PubMed/NCBI View Article : Google Scholar
41	Druzhkova I, Potapov A, Ignatova N, Bugrova M, Shchechkin I, Lukina M, Shimolina L, Kolesnikova E, Shirmanova M and Zagaynova E: Cell hiding in colorectal cancer: Correlation with response to chemotherapy in vitro and in vivo. Sci Rep. 14(28762)2024.PubMed/NCBI View Article : Google Scholar
42	Lugini L, Lozupone F, Matarrese P, Funaro C, Luciani F, Malorni W, Rivoltini L, Castelli C, Tinari A, Piris A, et al: Potent phagocytic activity discriminates metastatic and primary human malignant melanomas: A key role of ezrin. Lab Invest. 83:1555–1567. 2003.PubMed/NCBI View Article : Google Scholar