Cancer‑associated fibroblasts as cellular vehicles in endometrial cancer cell migration

Voon,Yap Chee; Omar,Intan Sofia; Wu,Ming-Heng; Said,Nur Akmarina B.M.; Chung,Ivy

doi:10.3892/ol.2021.13121

Cancer‑associated fibroblasts as cellular vehicles in endometrial cancer cell migration

Authors:
- Yap Chee Voon
- Intan Sofia Omar
- Ming-Heng Wu
- Nur Akmarina B.M. Said
- Ivy Chung
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Affiliations: Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, Graduate Institute of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan, R.O.C., Department of Pharmaceutical Life Science, Faculty of Pharmacy, University of Malaya, 50603 Kuala Lumpur, Malaysia
Published online on: November 4, 2021 https://doi.org/10.3892/ol.2021.13121
Article Number: 3
Copyright: © Voon et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Cell motility is a critical step in the metastasis cascade. However, the role of cancer‑associated fibroblasts (CAFs) in facilitating endometrial cancer (EC) cell motility remains unclear. The present study aimed to investigate the role of CAFs in EC motility in a 3D environment. A co‑culture model was established using an EC cell line (ECC‑1) and CAFs on a Matrigel^® matrix and compared to the respective individual monocultures. It was demonstrated that endometrial CAFs increased the motility of the EC cell line, compared with the monoculture. Using live cell imaging, CAFs were observed to form cell projections that served as contact guidance for ECC‑1 cell locomotion in the spheroid formation process. These effects were specific to CAFs, as fibroblasts isolated from benign endometrial tissue samples did not form cell projections. Molecular analysis revealed that RhoA/Rho‑associated, coiled‑coil containing protein kinase 1 (ROCK1) signaling activation partly contributed to CAF‑mediated ECC‑1 cell migration. The presence of Matrigel^® increased the mRNA expression of RhoA, and the mRNA and protein expression levels of its downstream effectors, ROCK1 and p‑MLC, respectively, in the ECC‑1 and CAF co‑culture, as well as the ECC‑1 and CAF monocultures. Interestingly, high phosphorylation levels of myosin light chain mediated the activation of RhoA/ROCK1 signaling in the ECC‑1 and CAF co‑culture. The ROCK1 inhibitor Y‑27632 attenuated the motility of tumor cells in ECC‑1 and CAF co‑cultures. However, similar treatment led to a significant inhibition in the motility of the CAF monoculture, but not the ECC‑1 monoculture. Moreover, tumor spheroid formation was inhibited due to a reduction in stress fiber formation in ECC‑1 and CAF co‑cultures. Altogether, these findings suggest that the regulation of the RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicles in order for EC cells to migrate and form spheroids in a 3D environment.

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1	American Cancer Society, . Key statistics for endometrial cancer. Journal. 2018.
2	World Cancer Research Fund International, . Endometrial cancer (cancer of the lining of the womb) statistics. Journal. 2018.
3	American Cancer Society, . Cancer facts & figures 2015. https://oralcancerfoundation.org/wp-content/uploads/2016/03/Us_Cancer_Facts.pdf
4	Bokhman JV: Two pathogenetic types of endometrial carcinoma. Gynecol Oncol. 15:10–17. 1983. View Article : Google Scholar : PubMed/NCBI
5	Amant F, Moerman P, Neven P, Timmerman D, Van Limbergen E and Vergote I: Endometrial cancer. Lancet. 366:491–505. 2005. View Article : Google Scholar : PubMed/NCBI
6	Bansal N, Yendluri V and Wenham RM: The molecular biology of endometrial cancers and the implications for pathogenesis, classification, and targeted therapies. Cancer Control. 16:8–13. 2009. View Article : Google Scholar : PubMed/NCBI
7	Baiden-Amissah REM, Annibali D, Tuyaerts S and Amant F: Endometrial cancer molecular characterization: The key to identifying high-risk patients and defining guidelines for clinical decision-making? Cancers (Basel). 13:39882021. View Article : Google Scholar : PubMed/NCBI
8	Cancer Genome Atlas Research Network, ; Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, Robertson AG, Pashtan I, Shen R, et al: Integrated genomic characterization of endometrial carcinoma. Nature. 497:67–73. 2013. View Article : Google Scholar : PubMed/NCBI
9	Welch DR and Hurst DR: Defining the hallmarks of metastasis. Cancer Res. 79:3011–3027. 2019. View Article : Google Scholar : PubMed/NCBI
10	Kurra V, Krajewski KM, Jagannathan J, Giardino A, Berlin S and Ramaiya N: Typical and atypical metastatic sites of recurrent endometrial carcinoma. Cancer Imaging. 13:113–122. 2013. View Article : Google Scholar : PubMed/NCBI
11	Ridley AJ: Rho GTPase signalling in cell migration. Curr Opin Cell Biol. 36:103–112. 2015. View Article : Google Scholar : PubMed/NCBI
12	Goubran HA, Kotb RR, Stakiw J, Emara ME and Burnouf T: Regulation of tumor growth and metastasis: The role of tumor microenvironment. Cancer Growth Metastasis. 7:9–18. 2014. View Article : Google Scholar : PubMed/NCBI
13	Cirri P and Chiarugi P: Cancer associated fibroblasts: The dark side of the coin. Am J Cancer Res. 1:482–497. 2011.PubMed/NCBI
14	Dvorak HF: Tumors: Wounds that do not heal-redux. Cancer Immunol Res. 3:1–11. 2015. View Article : Google Scholar : PubMed/NCBI
15	Kalluri R and Zeisberg M: Fibroblasts in cancer. Nat Rev Cancer. 6:392–401. 2006. View Article : Google Scholar : PubMed/NCBI
16	Subramaniam KS, Tham ST, Mohamed Z, Woo YL, Mat Adenan NA and Chung I: Cancer-associated fibroblasts promote proliferation of endometrial cancer cells. PLoS One. 8:e689232013. View Article : Google Scholar : PubMed/NCBI
17	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
18	Jain P, Worthylake RA and Alahari SK: Quantitative analysis of random migration of cells using time-lapse video microscopy. J Vis Exp. e35852012.PubMed/NCBI
19	Vega FM, Fruhwirth G, Ng T and Ridley AJ: RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. J Cell Biol. 193:655–665. 2011. View Article : Google Scholar : PubMed/NCBI
20	Baghban R, Roshangar L, Jahanban-Esfahlan R, Seidi K, Ebrahimi-Kalan A, Jaymand M, Kolahian S, Javaheri T and Zare P: Tumor microenvironment complexity and therapeutic implications at a glance. Cell Commun Signal. 18:592020. View Article : Google Scholar : PubMed/NCBI
21	Bussard KM, Mutkus L, Stumpf K, Gomez-Manzano C and Marini FC: Tumor-associated stromal cells as key contributors to the tumor microenvironment. Breast Cancer Res. 18:842016. View Article : Google Scholar : PubMed/NCBI
22	Orimo A, Tomioka Y, Shimizu Y, Sato M, Oigawa S, Kamata K, Nogi Y, Inoue S, Takahashi M, Hata T and Muramatsu M: Cancer-associated myofibroblasts possess various factors to promote endometrial tumor progression. Clin Cancer Res. 7:3097–3105. 2001.PubMed/NCBI
23	Teng F, Tian WY, Wang YM, Zhang YF, Guo F, Zhao J, Gao C and Xue FX: Cancer-associated fibroblasts promote the progression of endometrial cancer via the SDF-1/CXCR4 axis. J Hematol Oncol. 9:82016. View Article : Google Scholar : PubMed/NCBI
24	Neuzillet C, Tijeras-Raballand A, Ragulan C, Cros J, Patil Y, Martinet M, Erkan M, Kleeff J, Wilson J, Apte M, et al: Inter- and intra-tumoural heterogeneity in cancer-associated fibroblasts of human pancreatic ductal adenocarcinoma. J Pathol. 248:51–65. 2019. View Article : Google Scholar : PubMed/NCBI
25	Costa A, Kieffer Y, Scholer-Dahirel A, Pelon F, Bourachot B, Cardon M, Sirven P, Magagna I, Fuhrmann L, Bernard C, et al: Fibroblast heterogeneity and immunosuppressive environment in human breast cancer. Cancer Cell. 33:463–479.e10. 2018. View Article : Google Scholar : PubMed/NCBI
26	Subramaniam KS, Omar IS, Kwong SC, Mohamed Z, Woo YL, Mat Adenan NA and Chung I: Cancer-associated fibroblasts promote endometrial cancer growth via activation of interleukin-6/STAT-3/c-Myc pathway. Am J Cancer Res. 6:200–213. 2016.PubMed/NCBI
27	Arnold JT, Lessey BA, Seppälä M and Kaufman DG: Effect of normal endometrial stroma on growth and differentiation in Ishikawa endometrial adenocarcinoma cells. Cancer Res. 62:79–88. 2002.PubMed/NCBI
28	Shi M, Zhang H, Li M, Xue J, Fu Y, Yan L and Zhao X: Normal endometrial stromal cells regulate survival and apoptosis signaling through PI3K/AKt/Survivin pathway in endometrial adenocarcinoma cells in vitro. Gynecol Oncol. 123:387–392. 2011. View Article : Google Scholar : PubMed/NCBI
29	Luo N, Guan Q, Zheng L, Qu X, Dai H and Cheng Z: Estrogen-mediated activation of fibroblasts and its effects on the fibroid cell proliferation. Transl Res. 163:232–241. 2014. View Article : Google Scholar : PubMed/NCBI
30	Pineda MJ, Lu Z, Cao D and Kim JJ: Influence of cancer-associated endometrial stromal cells on hormone-driven endometrial tumor growth. Horm Cancer. 6:131–141. 2015. View Article : Google Scholar : PubMed/NCBI
31	Yang B, Chen R, Liang X, Shi J, Wu X, Zhang Z and Chen X: Estrogen enhances endometrial cancer cells proliferation by upregulation of prohibitin. J Cancer. 10:1616–1621. 2019. View Article : Google Scholar : PubMed/NCBI
32	Kim SA, Lee EK and Kuh HJ: Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial-mesenchymal transition in vitro. Exp Cell Res. 335:187–196. 2015. View Article : Google Scholar : PubMed/NCBI
33	Yu Y, Xiao CH, Tan LD, Wang QS, Li XQ and Feng YM: Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling. Br J Cancer. 110:724–732. 2014. View Article : Google Scholar : PubMed/NCBI
34	Baulida J: Epithelial-to-mesenchymal transition transcription factors in cancer-associated fibroblasts. Mol Oncol. 11:847–859. 2017. View Article : Google Scholar : PubMed/NCBI
35	Gaggioli C: Collective invasion of carcinoma cells: When the fibroblasts take the lead. Cell Adh Migr. 2:45–47. 2008. View Article : Google Scholar : PubMed/NCBI
36	Däster S, Amatruda N, Calabrese D, Ivanek R, Turrini E, Droeser RA, Zajac P, Fimognari C, Spagnoli GC, Iezzi G, et al: Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment. Oncotarget. 8:1725–1736. 2017. View Article : Google Scholar : PubMed/NCBI
37	Riffle S and Hegde RS: Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids. J Exp Clin Cancer Res. 36:1022017. View Article : Google Scholar : PubMed/NCBI
38	Zanoni M, Piccinini F, Arienti C, Zamagni A, Santi S, Polico R, Bevilacqua A and Tesei A: 3D tumor spheroid models for in vitro therapeutic screening: A systematic approach to enhance the biological relevance of data obtained. Sci Rep. 6:191032016. View Article : Google Scholar : PubMed/NCBI
39	Choe C, Shin YS, Kim SH, Jeon MJ, Choi SJ, Lee J and Kim J: Tumor-stromal interactions with direct cell contacts enhance motility of non-small cell lung cancer cells through the hedgehog signaling pathway. Anticancer Res. 33:3715–3723. 2013.PubMed/NCBI
40	Henriksson ML, Edin S, Dahlin AM, Oldenborg PA, Öberg Å, Van Guelpen B, Rutegård J, Stenling R and Palmqvist R: Colorectal cancer cells activate adjacent fibroblasts resulting in FGF1/FGFR3 signaling and increased invasion. Am J Pathol. 178:1387–1394. 2011. View Article : Google Scholar : PubMed/NCBI
41	Angelucci C, Lama G, Proietti G, Fabbri C, Masetti R, Sica S and Maulucci G: Breast cancer cells and fibroblasts in co-culture: Reciprocal influences on cell adhesion, membrane fluidity and migration. Ital J Anat Embryol. 116:122011.
42	Wolf K, Wu YI, Liu Y, Geiger J, Tam E, Overall C, Stack MS and Friedl P: Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat Cell Biol. 9:893–904. 2007. View Article : Google Scholar : PubMed/NCBI
43	Djamgoz MB, Coombes RC and Schwab A: Ion transport and cancer: From initiation to metastasis. Philos Trans R Soc Lond B Biol Sci. 369:201300922014. View Article : Google Scholar : PubMed/NCBI
44	MacDonald IC, Groom AC and Chambers AF: Cancer spread and micrometastasis development: Quantitative approaches for in vivo models. Bioessays. 24:885–893. 2002. View Article : Google Scholar : PubMed/NCBI
45	Spiering D and Hodgson L: Dynamics of the Rho-family small GTPases in actin regulation and motility. Cell Adh Migr. 5:170–180. 2011. View Article : Google Scholar : PubMed/NCBI
46	Liu J, Gao HY and Wang XF: The role of the Rho/ROCK signaling pathway in inhibiting axonal regeneration in the central nervous system. Neural Regen Res. 10:1892–1896. 2015. View Article : Google Scholar : PubMed/NCBI
47	Chiba Y, Goto K, Momata M, Kobayashi T and Misawa M: Induction of RhoA gene expression by interleukin-4 in cultured human bronchial smooth muscle cells. J Smooth Muscle Res. 46:217–224. 2010. View Article : Google Scholar : PubMed/NCBI
48	Chiba Y, Nakazawa S, Todoroki M, Shinozaki K, Sakai H and Misawa M: Interleukin-13 augments bronchial smooth muscle contractility with an up-regulation of RhoA protein. Am J Respir Cell Mol Biol. 40:159–167. 2009. View Article : Google Scholar : PubMed/NCBI
49	Villar-Cheda B, Dominguez-Meijide A, Joglar B, Rodriguez-Perez AI, Guerra MJ and Labandeira-Garcia JL: Involvement of microglial RhoA/Rho-kinase pathway activation in the dopaminergic neuron death. Role of angiotensin via angiotensin type 1 receptors. Neurobiol Dis. 47:268–279. 2012. View Article : Google Scholar : PubMed/NCBI
50	Etienne-Manneville S and Hall A: Rho GTPases in cell biology. Nature. 420:629–635. 2002. View Article : Google Scholar : PubMed/NCBI
51	Yoneda A, Multhaupt HA and Couchman JR: The Rho kinases I and II regulate different aspects of myosin II activity. J Cell Biol. 170:443–453. 2005. View Article : Google Scholar : PubMed/NCBI
52	Wang Y, Zheng XR, Riddick N, Bryden M, Baur W, Zhang X and Surks HK: ROCK isoform regulation of myosin phosphatase and contractility in vascular smooth muscle cells. Circ Res. 104:531–540. 2009. View Article : Google Scholar : PubMed/NCBI
53	Matsubara M and Bissell MJ: Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context. Oncotarget. 7:31602–31622. 2016. View Article : Google Scholar : PubMed/NCBI
54	Anderson S, DiCesare L, Tan I, Leung T and SundarRaj N: Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts. Exp Cell Res. 298:574–583. 2004. View Article : Google Scholar : PubMed/NCBI
55	Kolega J: Cytoplasmic dynamics of myosin IIA and IIB: Spatial ‘sorting’ of isoforms in locomoting cells. J Cell Sci. 111:2085–2095. 1998. View Article : Google Scholar : PubMed/NCBI
56	Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M and Narumiya S: Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 57:976–983. 2000.PubMed/NCBI
57	Tanihara H, Inoue T, Yamamoto T, Kuwayama Y, Abe H and Araie M; K-115 Clinical Study Group, : Phase 1 clinical trials of a selective Rho kinase inhibitor, K-115. JAMA Ophthalmol. 131:1288–1295. 2013. View Article : Google Scholar : PubMed/NCBI
58	Okumura N, Kinoshita S and Koizumi N: Application of Rho kinase inhibitors for the treatment of corneal endothelial diseases. J Ophthalmol. 2017:26469042017. View Article : Google Scholar : PubMed/NCBI
59	Mueller BK, Mack H and Teusch N: Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov. 4:387–398. 2005. View Article : Google Scholar : PubMed/NCBI
60	Garnock-Jones KP: Ripasudil: First global approval. Drugs. 74:2211–2215. 2014. View Article : Google Scholar : PubMed/NCBI
61	Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF, Harrington K and Sahai E: Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 9:1392–1400. 2007. View Article : Google Scholar : PubMed/NCBI
62	Rodriguez-Hernandez I, Cantelli G, Bruce F and Sanz-Moreno V: Rho, ROCK and actomyosin contractility in metastasis as drug targets. F1000Res 5 (F1000 Faculty Rev). 7832016. View Article : Google Scholar
63	Liu S, Goldstein RH, Scepansky EM and Rosenblatt M: Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone. Cancer Res. 69:8742–8751. 2009. View Article : Google Scholar : PubMed/NCBI