Abstract
Aim:
Recent evidence shows that localization of mRNAs and their protein products at cellular protrusions plays a decisive function in the metastasis of cancer cells. The aim of this study was to identify the variety of proteins encoded by protrusion-localized mRNAs and their roles in the metastasis and invasion of liver cancer cells.
Methods:
Highly metastatic hepatocellular carcinoma cell line HCCLM3 and non-metastatic hepatocellular carcinoma cell line SMMC-7721 were examined. Cell protrusions (Ps) were separated from cell bodies (CB) using a Boyden chamber assay; total mRNA population in CB and Ps fractions was analyzed using high-throughput direct RNA sequencing. The localization of STAT3 mRNA and protein at Ps was confirmed using RT-qPCR, RNA FISH, and immunofluorescence assays. Cell migration capacity and invasiveness of HCCLM3 cells were evaluated using MTT, wound healing migration and in vitro invasion assays. The interaction between Stat3 and growth factor receptors was explored with co-immunoprecipitation assays.
Results:
In HCCLM3 cells, 793 mRNAs were identified as being localized in the Ps fraction according to a cut-off value (Ps/CB ratio) >1.6. The Ps-localized mRNAs could be divided into 4 functional groups, and were all closely related to the invasive and metastatic properties. STAT3 mRNA accumulated in the Ps of HCCLM3 cells compared with non-metastatic SMMC-7721 cells. Treatment of HCCLM3 cells with siRNAs against STAT3 mRNA drastically decreased the cell migration and invasion. Moreover, Ps-localized Stat3 was found to interact with pseudopod-enriched platelet-derived growth factor receptor tyrosine kinase (PDGFRTK) in a growth factor-dependent manner.
Conclusion:
This study reveals STAT3 mRNA localization at the Ps of metastatic hepatocellular carcinoma HCCLM3 cells by combining application of genome-wide and gene specific description and functional analysis.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Yuen MF, Lai CL . Serological markers of liver cancer. Best Pract Res Clin Gastroenterol 2005; 19: 91–9.
Kew MC . Hepatocellular cancer. A century of progress. Clin Liver Dis 2000; 4: 257–68.
El-Serag HB, Mason AC, Key C . Trends in survival of patients with hepatocellular carcinoma between 1977 and 1996 in the United States. Hepatology 2001; 33: 62–5.
Chambers AF, Groom AC, MacDonald IC . Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2002; 2: 563–72.
Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science 2003; 302: 1704–9.
Schoumacher M, Goldman RD, Louvard D, Vignjevic DM . Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia. J Cell Biol 2010; 189: 541–56.
Wang Y, Ding SJ, Wang W, Jacobs JM, Qian WJ, Moore RJ, et al. Profiling signaling polarityin chemotactic cells. Proc Natl Acad Sci U S A 2007; 104: 8328–33.
Liao G, Mingle L, Van De Water L, Liu G . Control of cell migration through mRNA localization and local translation. Wiley Interdiscip Rev RNA 2015; 6: 1–15.
Mardarheh FK, Paul A, Kumper S, Sadok A, Paterson H, Mccarthy A, et al. Global analysis of mRNA, translation, and protein localization: Local translation is a key regulator of cell protrusions. Dev Cell 2015; 35: 344–57.
Mili S, Macara IG . RNA localization and polarity: from A(PC) to Z(BP). Trends Cell Biol 2009; 19: 156–64.
Shankar J, Messenberg A, Chan J, Underhill TM, Foster LJ, Nabi IR . Pseudopodial actin dynamics control epithelial-mesenchymal transition in metastatic cancer cells. Cancer Res 2010; 70: 3780–90.
Jia Z, Barbier L, Stuart H, Amraei M, Pelech S, Dennis JW, et al. Tumor cell pseudopodial protrusions. Localized signaling domains coordinating cytoskeleton remodeling, cell adhesion, glycolysis, RNA translocation, and protein translation. J Biol Chem 2005; 280: 30564–73.
Beckner ME, Chen X, An J, Day BW, Pollack IF . Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies. Lab Invest 2005; 85: 316–27.
Stuart HC, Jia Z, Messenberg A, Joshi B, Underhill TM, Moukhles H, et al. Localized Rho GTPase activation regulates RNA dynamics and compartmentalization in tumor cell protrusions. J Biol Chem 2008; 283: 34785–95.
Ozsolak F, Goren A, Gymrek M, Guttman M, Regev A, Bernstein BE, et al. Digital transcriptome profiling from attomole-level RNA samples. Genome Res 2010; 20: 519–25.
Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, et al. Direct RNA sequencing. Nature 2009; 461: 814–8.
Thomsen R, Lade Nielsen A . A Boyden chamber-based method for characterization of astrocyte protrusion localized RNA and protein. Glia 2011; 59: 1782–92.
Smit M, Leng J, Klemke RL . Assay for neurite outgrowth quantification. Biotechniques 2003; 35: 254–6.
Chartrand P, Singer RH, Long RM . Sensitive and highresolution detection of RNA in situ. Methods Enzymol 2000; 318: 493–506.
Li Y, Tang Y, Ye L, Liu B, Liu K, Chen J, et al. Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray. J Cancer Res Clin Oncol 2003; 129: 43–51.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B . Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008; 5: 621–8.
Jakobsen KR, Sørensen E, Brøndum KK, Daugaard TF, Thomsen R, Nielsen AL . Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells. J Mol Signal 2013; 8: 9.
Teng Y, Ross JL, Cowell JK . The involvement of JAK-STAT3 in cell motility, invasion, and metastasis. JAKSTAT 2014; 3: e 28086.
Zhang CH, Guo FL, Xu GL, Jia WD, Ge YS . STAT3 activation mediates epithelial-to-mesenchymal transition in human hepatocellular carcinoma cells. Hepatogastroenterology 2014; 61: 1082–9.
Turkson J, Jove R . STAT proteins: novel molecular targets for cancer drug discovery. Oncogene 2000; 19: 6613–26.
Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, et al. STAT3 as an oncogene. Cell 1999; 98: 295–303.
Turkson J . STAT proteins as novel targets for cancer drug discovery. Expert Opin Ther Targets 2004; 8: 409–22.
Betsholtz C . Insight into the physiological functions of PDGF through genetic studies in mice. Cytokine Growth Factor Rev 2004; 15: 215–28.
Heldin CH, Westermark B . Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999; 79: 1283–316.
Vignais ML, Sadowski HB, Watling D, Rogers NC, Gliman M . Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins. Mol Cell Biol 1996; 16: 1759–69.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (21302108), Shenzhen Municipal Government (JCYJ20140827150509058, 20150113A0410006, JSGG20141016150327538, and SZSTI CXB201104210013A) and Zhejiang Province Natural Science Foundation of China (LY16C070002).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Supplementary information is available at the Acta Pharmacologica Sinica's website.
Supplementary information
Supplementary Methods
Supplementary methods (DOC 39 kb)
Supplementary Information Table S1
The total number of mRNAs with RPKM values larger than 5 in both Ps and CB. (DOC 7363 kb)
Supplementary Information Table S2
793 mRNAs identified to be localized in the cell protrusions. (DOC 622 kb)
Supplementary Information Table S3
Top 50 protrusion-localized mRNAs in SMMC-7721 cells. (DOC 31 kb)
Rights and permissions
About this article
Cite this article
Liu, Yh., Jin, Jl., Wang, Yz. et al. Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro. Acta Pharmacol Sin 37, 805–813 (2016). https://doi.org/10.1038/aps.2015.166
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/aps.2015.166
Keywords
This article is cited by
-
Folylpolyglutamate synthetase mRNA G-quadruplexes regulate its cell protrusion localization and enhance a cancer cell invasive phenotype upon folate repletion
BMC Biology (2023)
-
FMRP regulates STAT3 mRNA localization to cellular protrusions and local translation to promote hepatocellular carcinoma metastasis
Communications Biology (2021)
-
Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging
Nature Communications (2018)