Cancer Res Treat.  2019 Jan;51(1):391-401. 10.4143/crt.2018.103.

NFATC3–PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines

• Jang JE
• Kim HP
• Han SW
• Jang H
• Lee SH
• Song SH
• Bang D
• Kim TY

• KMID: 2437629
• DOI: http://doi.org/10.4143/crt.2018.103

Abstract

PURPOSE
This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer (CRC) lines.
MATERIALS AND METHODS
We performed paired-end RNA sequencing of 28 CRC cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed.
RESULTS
One thousand three hundred eighty FT candidates were detected through bioinformatics filtering. We selected six candidate FTs, including four inter-chromosomal and two intrachromosomal FTs and each FT was found in at least one of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in two. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation.
CONCLUSION
These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

Keyword

Colorectal neoplasms; Fusion transcript; RNA-sequencing; NFATC3; PLA2G15

MeSH Terms

Cadherins
Cell Line*
Cell Movement
Cell Proliferation
Claudin-1
Colorectal Neoplasms*
Computational Biology
Fibronectins
Humans
RNA*
RNA, Messenger
RNA, Small Interfering
Sequence Analysis, RNA*
Vimentin
Cadherins
Claudin-1
Fibronectins
RNA
RNA, Messenger
RNA, Small Interfering
Vimentin

Figure

• 

  Fig. 1. A schematic of the fusion candidate filtering process.

• 

  Fig. 2. Identification of 6 fusion transcripts (FTs) in the colorectal cancer (CRC) cell lines. (A) Presence of PPP2CA–SKP1, CTNNBIP1–CLSTN1, AKAP13–PDE8A, NFATC3–PLA2G15, FZD2–RPS24, and KRT8–PKM2 FTs was confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) in CRC cell lines. (B) The NFATC3–PLA2G15 FT was detected in colon cancer tissues by RT-PCR. N, normal tissue; T, tumor tissue. (C) The NFATC3–PLA2G15 FT was validated in the colo-320 cell line using Sanger-sequencing. The FT breakpoint was passed from exon 9 of NFATC3 to exon 2 of PLA2G15. (D) NFATC3–PLA2G15 FT mRNA expression was observed in the colo-320 fusion-positive cell line and the colo-205 fusion-negative cell line by RT-PCR. Forward primers targeted NFATC3 exon 4, 6, and 8 (black arrows) and reverse primers targeted PLA2G15 exon 2 (red arrow). Distilled water (DW) was used for the negative control.

• 

  Fig. 3. The NFATC3–PLA2G15 fusion transcript is involved in regulation of epithelial–mesenchymal transition. (A) The colo320 cell line was transfected with NFATC3–PLA2G15 fusion transcript siRNA for 48 hours. (B, C) mRNA expression of mesenchymal markers (vimentin, twist, and fibronectin) and epithelial markers (E-cadherin, claudin-1, and FOXC2) were detected by quantitative real-time polymerase chain reaction (*p < 0.05). (D) Western blot analysis revealed E-cadherin, vimentin, and β-actin protein expression in the colo-320 cell line after treatment with the NFATC3–PLA2G15 fusion siRNA for 48 hours.

• 

  Fig. 4. The NFATC3–PLA2G15 fusion transcript affects invasiveness and has anchorage-independent abilities. (A, B) We conducted a 48-hour Matrigel invasion assay with colo-320 cells that were stably transfected with NFATC3–PLA2G15 siRNA and control cells treated with siRNA control (*p < 0.05). (C, D) We conducted a 2-week soft-agar independentanchorage assay on siRNA-treated colo-320 fusion-positive cell line and control cells treated with siRNA control (*p < 0.05).

• 

  Fig. 5. The NFATC3–PLA2G15 fusion transcript effected the regulation of cell number. (A, B) The NFATC3–PLA2G15 fusion transcript siRNA was transfected into the colo-320 cell line and cell confluence was observed at 48 hours. Cells were observed by microscopy after siRNA treatment in the colo-320 fusion positive cell line. (C, E) Cell counting assay. A cell counting assay was conducted after NFATC3–PLA2G15 fusion siRNA treatment in the colo-320 fusion positive cell line and SNU-1033 fusion negative cell line in a time-dependent manner. Viable cells were counted every 3 days (*p < 0.05). (D) The colo-320 cell line was transfected with NFATC3–PLA2G15 fusion transcript siRNA for 48 hours. (F) Western-blotting analysis revealed cyclin D, and p27 protein expression in the colo-320 and SNU-1033 cell lines after treatment with the NFATC3–PLA2G15 fusion siRNA for 48 hours.

Reference

References

1. Siegel R, Desantis C, Jemal A. Colorectal cancer statistics, 2014. CA Cancer J Clin. 2014; 64:104–17.

2. Allen TM. Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer. 2002; 2:750–63.

3. Maher CA, Palanisamy N, Brenner JC, Cao X, Kalyana-Sundaram S, Luo S, et al. Chimeric transcript discovery by paired-end transcriptome sequencing. Proc Natl Acad Sci U S A. 2009; 106:12353–8.

4. Maher CA, Kumar-Sinha C, Cao X, Kalyana-Sundaram S, Han B, Jing X, et al. Transcriptome sequencing to detect gene fusions in cancer. Nature. 2009; 458:97–101.

5. Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011; 12:87–98.

6. Kim M, Lee KH, Yoon SW, Kim BS, Chun J, Yi H. Analytical tools and databases for metagenomics in the next-generation sequencing era. Genomics Inform. 2013; 11:102–13.

7. Griswold IJ, MacPartlin M, Bumm T, Goss VL, O'Hare T, Lee KA, et al. Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib. Mol Cell Biol. 2006; 26:6082–93.

8. Bunting SF, Nussenzweig A. End-joining, translocations and cancer. Nat Rev Cancer. 2013; 13:443–54.

9. Mitelman F, Johansson B, Mertens F. The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer. 2007; 7:233–45.

10. Shaw AT, Hsu PP, Awad MM, Engelman JA. Tyrosine kinase gene rearrangements in epithelial malignancies. Nat Rev Cancer. 2013; 13:772–87.

11. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007; 448:561–6.

12. Antoniu SA. Crizotinib for EML4-ALK positive lung adenocarcinoma: a hope for the advanced disease? Evaluation of Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363(18):1693-703. Expert Opin Ther Targets. 2011; 15:351–3.

13. Bass AJ, Lawrence MS, Brace LE, Ramos AH, Drier Y, Cibulskis K, et al. Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion. Nat Genet. 2011; 43:964–8.

14. Seshagiri S, Stawiski EW, Durinck S, Modrusan Z, Storm EE, Conboy CB, et al. Recurrent R-spondin fusions in colon cancer. Nature. 2012; 488:660–4.

15. Nome T, Hoff AM, Bakken AC, Rognum TO, Nesbakken A, Skotheim RI. High frequency of fusion transcripts involving TCF7L2 in colorectal cancer: novel fusion partner and splice variants. PLoS One. 2014; 9:e91264.

16. Nome T, Thomassen GO, Bruun J, Ahlquist T, Bakken AC, Hoff AM, et al. Common fusion transcripts identified in colorectal cancer cell lines by high-throughput RNA sequencing. Transl Oncol. 2013; 6:546–53.

17. Lovf M, Nome T, Bruun J, Eknaes M, Bakken AC, Mpindi JP, et al. A novel transcript, VNN1-AB, as a biomarker for colorectal cancer. Int J Cancer. 2014; 135:2077–84.

18. Ku JL, Park JG. Biology of SNU cell lines. Cancer Res Treat. 2005; 37:1–19.

19. Li X, Zhu L, Yang A, Lin J, Tang F, Jin S, et al. Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos. Cell Stem Cell. 2011; 8:46–58.

20. Sengupta S, Jana S, Biswas S, Mandal PK, Bhattacharyya A. Cooperative involvement of NFAT and SnoN mediates transforming growth factor-beta (TGF-beta) induced EMT in metastatic breast cancer (MDA-MB 231) cells. Clin Exp Metastasis. 2013; 30:1019–31.

21. Wen H, Li Y, Malek SN, Kim YC, Xu J, Chen P, et al. New fusion transcripts identified in normal karyotype acute myeloid leukemia. PLoS One. 2012; 7:e51203.

22. Bond J, Tran Quang C, Hypolite G, Belhocine M, Bergon A, Cordonnier G, et al. Novel intergenically spliced chimera, NFATC3-PLA2G15, is associated with aggressive T-ALL biology and outcome. Mol Cancer Res. 2018; 16:470–5.

23. Hoey T, Sun YL, Williamson K, Xu X. Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins. Immunity. 1995; 2:461–72.

24. Oh-hora M, Rao A. The calcium/NFAT pathway: role in development and function of regulatory T cells. Microbes Infect. 2009; 11:612–9.

25. Branden LJ, Mohamed AJ, Smith CI. A peptide nucleic acidnuclear localization signal fusion that mediates nuclear transport of DNA. Nat Biotechnol. 1999; 17:784–7.

26. MacDonnell SM, Weisser-Thomas J, Kubo H, Hanscome M, Liu Q, Jaleel N, et al. CaMKII negatively regulates calcineurin-NFAT signaling in cardiac myocytes. Circ Res. 2009; 105:316–25.

27. Abe A, Kelly R, Shayman JA. The measurement of lysosomal phospholipase A2 activity in plasma. J Lipid Res. 2010; 51:2464–70.

28. Park JB, Lee CS, Jang JH, Ghim J, Kim YJ, You S, et al. Phospholipase signalling networks in cancer. Nat Rev Cancer. 2012; 12:782–92.

29. Nilsson LM, Sun ZW, Nilsson J, Nordstrom I, Chen YW, Molkentin JD, et al. Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation. Am J Physiol Cell Physiol. 2007; 292:C1167–78.

30. Medico E, Russo M, Picco G, Cancelliere C, Valtorta E, Corti G, et al. The molecular landscape of colorectal cancer cell lines unveils clinically actionable kinase targets. Nat Commun. 2015; 6:7002.