Epigenetic changes and nuclear factor-κB activation, but not microRNA-224, downregulate Raf-1 kinase inhibitor protein in triple-negative breast cancer SUM 159 cells

Labbozzetta,Manuela; Poma,Paola; Vivona,Nicoletta; Gulino,Alessandro; D'Alessandro,Natale; Notarbartolo,Monica

doi:10.3892/ol.2015.3787

Epigenetic changes and nuclear factor-κB activation, but not microRNA-224, downregulate Raf-1 kinase inhibitor protein in triple-negative breast cancer SUM 159 cells

Authors:
- Manuela Labbozzetta
- Paola Poma
- Nicoletta Vivona
- Alessandro Gulino
- Natale D'Alessandro
- Monica Notarbartolo
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Affiliations: Pharmacology Unit, Department of Health Sciences and Mother and Child Care ‘G. D'Alessandro’, University of Palermo, Palermo 90127, Italy, Tumor Immunology Unit, Department of Health Sciences and Mother and Child Care ‘G. D'Alessandro’, University of Palermo, Palermo 90127, Italy
Published online on: October 9, 2015 https://doi.org/10.3892/ol.2015.3787
Pages: 3807-3815

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Abstract

Raf-1 kinase inhibitor protein (RKIP) is a tumor suppressor and metastasis inhibitor, which enhances drug-induced apoptosis of cancer cells. Downregulation of RKIP may be significant in the biology of highly aggressive and drug‑resistant tumors, for example triple‑negative breast cancers (TNBCs). Potential causes for the low levels of RKIP expressed by SUM 159 TNBC cells were investigated in the present study. Bisulphite modification, methylation specific-polymerase chain reaction (PCR) and a TransAM NF-κB assay were performed and the results suggested that various mechanisms, including methylation of the gene promoter, histone deacetylation and nuclear factor‑κB (NF‑κB) activation, but not targeting by microRNA‑224 (miR/miRNA‑224), as determined by transfection of pre‑miR‑224 miRNA precursor or anti‑miR‑224 miRNA inhibitor, may downregulate RKIP in these cells. Furthermore, reverse transcription‑quantitative PCR, western blotting, 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulphophenyl)‑2H‑tetrazolium cell growth assay and flow cytometry revealed that in SUM 159 cells, the demethylating agent 5‑aza‑2'‑deoxycytidine (5‑AZA), the histone deacetylase inhibitor trichostatin A (TSA) and the NF‑κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) enhanced RKIP expression and resulted in significant cell growth inhibition and induction of apoptosis. 5‑AZA and TSA mainly produced additive antitumor effects, while the combination of DHMEQ and TSA exhibited significant synergy in cell growth inhibition and induction of apoptosis assays. Increasing evidence that aberrant activation of NF‑κB signaling is a frequent characteristic of TNBC highlights the fact that this transcription factor may be a useful target for treatment of such tumors. In addition to DHMEQ, proteasome inhibitors may also represent valuable therapeutic resources in this context. Notably, proteasome inhibitors, in addition to the inhibition of NF‑κB activation, may also restore RKIP levels by inhibiting proteasome degradation of the ubiquitinated protein. The current results contribute to the understanding of the molecular mechanisms of RKIP downregulation in TNBC and suggest possible novel therapeutic approaches for the treatment of these types of cancer.

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1	Schmadeka R, Harmon BE and Singh M: Triple-negative breast carcinoma: Current and emerging concepts. Am J Clin Pathol. 141:462–477. 2014. View Article : Google Scholar : PubMed/NCBI
2	Abramson VG, Lehmann BD, Ballinger TJ and Pietenpol JA: Subtyping of triple-negative breast cancer: Implications for therapy. Cancer. 121:8–16. 2014. View Article : Google Scholar : PubMed/NCBI
3	Minn AJ, Bevilacqua E, Yun J and Rosner MR: Identification of novel metastasis suppressor signaling pathways for breast cancer. Cell Cycle. 11:2452–2457. 2012. View Article : Google Scholar : PubMed/NCBI
4	Hao C, Wei S, Tong Z, Li S, Shi Y, Wang X and Zhu ZH: The effects of RKIP gene expression on the biological characteristics of human triple-negative breast cancer cells in vitro. Tumour Biol. 33:1159–1167. 2012. View Article : Google Scholar : PubMed/NCBI
5	Al-Mulla F, Bitar MS, Thiery JP, Zea TT, Chatterjee D, Bennett L, Park S, Edwards J and Yeung KC: Clinical implications for loss or diminution of Raf-1 kinase inhibitory protein and its phosphorylated form in ductal breast cancer. Am J Cancer Res. 3:446–464. 2013.PubMed/NCBI
6	Odabaei G, Chatterjee D, Jazirehi AR, Goodglick L, Yeung K and Bonavida B: Raf-1 kinase inhibitor protein: Structure, function, regulation of cell signaling and pivotal role in apoptosis. Adv Cancer Res. 91:169–200. 2004. View Article : Google Scholar : PubMed/NCBI
7	Tang H, Park S, Sun SC, Trumbly R, Ren G, Tsung E and Yeung KC: RKIP inhibits NF-kappaB in cancer cells by regulating upstream signaling components of the IkappaB kinase complex. FEBS Lett. 584:662–668. 2010. View Article : Google Scholar : PubMed/NCBI
8	Yousuf S, Duan M, Moen EL, Cross-Knorr S, Brilliant K, Bonavida B, LaValle T, Yeung KC, Al-Mulla F, Chin E and Chatterjee D: Raf kinase inhibitor protein (RKIP) blocks signal transducer and activator of transcription 3 (STAT3) activation in breast and prostate cancer. PLoS One. 9:e924782014. View Article : Google Scholar : PubMed/NCBI
9	Eves EM, Shapiro P, Naik K, Klein UR, Trakul N and Rosner MR: Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint. Mol Cell. 23:561–574. 2006. View Article : Google Scholar : PubMed/NCBI
10	Klysik J, Theroux SJ, Sedivy JM, Moffit JS and Boekelheide K: Signaling crossroads: The function of Raf kinase inhibitory protein in cancer, the central nervous system and reproduction. Cell Signal. 20:1–9. 2008. View Article : Google Scholar : PubMed/NCBI
11	Poma P, Labbozzetta M, Vivona N, Porcasi R, D'Alessandro N and Notarbartolo M: Analysis of possible mechanisms accounting for raf-1 kinase inhibitor protein downregulation in hepatocellular carcinoma. OMICS. 16:579–588. 2012. View Article : Google Scholar : PubMed/NCBI
12	Jazirehi AR and Bonavida B: Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: Implications in chemosensitization and therapeutic intervention. Oncogene. 24:2121–2143. 2005. View Article : Google Scholar : PubMed/NCBI
13	Hsu YL, Chen CY, Lin IP, Tsai EM, Kuo PL and Hou MF: 4-Shogaol, an active constituent of dietary ginger, inhibits metastasis of MDA-MB-231 human breast adenocarcinoma cells by decreasing the repression of NF-κB/Snail on RKIP. J Agric Food Chem. 60:852–861. 2012. View Article : Google Scholar : PubMed/NCBI
14	Chen TC, Hsu YL, Tsai YC, Chang YW, Kuo PL and Chen YH: Gemifloxacin inhibits migration and invasion and induces mesenchymal-epithelial transition in human breast adenocarcinoma cells. J Mol Med (Berl). 92:53–64. 2014. View Article : Google Scholar : PubMed/NCBI
15	Ren G, Baritaki S, Marathe H, Feng J, Park S, Beach S, Bazeley PS, Beshir AB, Fenteany G, Mehra R, et al: Polycomb protein EZH2 regulates tumor invasion via the transcriptional repression of the metastasis suppressor RKIP in breast and prostate cancer. Cancer Res. 72:3091–3104. 2012. View Article : Google Scholar : PubMed/NCBI
16	Huang L, Dai T, Lin X, Zhao X, Chen X, Wang C, Li X, Shen H and Wang X: MicroRNA-224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells. Biochem Biophys Res Commun. 425:127–133. 2012. View Article : Google Scholar : PubMed/NCBI
17	Chou TC and Talalay P: Analysis of combined drug effects: A new look at a very old problem. Trends Pharmacol Sci. 4:450–454. 1983. View Article : Google Scholar
18	Florena AM, Tripodo C, Guarnotta C, Ingrao S, Porcasi R, Martorana A, Lo Bosco G, Cabibi D and Franco V: Associations between Notch-2, Akt-1 and HER2/neu expression in invasive human breast cancer: A tissue microarray immunophenotypic analysis on 98 patients. Pathobiology. 74:317–322. 2007. View Article : Google Scholar : PubMed/NCBI
19	Notarbartolo M, Giannitrapani L, Vivona N, Poma P, Labbozzetta M, Florena AM, Porcasi R, Muggeo VM, Sandonato L and Cervello M: Frequent alteration of the Yin Yang 1/Raf-1 kinase inhibitory protein ratio in hepatocellular carcinoma. OMICS. 15:267–272. 2011. View Article : Google Scholar : PubMed/NCBI
20	Neve RM, Chin K, Fridlyand J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, et al: A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 10:515–527. 2006. View Article : Google Scholar : PubMed/NCBI
21	Schuierer MM, Bataille F, Weiss TS, Hellerbrand C and Bosserhof AK: Raf kinase inhibitor protein is down-regulated in hepatocellular carcinoma. Oncol Rep. 16:451–456. 2006.PubMed/NCBI
22	Minoo P, Zlobec I, Baker K, Tornillo L, Terracciano L, Jass JR and Lugli A: Loss of raf-1 kinase inhibitor protein expression is associated with tumor progression and metastasis in colorectal cancer. Am J Clin Pathol. 127:820–827. 2007. View Article : Google Scholar : PubMed/NCBI
23	Al-Mulla F, Hagan S, Al-Ali W, Jacob SP, Behbehani AI, Bitar MS, Dallol A and Kolch W: Raf kinase inhibitor protein: Mechanism of loss of expression and association with genomic instability. J Clin Pathol. 61:524–529. 2008. View Article : Google Scholar : PubMed/NCBI
24	Li DX, Cai HY, Wang X, Feng YL and Cai SW: Promoter methylation of Raf kinase inhibitory protein: A significant prognostic indicator for patients with gastric adenocarcinoma. Exp Ther Med. 8:844–850. 2014.PubMed/NCBI
25	Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR, Barker M, Buchanan D, Young J and Jass JR: Extensive DNA methylation in normal colorectal mucosa in hyperplatic polyposis. Gut. 55:1467–1474. 2006. View Article : Google Scholar : PubMed/NCBI
26	Connolly R and Stearns R: Epigenetics as a therapeutic target in breast cancer. J Mammary Gland Biol Neoplasia. 17:191–204. 2012. View Article : Google Scholar : PubMed/NCBI
27	Wu K and Bonavida B: The activated NF-kappaB-Snail-RKIP circuitry in cancer regulates both the metastatic cascade and resistance to apoptosis by cytotoxic drugs. Crit Rev Immunol. 29:241–254. 2009. View Article : Google Scholar : PubMed/NCBI
28	Umezawa K: Possible role of peritoneal NF-κB in peripheral inflammation and cancer: Lessons from the inhibitor DHMEQ. Biomed Pharmacother. 65:252–259. 2011. View Article : Google Scholar : PubMed/NCBI
29	Poma P, Notarbartolo M, Labbozzetta M, Sanguedolce R, Alaimo A, Carina V, Maurici A, Cusimano A, Cervello M and D'Alessandro N: Antitumor effects of the novel NF-kappaB inhibitor dehydroxymethyl-epoxyquinomicin on human hepatic cancer cells: Analysis of synergy with cisplatin and of possible correlation with inhibition of pro-survival genes and IL-6 production. Int J Oncol. 28:923–930. 2006.PubMed/NCBI
30	Iorio MV and Croce CM: MicroRNAs in cancer: Small molecules with a huge impact. J Clin Oncol. 27:5848–5856. 2009. View Article : Google Scholar : PubMed/NCBI
31	Fabbri M and Calin GA: Epigenetics and miRNAs in human cancer. Adv Genet. 70:87–99. 2010. View Article : Google Scholar : PubMed/NCBI
32	Inguglia L, Notarbartolo M, Poma P, Labbozzetta M and D'Alessandro N: Induction of apoptosis and chemosensitization by the histone deacetylase inhibitor trichostatin A in hepatocellular carcinoma cells: Molecular analysis and RKIP levels. Forum Immun Dis Ther. 2:127–135. 2011. View Article : Google Scholar
33	Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG and Davidson NE: Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells. Cancer Res. 61:7025–7029. 2001.PubMed/NCBI
34	Zhai FX, Liu XF, Fan RF, Long ZJ, Fang ZG, Lu Y, Zheng YJ and Lin DJ: RUNX3 is involved in caspase-3-dependent apoptosis induced by a combination of 5-aza-CdR and TSA in leukaemia cell lines. J Cancer Res Clin Oncol. 138:439–449. 2012. View Article : Google Scholar : PubMed/NCBI
35	Sethi S, Sarkar FH, Ahmed Q, Bandyopadhyay S, Nahleh ZA, Semaan A, Sakr W, Munkarah A and Ali-Fehmi R: Molecular markers of epithelial-to-mesenchymal transition are associated with tumor aggressiveness in breast carcinoma. Transl Oncol. 4:222–226. 2011. View Article : Google Scholar : PubMed/NCBI
36	Piao HL, Yuan Y, Wang M, Sun Y, Liang H and Ma L: α-catenin acts as a tumour suppressor in E-cadherin-negative basal-like breast cancer by inhibiting NF-κB signalling. Nat Cell Biol. 16:245–254. 2014. View Article : Google Scholar : PubMed/NCBI
37	Walker EJ, Rosenberg SA, Wands JR and Kim M: Role of raf kinase inhibitor protein in hepatocellular carcinoma. For Immunopathol Dis Therap. 2:195–204. 2011. View Article : Google Scholar : PubMed/NCBI