Tuomela Johanna M, Sandholm Jouko A, Kaakinen Mika, Hayden Katherine L, Haapasaari Kirsi-Maria, Jukkola-Vuorinen Arja, Kauppila Joonas H, Lehenkari Petri P, Harris Kevin W, Graves David E, Selander Katri S
Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.
Breast Cancer Res Treat. 2016 Jan;155(2):261-71. doi: 10.1007/s10549-016-3683-5. Epub 2016 Jan 18.
Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.
Toll样受体9（TLR9）是一种在癌症中广泛表达的细胞DNA受体。我们之前表明，合成的和自身来源的DNA片段在体外可诱导TLR9介导的乳腺癌细胞侵袭。我们在此研究了两种耐核酸酶的DNA片段的侵袭作用，一种是9聚体发夹结构，另一种是基于人类端粒序列的G-四链体DNA，二者均具有天然磷酸二酯主链。通过免疫荧光研究DNA的细胞摄取，用基质胶实验研究侵袭，用qPCR和蛋白质印迹研究mRNA和蛋白质表达，用酶谱分析研究蛋白酶活性。通过小干扰RNA抑制TLR9表达。尽管两种DNA均诱导TLR9介导的促侵袭性mRNA表达变化，但只有端粒G-四链体DNA显著增加细胞侵袭。这一作用被GM6001和抑肽酶抑制，提示由基质金属蛋白酶和丝氨酸蛋白酶介导。此外，与乳腺癌中存在的一种抗菌肽LL-37复合，可增加9聚体发夹结构和G-四链体DNA进入癌细胞的摄取。然而，与单独的DNA处理相比，DNA/LL-37复合物降低了侵袭。还对从新辅助化疗治疗的乳腺肿瘤中分离的DNA片段进行了侵袭研究。这种DNA在体外也诱导乳腺癌细胞侵袭。与合成DNA一样，将新辅助肿瘤来源的DNA与LL-37复合可降低这种侵袭作用。我们得出结论，9聚体发夹结构和G-四链体DNA片段是耐核酸酶的DNA结构，可作为诱导侵袭的TLR9配体。它们的细胞摄取和侵袭作用通过LL-37调节。尽管这种结构可能存在于化疗治疗的肿瘤中，但这一发现的临床意义需要进一步研究。
