Antisense inhibition of microRNA-21 and microRNA-221 in tumor-initiating stem-like cells modulates tumorigenesis, metastasis, and chemotherapy resistance in pancreatic cancer

Abstract

Our preliminary studies identified a small population side population (SP) cells in pancreatic cancer cells with stem cell-like properties, which were able to induce fast and aggressive tumor formation in nude mice. Gene expression analysis showed a significant difference in the expression of more than 1,300 genes in SP cells, among which a highly significant difference in microRNA expression of miR-21 and miR-221 between SP and NSP cells was identified. SP cells were identified and characterized by flow cytometry using Hoechst 33342 dye staining from a highly metastatic human pancreatic cancer cell line (L3.6pl). Antagomir transfection was performed using miRNA-21 and miRNA-221 antisense oligonucleotides (ASOs) and followed by detection of cell apoptosis, cell cycle progression, chemosensitivity, and invasion. Sorted SP cells from gemcitabine-resistant L3.6pl cells (L3.6plGres-SP) cells were orthotopically implanted in nude mice with or without miRNA-21 and miRNA-221 ASOs mono- and combination therapy. The administration of antagomir-21 and antagomir-221 significantly reduced the SP cell fraction, decreased SP cell differentiation, and downstream gene regulation, and thereby induced reduction of L3.6pl cell proliferation, invasion, and chemoresistance against gemcitabine and 5-Fluorouracil. Combination of ASOs therapy against miRNA-21 and miRNA-221 significantly inhibited primary tumor growth and metastasis compared to single antagomir treatment, especially, in L3.6plGres-SP-induced pancreatic tumor growth in vivo. These findings further indicate that the inhibition of miR-21 and miR-221 appear particularly suitable to target stem-like subpopulations and address their specific biological function to promote tumor progression in pancreatic cancer.

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Acknowledgments

The authors appreciate the preparation support of the project from Andrea Renner and Sevdije Gashi. We thank the technology support from Anneli Tischmacher. This research was supported by the FöFoLe Research Program (no. 570/548/636) of the University of Munich, Munich, Germany, SPP1190/2 “Tumor vessel interface” (BR 1614/8-2) of the German Research Society (DFG), and the German Research Society (DFG) grant BR 1614/7-1. YZ, LZ, QB, and YW were financially supported by LMU-CSC (The China Scholarship Council) scholarship. Animal experiment was performed in accordance with institutional and governmental guidelines and approval obtained from the ethics commission of the State of Bavaria (no. 55.2-1-54-2531-19-08).

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No potential conflicts of interest are disclosed.

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Correspondence to Yue Zhao or Peter Camaj.

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Fig. 1S

a Only 1/10 amount of L3.6plGres-SP cells were necessary to generate within the same time primary pancreatic tumors identical in tumor size compared to 1 × 106 L3.6plGres whole population cells. b There was no significant difference in tumor weight of primary tumors generated from 1 × 106 L3.6plGres whole cell population compared to 1 × 105 L3.6plGres-SP cells (p = 0.68). Primary pancreatic tumors generated within the same time from 1 × 105 L3.6plGres-NSP cells were significantly smaller than tumors generated from either 1 × 106 L3.6plGres whole population cells or 1 × 105 L3.6plGres-SP cells (L3.6plGres-NSP vs. L3.6plGres-SP, ***p = 0.0003). (DOCX 157 kb)

Fig. 2S

Histomorphological and immunohistochemical tissue analysis of cell proliferation, apoptosis, and angiogenesis using H&E, Ki67, TUNEL, and CD31 staining in primary pancreatic tumor generated from L3.6plGres whole population cells versus L3.6plGres-SP cells demonstrating no significant differences in the Ki67 and TUNEL indexes as well as MVD. (DOCX 252 kb)

Table 1S

Incidence of primary tumors and metastatic lesions generated from L3.6plGres, L3.6plGres-SP, and L3.6plGres-NSP cells. (DOCX 27 kb)

Table 2S

Demographic and histopathological parameters of 28 patients with pancreatic cancer who underwent radical surgery are described. The study groups were comparable in age, sex, grading of the tumor, and pTNM staging. However, there was no significant difference in grading, TNM staging, and lymphatic invasion between high and low expression of the different miRNAs. (DOCX 57 kb)

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Zhao, Y., Zhao, L., Ischenko, I. et al. Antisense inhibition of microRNA-21 and microRNA-221 in tumor-initiating stem-like cells modulates tumorigenesis, metastasis, and chemotherapy resistance in pancreatic cancer. Targ Oncol 10, 535–548 (2015). https://doi.org/10.1007/s11523-015-0360-2

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Keywords

  • miRNAs
  • Side population
  • Tumorigenesis
  • Metastasis
  • Chemotherapy resistance
  • Pancreatic cancer