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. 2012 Feb 6;14(1):R22.
doi: 10.1186/bcr3107.

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

Kaiji Hu  1 , Jennifer H Law, Abbas Fotovati, Sandra E Dunn
Affiliations

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

Kaiji Hu et al. Breast Cancer Res. .

Abstract

Introduction: Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.

Methods: A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.

Results: Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.

Conclusion: Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.

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Figures

Figure 1
Figure 1
Growth inhibition of the 28 active kinases on different breast cancer cells after siRNA silencing. (A) Percentage growth inhibition of SUM149 by the kinases after siRNA silencing at 5 nM for 72 hours. C, control without siRNA. *PLK1. (B) Percentage growth inhibition of BT474-M1 by the kinases. (C) Percentage growth inhibition of MDA-MB-231 by the kinases. (D) Percentage growth inhibition of HR5 by the kinases. Data are presented as mean ± SD of two independent tests. Kinases in the figure: CDC2L1, cell-division cycle 2-like 1 (PITSLRE proteins); CHEK1, CHK1 checkpoint homologue (S. pombe); CDC2, cell-division cycle 2, G1 to S and G2 to M; CSNK2A2, casein kinase 2, alpha prime polypeptide; DGUOK, deoxyguanosine kinase; GCK, glucokinase (hexokinase 4, maturity-onset diabetes of the young 2); IRAK1, interleukin-1 receptor-associated kinase 1; MAP3K4, mitogen-activated protein kinase kinase kinase 4; PDGFRA, platelet-derived growth factor receptor, α polypeptide; PFKFB2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2; PIK3C2G, phosphoinositide-3-kinase, class 2, γ polypeptide; PIK4CB, phosphatidylinositol 4-kinase, catalytic, β polypeptide; PHKG2, phosphorylase kinase, γ 2 (testis); PFTK1, PFTAIRE protein kinase 1; PLAU, plasminogen activator, urokinase; PLK1, polo-like kinase 1 (Drosophila); PRKCD, protein kinase C, delta; SKP2, S-phase kinase-associated protein 2 (p45); RPS6KA2, ribosomal protein S6 kinase, 90 kDa, polypeptide 2; TTK, TTK protein kinase; VRK2, vaccinia-related kinase 2; IHPK1, inositol hexaphosphate kinase 1; PKIG, protein kinase (cAMP-dependent, catalytic) inhibitor γ; MAPK8IP3, mitogen-activated protein kinase 8 interacting protein 3; PRKD2, protein kinase D2; PI4K2B, phosphatidylinositol 4-kinase type-II β; UCK1, uridine-cytidine kinase 1; LOC392265, similar to cell-division protein kinase 5 (Tau protein kinase II catalytic subunit) (TPKII catalytic subunit) (serine/threonine-protein kinase PSSALRE).
Figure 2
Figure 2
Effect of the active kinases on CD44high and the sorted CD44high/CD24-/low populations of SUM149. (A) Number of CD44high cells (per view field, ×10 magnification) of SUM149 after kinase silencing by siRNA at 5 nM for 72 hours. C, control without siRNA; Sc, scramble siRNA. *PLK1. (B) Growth inhibition of the sorted CD44high/CD24-/low population of SUM149 by siRNA silencing of different kinases at 5 nM for 72 hours. Data are presented as mean ± SD of two independent tests. Kinases in the figure: CDC2L1, cell-division cycle 2-like 1 (PITSLRE proteins); CHEK1, CHK1 checkpoint homologue (S. pombe); CDC2, cell-division cycle 2, G1 to S and G2 to M; CSNK2A2, casein kinase 2, alpha prime polypeptide; DGUOK, deoxyguanosine kinase; GCK, glucokinase (hexokinase 4, maturity-onset diabetes of the young 2); IRAK1, interleukin-1 receptor-associated kinase 1; MAP3K4, alpha mitogen-activated protein kinase kinase kinase 4; PDGFRA, platelet-derived growth factor receptor, alpha polypeptide; PFKFB2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2; PIK3C2G, phosphoinositide-3-kinase, class 2, gamma polypeptide; PIK4CB, phosphatidylinositol 4-kinase, catalytic, beta polypeptide; PHKG2, phosphorylase kinase, gamma 2 (testis); PFTK1, PFTAIRE protein kinase 1; PLAU, plasminogen activator, urokinase; PLK1, polo-like kinase 1 (Drosophila); PRKCD, protein kinase C, delta; SKP2: S-phase kinase-associated protein 2 (p45); RPS6KA2, ribosomal protein S6 kinase, 90 kDa, polypeptide 2; TTK, TTK protein kinase; VRK2, vaccinia-related kinase 2; IHPK1, inositol hexaphosphate kinase 1; PKIG, protein kinase (cAMP-dependent, catalytic) inhibitor gamma; MAPK8IP3, mitogen-activated protein kinase 8-interacting protein 3; PRKD2, protein kinase D2; PI4K2B, phosphatidylinositol 4-kinase type-II beta; UCK1, uridine-cytidine kinase 1; LOC392265, similar to cell-division protein kinase 5 (tau protein kinase II catalytic subunit) (TPKII catalytic subunit) (serine/threonine-protein kinase PSSALRE).
Figure 3
Figure 3
PLK1 expression, its association with CD44, and its activity after BI 2536 inhibition. (A) PLK1 expression in different breast cancer cells by immunoblotting. (B) PLK1 expression after PLK1 siRNA silencing in two TNBC (triple-negative breast cancer) cell lines, SUM149 and MDA-MB-231. (C) PLK1 is positively associated with CD44 expression with immunofluorescent assay. (D) Immunofluorescent images showing the accumulation of phospho-cyclin B1 in SUM149 cells after BI 2536 treatment (72 hours). DMSO, dimethyl sulfoxide; DAPI, 4ʹ,6-diamidino-2-phenylindole. (E) Quantitative analysis of phospho-cyclin B1 in SUM149 cells after BI 2536 treatment (72 hours).
Figure 4
Figure 4
Effect of PLK1 inhibition on breast cancer cell growth, apoptosis, and TICs. (A) Percentage of growth inhibition of different breast cancer cells by a PLK1 small-molecule inhibitor BI 2536 at different concentrations for 72 hours. C, control (medium only). D, dimethyl sulfoxide only. (B) Percentage of growth inhibition of SUM149 cells by BI 2536 at different concentrations for 10 days. (C) Percentage of growth inhibition of sorted CD44high/CD24-/low of SUM149 by BI 2536 for 72 hours. (D) Percentage of mammosphere reduction in SUM149 and MDA-MB-231 by BI 2536. *Significant difference from the control (Student t test, P < 0.05). (E) Percentage of apoptosis of different breast cancer cells caused by BI 2536 after 72 hours. (F) Time-course apoptosis of SUM149 treated with BI 2536 for 6, 24, and 48 hours. (G) Apoptotic images of the cells after BI 2536 treatment, as revealed by HCS (high-content screening) instrument (×10) with phospho-H2AX antibody or PI (propidium iodide) intake. Data are presented as mean ± SD of two independent tests. TICs, tumor-initiating cells.
Figure 5
Figure 5
Effect of combined treatment of Taxol, Dox, or 5FU followed by BI 2536 on SUM149 cells. (A) Taxol, Dox, and 5FU inhibited the growth of SUM149 after 72 hours. C, control (medium only). (B) Drug treatment led to a higher percentage of CD44high/CD24-/low TIC (tumor-initiating cell) subpopulation in surviving SUM149 cells after 72 hours. Top image panel: CD44high/CD24-/low cells (arrows) after 5FU treatment (0.1 mM, 72 hours) as viewed by HCS (high-content screening) instrument (×10). (C) Combined treatment with the drugs (Taxol, Dox, or 5FU) for 72 hours, followed by BI 2536 for another 96 hours, significantly reduced cell growth, even though the drugs induced a higher percentage of the CD44high/CD24-/low subpopulation. All data are presented as mean ± SD of two independent tests. DMSO, dimethyl sulfoxide; Taxol, paclitaxel; Dox, doxorubicin; 5FU, 5-fluorouracil.
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References

    1. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenchikov A, Williams C, Zhu SX, Lønning PE, Børreson-Dale AL, Brown PO, Botstein D. Molecular portraits of human breast tumours. Nature. 2000;406:747–752. doi: 10.1038/35021093. - DOI - PubMed
    1. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lønning P, Børresen-Dale AL. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001;98:10869–10874. doi: 10.1073/pnas.191367098. - DOI - PMC - PubMed
    1. Dent R, Trudeau M, Pritchard KL, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429–4434. doi: 10.1158/1078-0432.CCR-06-3045. - DOI - PubMed
    1. Chacon RD, Costanzo MV. Triple negative breast cancer. Breast Cancer Res. 2010;12(Suppl 2):S3. - PMC - PubMed
    1. de Ruijter TC, Veeck J, de Hoon JPJ, van Engeland M, Tjan-Heijnen VC. Characteristics of triple-negative breast cancer. J Cancer Res Clin Oncol. 2011;137:183–192. doi: 10.1007/s00432-010-0957-x. - DOI - PMC - PubMed

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