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. 2016 Oct 31;6:36132.
doi: 10.1038/srep36132.

A Dual Specificity Kinase, DYRK1A, as a Potential Therapeutic Target for Head and Neck Squamous Cell Carcinoma

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Free PMC article

A Dual Specificity Kinase, DYRK1A, as a Potential Therapeutic Target for Head and Neck Squamous Cell Carcinoma

Aneesha Radhakrishnan et al. Sci Rep. .
Free PMC article

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Abstract

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

Figures

Figure 1
Figure 1. Inhibition of DYRK1A reduces cellular proliferation in HNSCC.
(a) Western blot analysis shows the expression profile of DYRK1A in a panel of HNSCC cell lines – JHU-O11, JHU-O22, JHU-O28, JHU-O29, FaDu and CAL 27 compared to normal oral keratinocytes OKF6/TERT1. (b) Immunohistochemical validation of DYRK1A in HNSCC tissue - representative sections from normal and HNSCC cases were stained with anti-DYRK1A antibody. (c) Western blot analysis depicting DYRK1A expression in HNSCC cell lines upon transfection with DYRK1A siRNA. β-actin was used as a loading control. (d) Cellular proliferation of HNSCC cells upon siRNA mediated silencing of DYRK1A (*p < 0.05).
Figure 2
Figure 2. Inhibition of DYRK1A affects the colony forming ability of the HNSCC cells.
(a) Colony formation assay following siRNA mediated knockdown of DYRK1A or control siRNA in a panel of HNSCC cell lines, as indicated. Colonies formed were visualized after staining with methylene blue. (b) A graphical representation of the colony forming ability of the HNSCC cells upon DYRK1A silencing (*p < 0.05). (c) Colony forming ability of the HNSCC cells upon inhibition of DYRK1A using harmine or control (DMSO), in the indicated panel of HNSCC cells. (d) A graphical representation of the colony forming ability of HNSCC cells upon harmine treatment (*p < 0.05). Representative images were photographed at a magnification (2.5x).
Figure 3
Figure 3. Inhibition of DYRK1A reduces the invasive ability of the HNSCC cells.
(a) HNSCC cells were transfected with DYRK1A specific siRNA and/or scramble siRNA and invasion assays were carried out using in a transwell system using Matrigel-coated filters and the number of cells that migrated to the lower chamber was counted. Cells that migrated are visualized following methylene blue staining in a panel of HNSCC cell lines as indicated and invaded cells were photographed. (b) Graphical representation of invasive ability of HNSCC cells upon DYRK1A silencing (*p < 0.05). (c) HNSCC cells were treated with DYRK1A inhibitor (harmine) or vehicle control (DMSO) and invaded cells were photographed. (d) Graphical representation of invasive ability of DYRK1A upon inhibition with harmine (*p < 0.05). Representative images were photographed at a magnification (10x).
Figure 4
Figure 4
Inhibition of DYRK1A suppresses tumor growth in vivo (a) CAL 27 (2 × 106) cells were injected into the flanks of female nude mice (n = 10) and tumor growth kinetics is represented for a period of 25 days. *p = 0.02 (b) Representative pictures of tumors from vehicle and harmine treated groups. (c) Bar graph representing the tumor weights (**p < 0.05). (d) Expression of Ki67 (Alexa Fluor 594) in xenograft tissue sections was determined by immunofluorescence. Cell nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI).
Figure 5
Figure 5. Inhibition of DYRK1A induces apoptosis in vitro and in vivo.
Apoptosis was measured in CAL 27 (a) and JHU-O28 (b) cells using Annexin V/PI staining. (c) Western blot analysis was carried out for the indicated proteins using CAL 27 and JHU-O28 cells treated with harmine or DMSO (control). β-actin was used as a loading control. (d) Western blot analysis was carried out using cellular lysates of xenograft tissue (Harmine and DMSO treated) for the indicated proteins. β-actin was used as a loading control.
Figure 6
Figure 6. Inhibition of DYRK1A leads to activation of FOXO3A in HNSCC.
HNSCC cell lines JHU-O28, FaDu, JHU-O11, JHU-O29 (a) and CAL 27 (b) were treated with DYRK1A inhibitor harmine. Immunoblot analysis of p-AKT (Ser473), Total AKT, p-FOXO3A (Ser253) and Total FOXO3A was performed. β-actin was used as loading control. (b) Western blot analysis was performed using cellular lysates from xenograft tissue (Harmine and DMSO treated) for AKT (Ser473), Total AKT, p-FOXO3A (Ser253) and Total FOXO3A. (c) CAL 27 cells were treated with DYRK1A siRNA or control siRNA and Western blot analysis was carried for the indicated proteins.

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