Discoidin Domain Receptor-1 (DDR1) is Involved in Angiolymphatic Invasion in Oral Cancer

Abstract

The discoidin domain receptor-1 (DDR1) is a non-integrin collagen receptor recently implicated in the collective cell migration of other cancer types. Previously, we identified an elevated expression of DDR1 in oral squamous cell carcinoma (OSCC) cells. Through the data mining of a microarray dataset composed of matched tumor-normal tissues from forty OSCC patients, we distilled overexpressed genes statistically associated with angiolymphatic invasion, including DDR1, COL4A5, COL4A6 and PDPN. Dual immunohistochemical staining further confirmed the spatial locations of DDR1 and PDPN in OSCC tissues indicative of collective cancer cell invasion. An elevated DDR1 expression at both the transcription and protein level was observed by treating keratinocytes with collagen of fibrillar or basement membrane types. In addition, inhibition of DDR1 kinase activity in OSCC TW2.6 cells disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues. Taken together, these results suggest that collagen deposition in the affected tissues followed by DDR1 overexpression could be central to OSCC tumor growth and angiolymphatic invasion. Thus, DDR1 inhibitors are potential therapeutic compounds in restraining oral cancer, which has not been previously explored.

Keywords: angiolymphatic invasion (ALI); collective cancer cell migration; discoidin domain receptor-1 (DDR1); oral squamous cell carcinoma (OSCC).

Figure A1

Integrated associations of angiolymphatic invasion phenotype (column C) of NCKU-OrCA-40TN (column B: 40T in blue; 40N in red), with gene expression of DDR1, PDPN, COL4A5, COL4A6, MMP7, and TGFBI by using UCSC Xena platform [59]. LYVE1 (lymphatic vessel marker), PECAM1 (CD31, vascular vessel marker), and PIK3CA are included as controls.

Figure 1

Upregulation of DDR1 and co-localization of DDR1 and PDPN in the invasion front of OSCC tissues with angiolymphatic invasion (ALI). (a) Heat map shows top 100 genes differentially expressed in ALI-Yes (n = 18) and ALI-No (n = 22). Expression values of high, moderate, low, and lowest are represented as red, pink, light blue, and dark blue, respectively [32]. DDR1, COL4A6, PDPN, and MMP7 are marked with orange dots. (b) Enriched gene ontology terms (left panel, FDR < 0.05) and selected differentially expressed genes (right panel, fold change > 1.5) in ALI. COL4A5, COL4A6, DDR1, MMP7 and PDPN are denoted with orange dots. (c) Representative immunohistochemistry images of OSCC tissues stained for DDR1 (brown) and PDPN (green). Scale bars, 50 μm.

Figure 2

Correlation of collagen and DDR1 expression in vivo and in vitro. (a) Pearson correlation analysis between the expression levels of DDR1 and that of collagen type I (upper panel) and type IV (lower panel) in the 40 tumor tissues of NCKU-OrCA-40TN. DDR1 is positively correlated with COL4A5 and –A6, but not COL1A1 or –A2. (b) (upper) RT-qPCR assay of DDR1 mRNA levels in human keratinocytes treated with collagen (Col I or Col IV, 10 μg/mL) for 12 h. Data are presented as mean ± SD from triplicate assays. Statistical evaluation was performed with Student’s t-test. * p < 0.05; ** p < 0.01. Similar results were obtained from two experiments; one set of data is shown (lower). Western blot analysis of DDR1 protein levels in human keratinocytes treated with type I or type IV collagen for 24 h α-tubulin served as an internal control.

Figure 3

Constitutive phosphorylation of DDR1 in OSCC cells. (a) Western blot analysis of relative DDR1 expression levels in HEK293, CGHNK2, and six OSCC cells. Thirty μg protein lysates from each sample were used. α-tubulin served as an internal control. n.s., a nonspecific polypeptide cross-reactive to α-DDR1 antibody. (b) Total protein extracts of T-47D and OSCC cells were immunoprecipitated with the DDR1 antibody followed by western blot analysis using antibodies against p-Tyr or DDR1. The ratio of phosphorylated DDR1 intensity to that of total DDR1 was quantitated using ImageJ, with T-47D set to one. Similar results were obtained from two independent experiments; one set of data is shown. (c) T-47D and OSCC cells were cultured in a serum starved medium for 24 h followed by collagen stimulation (Col I or Col IV, 10 μg/mL) for an additional 24 h. The tyrosine-phosphorylation state of DDR1 was determined by immunoprecipitation-western blot analysis as described in (b).

Figure 4

DDR1 kinase inhibitors suppressed OSCC cell growth and clonogenicity. Drug sensitivity (left part in each panel) was determined by treating cells with 2–10 μM imatinib. (a) 0.01–1 μM dasatinib, (b) or 0.024–15 μM DDR1-IN-1 (c) for 3 d, followed by WST-1 proliferation assay. VC, vehicle control (H₂O for imatinib; DMSO for dasatinib and DDR1-IN-1). For each treatment, data are presented as mean ± SD from quadruplicate wells. Breast cancer cell line T-47D served as a control. Similar results were obtained from two independent experiments; one set of data is shown. Clonogenic survival assay (right part in each panel) was determined by treating cells in quadruplicate wells with 10 μM imatinib, (a) 1 μM dasatinib (b) or 10 μM DDR1-IN-1 (c) for 24 h, followed by cultivating cells in drug-free media for an additional 10–14 d. Representative photographs of crystal violet staining for each treatment are shown. Colony counts from two independent experiments are presented as mean ± SD, with the VC group set to 100%. * p < 0.05.

Figure 5

Kinase activity of DDR1 is involved in cohesive cell cluster formation. (a) Confocal-microscopic cell images of Ser19-phosphorylated myosin light chain (pMLC (S19)) staining (green) in the indicated OSCC cell lines. Nuclei were counterstained with DAPI (blue). Multi-cell cohesiveness, as evident by decreased pMLC (S19) staining at cell–cell contacts, was only observed in TW2.6 cells. (b) Confocal images of pMLC (S19) staining in A-431 (left) and TW2.6 (right) cells treated with vehicle control (VC) or DDR1 inhibitors (10 μM imatinib, 0.1 μM dasatinib, or 10 μM DDR1-IN-1). (c) Confocal images of CDH1 staining (red) in A-431 and TW2.6 treated with VC or DDR1 inhibitors. Scale bars, 20 μm.

Figure 6

Kinase activity of DDR1 is involved in spheroid invasion. (a) Representative bright-field images of two-day TW2.6 cell spheroids embedded in collagen I gel matrix with vehicle control (VC) or DDR1 inhibitors (10 μM imatinib, 0.1 μM dasatinib, 10 μM DDR1-IN-1). In each assay, a protrusion with >100 μm extension from its spheroid central body was counted as a branch; a spheroid with at least two branches was scored positive for collective invasion. The results of two independent experiments are summarized on the right. The fraction on each graph bar denotes positive (numerator) and total (denominator) numbers of spheroids examined. (b) Bright-field images of three-day TW2.6 and OC3 spheroids embedded in collagen I and IV gel matrix with VC or 10 μM DDR1-IN-1. (c) Bright-field images of nine-day TW2.6 and OC3 spheroids embedded in collagen I and IV gel matrix with VC only.

Figure 7

Suppression of collective TW2.6 cell invasion by DDR1-IN-1 in vivo. (a) (left) Schematic workflow of animal study. (right) Box plots show neither body weight nor tumor weight of mice in the control (PBS) and DDR1-IN-1 groups is statistically different. (b) Representative immunohistochemistry images of indicated TW2.6 xenograft tissues stained for CDH1 (brown) and PDPN (green). (c) Representative hematoxylin and eosin (H&E) stained xenograft sections with angiolymphatic invasion (ALI+) or perineural invasion (PNI+). Scale bars, 50 μm. Quantitative comparisons of ALI/PNI between PBS and DDR1-IN-1 mice are summarized on the right.