Fig 1.
LMO4, LDB1, SSBP2, and SSBP3 abundance was highly correlated in a panel of human oral cavity carcinoma cell lines.
The relative abundance of LMO4, LDB1, SSBP2, and SSBP3 in a panel of 10 human oral cavity carcinomas correlated significantly by immunoblot analysis. (A) LMO4 and LDB1 abundance were significantly correlated (r2 = 0.90, P < 0.0001, n = 10). (B) SSBP and LDB1 abundance were significantly correlated (black triangles, SSBP2, r2 = 0.95, P < 0.0001, n = 10; white triangles, SSBP3, r2 = 0.94, P < 0.0001, n = 10). (C) SSBP and LMO4 abundance were significantly correlated (black triangles, SSBP2, r2 = 0.80, P < 0.0005, n = 10; white triangles, SSBP3: r2 = 0.84, P < 0.0002, n = 10).
Fig 2.
LMO4, LDB1, SSBP2, and SSBP3 expression was concordant in human oro-pharyngeal carcinomas and lymph nodes involved by metastasis.
(A) LMO4, (C) LDB1, (E) SSBP2, and (G) SSBP3 expression was enriched at the invasive edge (thick arrows) of tumors compared to more central locations (thin arrows). (B) LMO4, (D) LDB1, (F) SSBP2, and (H) SSBP3 expression was detected in lymph nodes for all four carcinomas in which paired samples were available and exhibited a similar intracellular and intra-tumor distribution to the tumor primary. Control sections stained in parallel with secondary antibody alone or an isotype control antibody and secondary antibody were negative for histochemical reaction product (data not shown).
Fig 3.
LDB1 and LMO4 protein abundance were reduced and cellular proliferation inhibited in LDB1 knockout cells.
(A) LDB1 and, to a lesser extent, LMO4 protein abundance were reduced in VU-SCC-1729 cells in which LDB1 was inactivated by CRISPR/Cas9 gene targeting (VU-SCC-1729 exon 2) but not in cells in which gene targeting was less effective (VU-SCC-1729 exon 1). (B) Cells in which the LDB1 gene was inactivated (VU-SCC-1729-2:7) accumulated in significantly and progressively lower numbers relative to VU-SCC-1729 cells, starting on day 2 of culture (P = 0.053). +, vector control cells.
Fig 4.
Loss of LDB1 expression in VU-SCC-1729 cells significantly reduced cellular invasiveness in two experimental models.
(A) Invasion through reconstituted basement membrane (Matrigel) was evaluated for VU-SCC-1729 cells, vector control cells (+),VU-SCC-1729-1:2 cells in which LDB1 was only modestly knocked down, and VU-SCC-1729-2:7 cells in which LDB1 protein expression was almost completely ablated. (B) Invasion into a mixture of fibroblasts and extracellular matrix was evaluated in an organotypic reconstruct assay for parental and VU-SCC-1729-2:7 cells. The highly invasive VU-SCC-1729 cell line exhibited significant penetration of the fibroblast-collagen matrix by single cells (thick arrows) and cell clusters (thin arrows). The invasiveness of LDB1 knockout cells was significantly impaired, with only rare individual cells (thick arrows) and no clusters of cells penetrating the fibroblast layer. *, P < 0.01; ***, P < 0.0001.
Fig 5.
Loss of LDB1 expression in VU-SCC-1729 cells significantly reduced tumor growth in nude mice through an effect on cell proliferation.
(A) Tumor growth in nude mouse xenografts was compared in VU-SCC-1729 cells and VU-SCC-1729-2:7 cells in which LDB1 protein expression was ablated. A difference in tumor size was first detected on day 10 (P = 0.002), which progressively increased from day 13 (P = 0.00096) to day 20 (P = 0.0000194) to day 23 (P = 0.0000345). (B) Caspase-3 staining for apoptosis was not significantly different between LDB1 KO and parental tumors (P = 0.2406). Aggregate data on caspase-3 staining are presented to the right of the image. (C) PCNA staining for proliferation was significantly reduced in LDB1 KO tumors compared to tumors derived from parental VU-SCC-1729 cells (P = 0.00028). Aggregate data on PCNA staining are presented to the right of the image.
Fig 6.
Loss of LDB1 expression in VU-SCC-1729 cells significantly reduced angiogenesis in tumor xenografts in nude mice.
Endothelial cell numbers were compared in nude mouse xenografts from VU-SCC-1729 cells and VU-1729-2:7 cells in which LDB1 protein expression was eliminated (LDB1 KO). Endothelial cells were stained with antibody to vWF (green immunofluorescence) and nuclei counterstained with DAPI (blue immunofluorescence). The number of vWF-expressing endothelial cells was significantly reduced in LDB1 KO compared to control tumors (P = 0.00196).
Fig 7.
Biological processes highlighted by iPathway Guide from differentially expressed genes in LDB1 knockout compared to wild-type VU-SCC-1729 cells.
Numbers represent percentage of genes in each category differentially expressed in KO vs. wild-type cells using a threshold of 0.05 for statistical significance and 2 for absolute log expression change. P values for the pathways identified were: ECM-receptor interaction (P = 0.0000527), pathways in cancer (P = 0.001), TGF-β signaling (P = 0.002), transcriptional misregulation in cancer (P = 0.002), and PI3K-Akt signaling (P = 0.003).