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. 2016 Dec 1;383(1):106-114.
doi: 10.1016/j.canlet.2016.09.014. Epub 2016 Sep 28.

Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation

Tyler F Hayes  1 , Nathan Benaich  1 , Stephen J Goldie  2 , Kalle Sipilä  1 , Ashley Ames-Draycott  1 , Wenjun Cai  3 , Guangliang Yin  3 , Fiona M Watt  4
Affiliations

Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation

Tyler F Hayes et al. Cancer Lett. .

Abstract

Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an in vitro model that accurately reflects this heterogeneity, we generated a panel of HPV-negative OSCC cell lines. By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1 (an atypical cadherin) and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour.

Keywords: Caspase 8; FAT1; Oral squamous cell carcinoma; Whole exome sequencing.

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Figures

Fig. 1
Fig. 1
Genomic analysis of OSCC lines and TCGA HNSCC tumours. A–C) Somatic mutation rates (A), nucleotides transition and transversion frequencies (B), clinical characteristics and social histories (C) of cell lines and patients from which they were derived. D) Somatic single nucleotide variants (SNVs) and insertion/deletions (indels) in significantly mutated genes. Genes are ranked in order of significance from TCGA (6). E) Unsupervised clustering of TCGA HNSCC samples according to significance of mutated KEGG pathways. Rows are individual tumours; columns are KEGG pathways. OSCC are shown separately from other HNSCC. F) Unsupervised clustering of TCGA OSCC samples ranked according to frequency of mutated KEGG pathways (descending rank order) in OSCC cell lines. Rows are pathways and columns are individual tumours. (E, F) Colour scale is based on –log2(P) values.)
Fig. 2
Fig. 2
Identification of CASP8 and FAT1 as putative HNSCC/OSCC drivers. A) Genetic alterations in putative driver genes identified by exome sequencing and literature review. Samples are arranged to emphasize mutual exclusivity among mutations. Samples without genetic alterations in these genes are not shown. B) Pair associations of genetic alterations among putative driver genes in TCGA HNSCC project. P value was determined by Fisher's Exact test. C.O. = co-occurrence, M.E. = mutually exclusive. C) Genetic alterations in CASP8 and FAT1 in TCGA HNSCC and OSCC cell line datasets. LAGD = laminin a-g domain; EGFLR = EGF-like repeat; asterisk indicates mutation present in both TCGA and cell line datasets. D) mRNA expression of CASP8 and FAT1 in OSCC cell lines. P value was determined by unpaired Student's t-test. *P < 0.01–0.05. No significant difference was detected between presence of CASP8 mutations and CASP8 mRNA expression. OK and CRI-005 are two strains of primary oral keratinocytes. mut = mutant, wt = wild type. E) CASP8 mRNA levels in tumours with mutant and wild type CASP8 (left hand panel) and FAT1 mRNA levels in tumours with mutant and wild type FAT1 (right hand panel) stratified by putative copy-number alterations. Data from TCGA (6).
Fig. 3
Fig. 3
Phenotypic impact of CASP8 and FAT1 mutations and siRNA knockdown in OSCC lines. A) Phase-contrast microscopy of OSCC lines with (mt) and without (wt) predicted inactivating mutations in FAT1, and effects of FAT1 siRNA on SJG-33 (wt for FAT1). B) FAT1 mRNA expression in OSCC lines plated at low and high density for 48–72 h. OK = primary oral keratinocytes. C, D) Impact of CASP8 and FAT1 knockdown on clonal growth. (C) Triplicate dishes of SJG-17 cells. (D) Percent well area covered by cell clones. Significant differences from non-targeting control siRNA (siSCR) are shown. A–D) Cells were cultured on feeders. E, F) Impact of CASP8 and FAT1 knockdown on cell migration. (E) Representative images of cells at start (0 h) or 24 h later. In high magnification view, arrow indicates migratory cell front; asterisks indicate individual migrating cells. (F) Migration rate, defined as fold change in area of central detection zone at 16–24 h. Data are presented as mean ± SEM. (E, F) Cells were plated in the absence of feeders. (D, F) P values were determined by ANOVA and Tukey's test. *P < 0.01–0.05, **P < 0.001–0.01, ***P < 0.0001–0.001.
Fig. 4
Fig. 4
Impact of CASP8 and FAT1 siRNA knockdown on proliferation, differentiation and apoptosis. A) Proliferation was assessed by Ki67 immunostaining. Significant differences from non-targeting control siRNA (siSCR) are shown. wt = wild type, mut = mutant, OK = primary oral keratinocytes. B) Terminal differentiation was assessed by transglutaminase 1 (TGM1) immunostaining. A, B) Plasma membranes and nuclei were counterstained with CellMask™ Deep Red and DAPI. C) Cells were incubated with Staurosporine, and apoptosis was measured by Caspase-3 (CASP3) immunostaining. (A–C) Representative images are shown for SJG-33 (A), control O.K. and SJG-6 (B), siCASP8 and siCASP8/FAT1 SJG-41 (B) and SJG-17 (C). Data are presented as mean ± SEM. P value was determined by ANOVA and Tukey's test. *P < 0.01–0.05, **P < 0.001–0.01, ***P < 0.0001–0.001. Scale bars 100 μm.
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