Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.
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NCBI Reference Sequence
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We thank J.G. Rheinwald (Dana-Farber/Harvard Cancer Center) for his generous gift of SCC cell lines. We thank A.E. Oro, H.Y. Chang, M. Diehn, M.P. Scott, S.E. Artandi, G.R. Crabtree and T. Waldman as well as A. Zehnder, X. Bao, B.K. Sun, R.J. Flockhart and V. Lopez-Pajares for presubmission review and helpful comments. This work was supported by the US Veterans Affairs Office of Research and Development and by US National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS) grant AR43799 to P.A.K. C.S.L. is the recipient of career award K08 AR064732 from the NIH/NIAMS. A.S. is supported by NIH/National Institute of General Medical Science (NIGMS) grant GM074728 and American Cancer Society grant 120161-RSG. W.L.J. is the recipient of National Science Foundation (NSF) graduate research fellowship grant DGE-114747.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Comparison of mutation frequency in TP53, CDKN2A and HRAS between data in the COSMIC database and this study.
(a) Kinastrin is expressed in a broad variety of tissues. Kinastrin expression was extracted from the NextBio Body Atlas application (PLoS One 5, e13066, 2010). The dotted line indicates median expression. (b) Kinastrin expression in progenitor human primary keratinocytes11. The average FPKM of all expressed genes (FPKM > 1.5) in this population is shown for reference. (c) Kinastrin is expressed in normal skin and SCCs. Kinastrin expression was assayed in 76 cases of cutaneous SCC and 5 normal skin samples; representative images are shown. Scale bar, 25 μm.
KNSTRN p.Ser24Phe mutations were confirmed by Sanger sequencing. Sequence chromatograms from a representative SCC and normal skin (NL) sample from the same individual are shown.
(a) KNSTRN mutation frequency by cancer type (Cancer Discov. 2, 401–404, 2012). (b) Distribution of mutations in the kinastrin coding sequence (Cancer Discov. 2, 401–404, 2012). The labeled mutations disrupt sister chromatid cohesion in normal cells. SXLP, SXLP motif; CC, coiled-coil region.
Western blot analysis of early-passage primary human keratinocytes following transduction with the three kinastrin isoforms. Endogenous kinastrin expression is shown in lane 1; only isoform 3 is present. Lanes 6 and 7 show the enforced protein expression used to assess sister chromatid cohesion and DNA content. Numbers indicate molecular mass in kilodaltons.
(a) Western blot analysis of SCC-13 cells following transduction with N-terminally tagged (Flag-HA-(poly)His) kinastrin p.Ser24Phe versus wild-type kinastrin. The lower- and higher-molecular-weight bands represent endogenous and enforced kinastrin expression, respectively. (b) Quantification of unpaired chromatids. *P = 0.013, **P = 0.008; ns, not significant.
Supplementary Figure 7 Cancer-associated KNSTRN mutations disrupt chromatid cohesion in normal cells.
(a) Western blot analysis of early-passage primary human keratinocytes following transduction with KNSTRN p.Arg11Lys, p.Pro26Ser, p.Pro28Ser and p.Ala40Glu versus wild-type kinastrin. (b) Quantification of unpaired chromatids. *P = 0.05, **P < 0.03.
Early-passage, pooled primary human foreskin keratinocytes were transduced with dominant-negative p53 and LacZ (CTR), wild-type kinastrin (WT) or kinastrin p.Ser28Phe (S24F). Cells were grown in paclitaxel for 68 h to induce aneuploidy. (a) Percentage of primary human keratinocytes with DNA content >4N. Data shown represents the mean ± s.d. for biological duplicates with technical triplicates. *P = 0.0014, **P = 0.0004; ns, not significant. (b) Representative plots of cell cycle analysis are shown.
Early-passage primary human keratinocytes were transduced with LacZ (CTR), wild-type kinastrin (WT) or kinastrin p.Ser28Phe (S24F). (a) Equal numbers of cells were seeded in duplicate, and cell growth assays were performed in technical triplicates at each of the time points shown. (b) Asynchronously proliferating cells were stained with propidium iodide and assayed by flow cytometry in technical duplicates.
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