Extended Data Fig. 7: Loss of p16 or RB is associated with increased metastatic proclivity.

a, Western blot analysis of KP;Rb^TR/TR and KP (NKX2-1⁻HMGA2⁺ (T_Met) and NKX2-1⁺HMGA2⁻ (T_nonMet)) tumour-derived cell lines examining cyclin D1 and p16 expression. HSP90 was used as loading control. b, RNA sequencing reads at the Cdkn2a locus for NKX2-1⁺HMGA2⁻ KP (n = 2) and NKX2-1⁻HMGA2⁺ KP (n = 2) cell lines. Reads from exon 1α encoding p16 (left) and exon 1β encoding Arf (right) are shown. c, Representative Sashimi plots comparing the number of p16 and Arf exon-spanning reads from the Cdkn2a locus. Plots are shown for a representative NKX2-1⁺HMGA2⁻ tumour (red), NKX2-1⁻HMGA2⁺ tumour (blue) and metastasis (green). The number of reads that span each exon–exon junction is displayed. The range of minimum to maximum read count for the given plot is displayed in the top left corner. d, Quantification of the ratio of p16 to Arf reads from the Cdkn2a locus. RNA sequencing results examining NKX2-1⁺HMGA2⁻ tumours (n = 8), NKX2-1⁻HMGA2⁺ tumours (n = 8) and extrapulmonary metastases (n = 19) were obtained from the GEO (accession GSE84447)²⁵. Significance for each comparison was determined by two-tailed unpaired Student’s t-test. NKX2-1⁺HMGA2⁻ versus NKX2-1⁻HMGA2⁺, P = 0.0682; NKX2-1⁻HMGA2⁺ versus metastases, P = 0.0504; NKX2-1⁺HMGA2⁻ versus metastases, P = 0.0006. Data are represented by box and whisker plots, with the line indicating the median and whiskers indicating the minimum and maximum values. e, Western blot analysis of KP and KP;Rb^TR/TR tumour-derived cell lines for NKX2-1, HMGA2 and RB. Actin was used as loading control. f, g, Analysis of subcutaneous tumour growth (f) and associated lung metastases (g) of KP and KP;Rb^TR/TR tumour-derived cell lines from e. Symbols represent individual mice injected with either one of two NKX2-1⁻HMGA2⁺ KP cell lines (n = 4 and 5 mice per cell line), two NKX2-1⁺HMGA2⁺ KP;Rb^TR/TR cell lines (n = 3 and 4 mice per cell line) or two NKX2-1⁻HMGA2⁺ KP;Rb^TR/TR cell lines (n = 4 mice per cell line). Significance was determined by unpaired two-tailed Student’s t-test with Welch’s correction. f, Primary tumour weight. NKX2-1^–HMGA2⁺ KP versus NKX2-1⁺HMGA2⁺ KP;Rb^TR/TR (P = 0.2508) and KP versus NKX2-1⁻HMGA2⁺ KP;Rb^TR/TR (P = 0.2727). g, Lung metastases. KP versus NKX2-1⁺HMGA2⁺ KP;Rb^TR/TR (P = 4.8 × 10⁻⁵) and KP versus NKX2-1⁻HMGA2⁺ KP;Rb^TR/TR (P = 0.0027). Data are mean ± s.d. h, H&E photomicrographs of representative metastases from KP;Rb^TR/TR cell line allografts. Insets show magnification of boxed areas. Scale bars, 100 μm. i, Liver metastases after intrasplenic injection of KP and KP;Rb^TR/TR tumour-derived cell lines (top) from Extended Data Fig. 5i. Symbols represent individual mice injected with a NKX2-1⁺HMGA2⁻ KP cell line (n = 2 mice), a NKX2-1⁻HMGA2⁺ KP cell line (n = 5 mice), two NKX2-1⁺HMGA2⁺ KP;Rb^TR/TR cell lines (n = 4 mice for each cell line) or two NKX2-1⁻HMGA2⁺ KP;Rb^TR/TR cell lines (n = 5 mice for each cell line). Significance was determined by two-tailed unpaired Student’s t-test with Welch’s correction. P = 0.0007. Data are mean ± s.d.

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