Extended Data Fig. 10: Activation of the IGF1R–AKT–PCK1–INSIG1/2 signalling cascade is required for the growth of liver tumours and correlates with poor prognosis for HCC.
From: The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis
a, b, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90A) (a) or INSIG1(S207A)/INSIG2(S151A) double mutants (b) were stably transfected with or without Flag–IGF1R(V922E) and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 28 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). *P =0.011 (a), 0.013 (b), **P <0.001 (two-tailed t-test). c, IHC analyses of xenografted tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. d, e, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90A) (d) or INSIG1(S207A)/INSIG2(S151A) double mutants (e) were stably transfected with or without Flag–myr-AKT1 and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 28 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). *P = 0.015 (d), 0.010 (e), **P < 0.001 (two-tailed t-test). f, IHC analyses of xenografted tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. g, h, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) (g) or INSIG1(S207D)/INSIG2(S151D) double mutants (h) were stably transfected with Flag–IGF1R(L1003R) and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumour growth 28 days after injection. The arrows indicate tumours. Tumour volumes were calculated (right). Data are mean ± s.d. (n = 7). *P = 0.003 (g), 0.005 (h), **P < 0.001 (two-tailed t-test). i–n, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) (i–k) or INSIG1(S207D)/INSIG2(S151D) double mutants (l–n) were stably transfected with or without Flag–IGF1R(L1003R) and subcutaneously injected into the left flanks of athymic nude mice (n = 7 per group). The resulting tumours were resected 28 days after injection (i, l). The growth of xenografted tumours in the mice was measured (j, m). The tumours were weighed (k, n). Data are mean ± s.d. (n = 7). *P= 0.002 (j), 0.011 (k), 0.009 (m), 0.016 (n), **P < 0.001 compared with the PCK1 wild-type + IGF1R(L1003R) group (j, k) or compared with the INSIG1/2 wild-type + IGF1R(L1003R) group (m, n) (two-tailed t-test). o–q, Huh7 cells (1 × 106) with or without knock-in expression of PCK1(S90D) or INSIG1(S207D)/INSIG2(S151D) double mutants were stably transfected with or without Flag–IGF1R(L1003R) and intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized 28 days after injection. IHC analyses of xenografted tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. r–t, Wild-type pT3-EF1α-Flag-PCK1 (or pT3-EF1α-Flag-PCK1(S90A)), pT3-EF1α-HA-myr-AKT1 and pT3-EF1α-V5-c-Met, along with the sleeping beauty transposase (SB), were stably expressed in the mouse liver using hydrodynamic transfection into FVB/N mice (n = 7 per group). After 14 weeks, the mice were euthanized and representative liver tumours are shown (r, left). The average tumour volumes were measured (r, right) (n = 7 per group). **P < 0.001 (two-tailed t-test). s, IHC analyses of tumour samples were performed with an anti-Ki67 antibody (left). Ki67-positive cells were quantified in 10 microscopic fields (right). **P < 0.001 (two-tailed t-test). t, IHC analyses of liver tumours from nude mice were performed with the indicated antibodies. The regions in white boxes are shown at higher magnification below. u, IHC staining of 30 human HCC and matched non-tumour tissue samples was performed with the indicated antibodies. Representative images of two cases are shown. The regions in white boxes are shown at higher magnification below. v, Representative immunoblotting analyses of two cases of human HCC and matched non-tumour tissue samples was performed with the indicated antibodies. w, The indicated staining scores for PCK1(pS90), INSIG1(pS207)/INSIG2(pS151) and SREBP1 expression levels in HCC and matched non-tumour liver samples (n = 30) were compared using a paired t-test (two-tailed). Data are mean ± s.d. **P < 0.001 compared with the non-tumour adjacent tissue. x, Representative immunoblotting analyses of four different human HCC samples were performed with the indicated antibodies. y, IHC staining of human HCC samples with the indicated antibodies was scored, and correlation analyses were performed. A Pearson correlation test was used (two-tailed) (n = 40). Note that the scores for some samples overlap. z, Mechanism for PCK1-promoted activation of SREBP1 and lipogenesis. PEP, phosphoenolpyruvate; OAA, oxaloacetate; HC, hydroxycholesterol; bHLH, basic helix-loop-helix protein; Reg, regulatory domain of SREBP. All experiments were repeated three times independently with similar results.
