SOAT1 promotes mevalonate pathway dependency in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Figure 1.

SOAT1 expression increases during PDAC progression. (A) RNA-seq analysis of murine normal N (n = 7), PanIN P (n = 6), tumor T (n = 12), and metastatic M (n = 9) pancreatic organoids showing genes involved in cholesterol biosynthesis (black), transport and catabolism (green), and homeostasis (orange). The color scheme of the heat map represents Z-score distribution. (B) Western blot analysis of the inactive SREBP2 precursor (SREBP2-p) and the mature SREBP2 protein (SREBP2-m) in a panel of N (n = 2), P (n = 3), T (n = 3), and tumor-matched M (n = 3) organoids. ACTIN, loading control. (C) Cholesterol ester assays for N (n = 4), P (n = 4), T (n = 4), and tumor-matched M (n = 4) organoids. Results show mean ± SEM of two biological replicates (two technical replicates each). **, P < 0.01; ***, P < 0.001, paired Student’s t test. (D) qPCR analysis of Soat1 in murine pancreatic N (n = 2), P (n = 3), T (n = 3), and tumor-matched M (n = 3) organoids. Results show mean ± SEM. *, P < 0.05; **, P < 0.01, paired Student’s t test between matched T and M organoids; unpaired Student’s t test between N, P, and M organoids. (E) Western blot analysis of SOAT1 in N (n = 2), P (n = 3), T (n = 3), and tumor-matched M (n = 3) organoids shown in B. HSP90, loading control. (F) qPCR analysis of Soat1 in C57BL/6J normal pancreata (n = 3), KPC tumors (n = 3), and KPC metastases (n = 2). Results show mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test. (G) Representative RNA ISH of Soat1 in C57BL/6J normal pancreas (n = 3), KPC tumor (n = 3), and matched metastasis (n = 3). Inserts: magnification. Scale bars, 200 µm. Molecular weights in kilodaltons.

Figure S1.

SOAT1 expression increases during PDAC progression. (A) RNA-seq analysis of Srebf2 in murine N (n = 7), P (n = 6), T (n = 6), and tumor-matched M (n = 6) pancreatic organoids. Results show mean ± SEM. *, P < 0.05; ***, P < 0.001, paired Student’s t test between matched T and M organoids; unpaired Student’s t test between N, P, and M organoids. (B) qPCR analysis of Srebf2, Hmgcr, Hmgcs1, Sqle, and Fdps in M3L organoids with or without expression of two different shRNAs targeting Srebf2. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (C) Cholesterol assays for N (n = 4), P (n = 4), T (n = 4), and tumor-matched M (n = 4) organoids. Results show mean ± SEM of two biological replicates (two technical replicates each). No statistical difference was found, as calculated by paired Student’s t test. (D) RNA-seq analysis of Soat1 in N (n = 7), P (n = 6), T (n = 6), and tumor-matched M (n = 6) organoids. Results show mean ± SEM. ***, P < 0.001, paired Student’s t test between matched T and M organoids; unpaired Student’s t test between N, P, and M organoids. (E) Western blot analysis of SOAT1 (n = 2) in C57BL/6J normal pancreata (n = 2), KC tumors (n = 2), KPC tumors (n = 2), and KPC metastases (n = 2). Ponceau staining, loading control. Molecular weights in kilodaltons. (F) Representative RNA ISH for Srebf2 in C57BL/6J normal pancreas (n = 3), KPC tumor (n = 3), and matched metastasis (n = 3). Inserts: magnification. Scale bars, 200 µm. (G) RNA expression analysis of SOAT1, SREBF2, LDLR, and HMGCR in human PDAC (T, n = 179) and normal pancreas (N, n = 171) from the GEPIA tool.

Figure 2.

SOAT1 expression is dependent on p53 status. (A) Copy number variation analysis of Soat1 in murine N (n = 3), P (n = 3), T (n = 11), and M (n = 11) pancreatic organoids. (B) DNA gel showing Trp53 genetic status in metastatic (n = 3, M30, M29, and M28) and tumor (n = 2, T69B and T69A) PDAC organoids. T69B and T69A were generated from two primary tumors of the same KPC mouse. mut, mutant. (C) Western blot analysis of SOAT1 and p53 in T69B and T69A organoids (n = 2). HSP90, loading controls. (D) qPCR analysis of Soat1 in T69A and T69B organoids. Results show mean ± SD of two technical replicates. ***, P < 0.001, paired Student’s t test. (E) qPCR analysis of Soat1 in T6 and T23 organoids with or without p53 LOH. Results show mean ± SD of two technical replicates. ***, P < 0.001, paired Student’s t test. (F) Western blot analysis of SOAT1 and p53 in T6 and T23 organoids with or without p53 LOH. HSP90, loading controls. (G) DNA sequencing results showing Trp53 mutation following base-editing in P7 organoids. (H) qPCR analysis of Soat1 in P5 and P7 organoids with p53^WT/WT or p53^R270C/LOH. Results show mean ± SD of three technical replicates. ***, P < 0.001, paired Student’s t test. (I) Western blot analysis of SOAT1 and p53 in P5 and P7 organoids with p53^WT/WT or p53^R270C/LOH. HSP90, loading controls. (J) qPCR analysis of SOAT1 in a panel of human PDAC organoids with either mutant or WT p53. Results show mean ± SD of three technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. Human PDAC organoids were derived from resections of primary tumors (hT), fine needle aspirates/biopsies of primary tumors (hF) or metastases (hM). (K) Western blot analysis of SOAT1 in a panel of human PDAC organoids with either mutant or WT p53 (n = 2). HSP90, loading control. (L) qPCR analysis of Soat1 in KPC T (T3a, T6, and T23) and M (M1, M3L, and M10) organoids and in KPPC T^−/− (T91, T113, and T118) organoids. Results show mean ± SD of three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test. (M) Western blot analysis of SOAT1 in T (n = 3), M (n = 3), and T^−/− (n = 3) organoids. HSP90, loading control. (N) Western blot analysis of p53 in P5 and P7 organoids with p53^WT/WT or p53^−/−. HSP90, loading control. (O) qPCR analysis of Soat1 in P5 and P7 organoids with p53^WT/WT or p53^−/−. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (P) Western blot analysis of SOAT1 in P5 and P7 organoids with p53^WT/WT, p53^−/− or p53^R270C/LOH. HSP90, loading control. Molecular weights in kilodaltons.

Figure S2.

SOAT1 expression is dependent on p53 status. (A) Representative H3K27ac ChIP-seq profiles at the Soat1 locus in matched T (n = 6) and M (n = 6) organoids. (B) qPCR analyses of Soat1 in T6, T69A, and M3L organoids with or without expression of two different shRNAs targeting Srebf2. Results show mean ± SD of two technical replicates. No statistical difference was found, as calculated by paired Student’s t test. (C) Representative IHC of p53 in C57BL/6J normal pancreas (n = 3), KPC tumor (n = 3), and metastasis (n = 3). Inserts: magnification. Scale bars, 200 µm. (D) qPCR analysis of Cdkn1a and Mdm2 in T69B and T69A organoids. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (E) DNA gel showing Trp53 genetic status in T6 and T23 organoids with or without p53 LOH. mut, mutant. (F) qPCR analysis of Cdkn1a and Mdm2 in T6 and T23 organoids with or without p53 LOH. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (G) Representative H3K27ac ChIP-seq profiles at the Soat1 locus in T6 and T23 organoids with or without p53 LOH. (H) Representative bright-field images of P7 with p53^WT/WT or p53^R270C/LOH in complete media or complete media containing 10 µM Nutlin-3a. Scale bars, 800 µm. (I) DNA sequencing results showing Trp53 mutation following base-editing in P5 P organoids. (J) qPCR analysis of Cdkn1a and Mdm2 in P5 and P7 organoids with p53^WT/WT or p53^R270C/LOH. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (K) qPCR analysis of Trp53 in T (T3a, T6, and T23), M (M1, M3L, and M10), and KPPC T^−/− (T91, T113, and T118) organoids. Results show mean ± SD of three biological replicates. No Trp53 transcript was detected in the three T^−/− organoid lines. (L) qPCR analysis of Trp53 and Soat1 in M3L and M10 organoids with or without expression of an shRNA targeting Trp53. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (M) Western blot analysis of p53 and SOAT1 in M3L and M10 organoids with or without expression of an shRNA targeting Trp53 (n = 2). HSP90, loading controls. Molecular weights in kilodaltons. (N) qPCR analysis of TP53 and SOAT1 in hM1A organoids with or without expression of an shRNA targeting TP53. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (O) qPCR analysis of Hmgcr, Hmgcs1, Sqle, and Fdps in M3L organoids with or without expression of an shRNA targeting Trp53. Results show mean ± SD of two technical replicates. No statistical difference was found, as calculated by paired Student’s t test.

Figure 3.

SOAT1 loss significantly impairs PDAC progression. (A) Proliferation curves of murine M3L organoids with Soat1 WT or KO. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (B) Images of tumors derived from M3L OGO models with Soat1 WT (n = 5) or KO (n = 5) in nu/nu mice on day 48 after transplantation. (C) Quantification of tumor volumes from B. Results show mean ± SEM of five biological replicates per cohort. *, P < 0.05, unpaired Student’s t test. (D) Quantification of mice with metastases for the experiment shown in B. (E) Survival curves of M3L OGO models with Soat1 WT (n = 12) or KO (n = 11) in nu/nu mice. ***, P < 0.001. OS, overall survival. (F) Proliferation curves of M3L organoids with Soat1 WT or KO in complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point.

Figure S3.

SOAT1 loss significantly impairs PDAC progression. (A) Western blot analysis of SOAT1 in M3L organoids with Soat1 WT or KO (n = 2). HSP90, loading control. Molecular weights in kilodaltons. (B) qPCR analysis of Soat1 in T69A organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (C) Western blot analysis of SOAT1 in T69A organoids with or without expression of an shRNA targeting Soat1 (n = 2). HSP90, loading control. Molecular weights in kilodaltons. (D) Proliferation curves of T69A organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of five technical replicates. **, P < 0.01, unpaired Student’s t test calculated for the last time point. (E) qPCR analysis of Soat1 in T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of three technical replicates. **, P < 0.01, paired Student’s t test. (F) Proliferation curves of T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (G) Representative H&E stain of M3L OGO models with Soat1 WT or KO (n = 3). Scale bars, 400 µm. (H) Representative Masson's trichrome stain of M3L OGO models with Soat1 WT or KO (n = 3). Scale bars, 400 µm. (I) Images of T6^R172H/LOH OGO models with or without expression of an shRNA targeting Soat1 in nu/nu mice at day 24 after transplantation (n = 5 per cohort). (J) Quantification of tumor volumes of T6^R172H/LOH OGO models shown in I. Results show mean ± SEM of five biological replicates per cohort. *, P < 0.05, unpaired Student’s t test. (K) Number of mice with metastases for the experiment shown in I. (L) Representative H&E stain of T6^R172H/LOH OGO models with or without expression of an shRNA targeting Soat1 (n = 3). Scale bars, 200 µm. (M) Representative Masson’s trichrome stain of T6^R172H/LOH OGO models with or without expression of an shRNA targeting Soat1 (n = 3). Scale bars, 200 µm. (N) Representative RNA ISH of Soat1 in T6^R172H/LOH OGO models with or without expression of an shRNA targeting Soat1 (n = 3). Inserts: magnification. Scale bars, 200 µm. (O) Representative bright-field images of M3L organoids with or without Soat1 KO in complete media or complete media containing 50 µM cholesterol at 96 h (n = 2). Scale bars, 800 µm. (P) Proliferation curves of T69A organoids with or without expression of an shRNA targeting Soat1 in complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (Q) Proliferation curves of M3L organoids with or without Soat1 KO in complete media containing 0.5% LDL. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point.

Figure S4.

p53 LOH sensitizes tumor cells to SOAT1 deficiency. (A) qPCR analysis of Soat1 in N10 N organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (B) Proliferation curves of N10 organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (C) qPCR analysis of Soat1 in normal N12 organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (D) Proliferation curves of N12 organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (E) qPCR analysis of Soat1 in T23^R172H/WT organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (F) qPCR analysis of Soat1 in T6^R172H/WT organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of three technical replicates. **, P < 0.01, paired Student’s t test. (G) Western blot analysis of SOAT1 in T6^R172H//WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 (n = 2). HSP90, loading control. Molecular weights in kilodaltons. (H) Proliferation curves of T6^R172H/WT organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (I) qPCR analysis of Soat1 in T23^R172H/LOH organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (J) Proliferation curves of T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (K) qPCR analysis of Soat1 in P5 P organoids with p53^WT/WT with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (L) qPCR analysis of Soat1 in P7 organoids with p53^WT/WT with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (M) Proliferation curves of P7 organoids with p53^WT/WT with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (N) qPCR analysis of Soat1 in P5 organoids with p53^R270C/LOH with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (O) qPCR analysis of Soat1 in P7 organoids with p53^R270C/LOH with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (P) Proliferation curves of P7 organoids with p53^R270C/LOH with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (Q) qPCR analysis of Soat1 in T91^−/− organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (R) qPCR analysis of Soat1 in T113^−/− organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (S) Proliferation curves of T113^−/− organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (T) DNA gel showing Trp53 genetic status in T8 organoids and in DAPI⁻ CD45⁻ CD31⁻ PDPN⁻ EpCAM⁺ epithelial cancer cells sorted from T8 OGO models. mut, mutant. (U) Representative IHC of p53 in T8 OGO models (n = 2). Insert: magnification. Scale bar, 200 µm. (V) qPCR analysis of Soat1 in T8 organoids with or without expression of an shRNA targeting Soat1. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (W) Western blot analysis of SOAT1 in T8 organoids with or without expression of an shRNA targeting Soat1 (n = 2). HSP90, loading control. Molecular weights in kilodaltons. (X) Proliferation curves of T8 organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point.

Figure 4.

p53 LOH sensitizes tumor cells to SOAT1 deficiency. (A) Proliferation curves of T23^R172H/WT organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (B) Proliferation curves of T23^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (C) Proliferation curves of P5^WT/WT organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (D) Proliferation curves of P5^R270C/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (E) Proliferation curves of T91^−/− organoids with or without expression of an shRNA targeting Soat1 in complete media or complete media containing 50 µM cholesterol. Results show mean ± SD of five technical replicates. No statistical difference was found, as calculated by unpaired Student’s t test for the last time point. (F) Quantification of tumor volumes of T8 OGO models with (n = 5) or without (n = 4) expression of an shRNA targeting Soat1 in NOD scid gamma mice on day 58 after transplantation. Results show mean ± SEM. ***, P < 0.001, unpaired Student’s t test. (G) Quantification of mice with metastases for the experiment shown in F. (H) Representative H&E stain of T8 OGO models with or without expression of an shRNA targeting Soat1 for the experiment shown in F (n = 2). Scale bars, 800 µm. (I) Representative RNA ISH of Soat1 in T8 OGO models with or without expression of an shRNA targeting Soat1 for the experiment shown in F (n = 2). Inserts: magnification. Scale bars, 200 µm. (J) Representative H&E stain of T91^−/− OGO models with or without expression of an shRNA targeting Soat1 in nu/nu mice on day 73 after transplantation (n = 2). Scale bars, 200 µm. (K) Representative RNA ISH of Soat1 in T91^−/− OGO models for the experiment shown in J (n = 2). Inserts: magnification. Scale bars, 200 µm. (L) Quantification of tumor volumes of T91^−/− OGO models with (n = 4) or without (n = 5) expression of an shRNA targeting Soat1 for the experiment shown in J. Results show mean ± SEM. No statistical difference was found, as calculated by unpaired Student’s t test. (M) Quantification of mice with metastases for the experiment shown in L.

Figure 5.

SOAT1 expression sustains the mevalonate pathway in PDAC. (A) Cholesterol ester assays for M3L organoids with Soat1 WT or KO in complete media or complete media containing 50 µM cholesterol for 4 h. Results show mean ± SD of two technical replicates. **, P < 0.01; ***, P < 0.001, paired Student’s t test. (B) Cholesterol assays for M3L organoids with Soat1 WT or KO in complete media or complete media containing 50 µM cholesterol for 4 h. Results show mean ± SD of two technical replicates. **, P < 0.01, paired Student’s t test. (C) RNA-seq analysis of M3L organoids with Soat1 WT (n = 2) or KO (n = 2) in complete media containing 50 µM cholesterol for 24 h showing genes involved in cholesterol biosynthesis. The color scheme of the heat map represents Z-score distribution. (D) GSEA plot for cholesterol biosynthesis in M3L organoids with Soat1 KO in complete media or complete media containing 50 µM cholesterol for 24 h. NES, normalized enrichment score. (E) Western blot analysis of the inactive SREBP2 precursor (SREBP2-p) and the mature SREBP2 protein (SREBP2-m) in M3L organoids with or without Soat1 KO in complete media or complete media containing 50 µM cholesterol for 24 h (n = 2). ACTIN, loading control. (F) qPCR analysis of Ldlr, Fdps, Hmgcs1, Sqle, and Hmgcr in T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media for 24 h. Results show mean ± SD of three technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, paired Student’s t test. (G) Western blot analysis of the inactive SREBP2 precursor (SREBP2-p) and the mature SREBP2 protein (SREBP2-m) in T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media for 24 h (n = 2). ACTIN, loading control. (H) IC₅₀ curves and values of T6^R172H/WT, T6^R172H/LOH, T23^R172H/WT, and T23^R172H/LOH organoids in complete media for 72 h with 1 × 10⁻¹⁰ M to 1 × 10⁻⁴ M Simvastatin. Results show mean ± SD of five technical replicates. (I) Proliferation curves of T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Srebf2 in complete media. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (J) Western blot analysis of membrane and total RAS, RHO (A, B, and C), RAC1, and CDC42 in M3L organoids with or without Soat1 KO in complete media or complete media containing 50 µM cholesterol for 48 h (n = 2). (K) Proliferation curves of M3L organoids with Soat1 WT or KO in complete media containing 50 µM cholesterol with or without 25 µM FPP. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (L) Western blot analysis of membrane and total RAS and RHO (A, B, and C) in M3L organoids with or without Soat1 KO in complete media containing 50 µM cholesterol with or without 25 µM FPP for 48 h (n = 2). Molecular weights in kilodaltons.

Figure S5.

SOAT1 expression sustains the mevalonate pathway in PDAC. (A) Box plots of RNA-seq expression in M3L organoids with Soat1 WT (n = 2) or KO (n = 2) in complete media for 24 h showing genes involved in cholesterol metabolism. Box sizes reflects expression ranges ± SD between two technical replicates. (B) GSEA plot for cholesterol biosynthesis in M3L organoids with Soat1 WT in complete media or complete media containing 50 µM cholesterol for 24 h. NES, normalized enrichment score. No statistical difference was found based on NES and FDR. (C) Cholesterol ester assays for T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media for 4 h. Results show mean ± SD of two technical replicates. No statistical difference was found, as calculated by paired Student’s t test. (D) Cholesterol assays for T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media for 4 h. Results show mean ± SD of two technical replicates. No statistical difference was found, as calculated by paired Student’s t test. (E) qPCR analysis of Ldlr, Fdps, Hmgcs1, Sqle, and Hmgcr in T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media containing 50 µM cholesterol for 24 h. Results show mean ± SD of three technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, paired Student’s t test. (F) Cholesterol ester assays for T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media containing 50 µM cholesterol for 4 h. Results show mean ± SD of two technical replicates per cohort. **, P < 0.01, paired Student’s t test. (G) Cholesterol assays for T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media containing 50 µM cholesterol for 4 h. Results show mean ± SD of two technical replicates per cohort. *, P < 0.05, paired Student’s t test. (H) Western blot analysis of the inactive SREBP2 precursor (SREBP2-p) and mature SREBP2 protein (SREBP2-m) in T6^R172H/WT and T6^R172H/LOH organoids with or without expression of an shRNA targeting Soat1 in complete media containing 50 µM cholesterol for 24 h (n = 2). ACTIN, loading control. (I) IC₅₀ curves and values of N6, N10, T69B, T69A, and M3L organoids in complete media for 72 h with 1 × 10⁻¹⁰ M to 1 × 10⁻⁴ M Simvastatin. Results show mean ± SD of five technical replicates. (J) qPCR analysis of Srebf2, Ldlr, Fdps, Hmgcs1, Sqle, and Hmgcr in T6^R172H/WT organoids with or without expression of an shRNA targeting Srebf2 in complete media. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (K) qPCR analysis of Srebf2, Ldlr, Fdps, Hmgcs1, Sqle, and Hmgcr in T6^R172H/LOH organoids with or without expression of an shRNA targeting Srebf2 in complete media. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (L) qPCR analysis of Srebf2, Ldlr, Fdps, Hmgcs1, Sqle, and Hmgcr in T23^R172H/WT organoids with or without expression of an shRNA targeting Srebf2 in complete media. Results show mean ± SD of two technical replicates. *, P < 0.05; **, P < 0.01, paired Student’s t test. (M) qPCR analysis of Srebf2, Ldlr, Fdps, Hmgcs1, Sqle, and Hmgcr in T23^R172H/LOH organoids with or without expression of an shRNA targeting Srebf2 in complete media. Results show mean ± SD of two technical replicates. *, P < 0.05, paired Student’s t test. (N) Proliferation curves of T23^R172H/WT and T23^R172H/LOH organoids with or without expression of an shRNA targeting Srebf2 in complete media. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. (O) Western blot analysis of E-CADHERIN and ACTIN in total (Tot), cytoplasmic (Cyto) and membrane (Membr) protein fractions in M3L organoids with or without Soat1 KO in complete media for 48 h. (P) Western blot analysis of E-CADHERIN and ACTIN in total (Tot), cytoplasmic (Cyto) and membrane (Membr) protein fractions in M3L organoids with or without Soat1 KO in complete media containing 50 µM cholesterol for 48 h. (Q) Western blot analysis of phosphorylated ERK (p-ERK) and total ERK in M3L organoids with Soat1 WT or KO in complete media or complete media containing 50 µM cholesterol for 48 h (n = 2). HSP90, loading control. (R) Proliferation curves of M3L organoids with Soat1 WT or KO in complete media with or without 25 µM FPP. Results show mean ± SD of five technical replicates. ***, P < 0.001, unpaired Student’s t test calculated for the last time point. Molecular weights in kilodaltons.

Figure 6.

SOAT1 abrogates cholesterol feedback inhibition to promote mevalonate pathway dependency in PDAC. Model illustrating the role of SOAT1 in sustaining the activity of the mevalonate pathway. (A and B) Typically, compared with normal and preneoplastic pancreatic cells (A), SOAT1 levels, SREBP2 activity, and the expression of mevalonate pathway genes are up-regulated in PDAC cells with mutant p53 and LOH of WT p53 (B). (C and D) SOAT1 abrogation and consequential mevalonate pathway inhibition do not affect the proliferation of normal and preneoplastic pancreatic cells (C), whereas they significantly impair the proliferation of PDAC cells with mutant p53 that have undergone p53 LOH (D). SREBP2-p, inactive SREBP2 precursor. SREBP2-m, mature SREBP2 protein.