SCAP supports PDAC development and loss of SCAP prolongs survival in the KPC mouse model of pancreas cancer. A, Mice of the indicated genotype were aged until death. The figure shows the cumulative incidence curve of death due to PDAC for each genotype group. For the analysis, we considered death due to other causes (i.e., not due to PDAC) as a competing event. The Gray test was used for pairwise comparisons. B, Incidence of adverse event (death due to PDAC) in KPC cohorts from A either pooled (left figure) or stratified by sex (right figure). C, Incidence of PanINs and invasive PDAC identified histologically in KPC cohorts from A. D, Representative hematoxylin and eosin–stained sections of formalin-fixed pancreas tissue from KPC, KPCSfl/+ and KPCSfl/fl cohorts in A showing a magnification (2–4×) image (top row) and a higher magnification (20×) image (bottom row) of the changes in the pancreas. The C mouse is used as a control. E, Figure shows the survival curves by genotype group. Mice of the indicated genotypes were aged until death at which time histology was performed to determine the presence of PanINs and invasive PDAC. For this analysis, we treated time to invasive PDAC as interval-censored data. The permutation test was used for pairwise comparisons.

Table S1. Primer sequences used for quantitative PCR

SUPPLEMENTARY MATERIALS AND METHODS

FIGURE S2 – Mouse genotyping methods and survival study data

FIGURE S6 – Site-1 protease activation of the SREBP pathway is required for PDAC cell growth in low serum conditions.

Table S3. Detailed cause of death and observed lesions in KPC mouse cohorts

SREBP pathway is broadly required for cancer cell growth and is activated in human PDAC tumors. A–C, The essentiality of SCAP, SREBF1, and SREBF2 across cancer cell lines was examined using the Cancer Dependency Map project database (Public Chronos 23Q4). The mean Chronos dependency scores are shown for multiple organ systems, including the pancreas (red). Negative scores indicate gene essentiality. D, Expression of SREBP target genes in PDAC tumor tissue compared with normal tissue is shown from two Oncomine data sets Pei and Badea (33). Blue, lower gene expression; red, higher gene expression; black, P > 0.05. Genes are grouped and color-coded by bioinformatic process (https://www.genepattern.org/#gsc.tab=0). E, The association of SREBP target genes with survival from patients with PDAC was queried using the Kaplan–Meier Plotter tool (https://kmplot.com/analysis/) and RNA sequencing datasets (n = 177 patients for each curve).

SREBP pathway activation is required for PDAC cell growth in low-serum conditions. A, PDAC cells (Pa03c and Pa16c) were cultured in either 10% or 1% FBS with indicated concentrations of 25-hydroxycholesterol (25-HC) for 72 hours, and cell growth was determined using an MTS assay with data normalized to 10% FBS untreated cells. Data are representative of two biological replicates with three technical replicates for each biological replicate. Error bar denotes SD. B, Immunoblot analysis of nuclear extracts from human Pa03c and Pa16c cells cultured in either 10% FBS, 1% FBS, or 1% FBS-containing 25-hydroxycholesterol (2.5 μmol/L) for 16 hours. Blots were probed for either SREBP1-N or SREBP2-N (arrow), and lysine-specific histone demethylase 1A (KDM1A) served as a loading control. The result is representative of two biological replicates. C, Pa03c and Pa16c cells were cultured under the same conditions as in B, and quantitative RT-PCR was performed for target genes of SREBP1 (SCD, FASN, and INSIG1) and SREBP2 (HMGCR, HMGCS1, and LDLR). GAPDH served as a control. Data are representative of two biological replicates with three technical replicates for each biological replicate. Error bar denotes standard deviation. Statistical significance was determined using one-way ANOVA and Tukey HSD test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. D, Immunoblot analysis of whole cell lysates from Pa03c and Pa16c cells cultured as in B for indicated SREBP target protein expression. The asterisk indicates a nonspecific band present in Pa03c and Pa16c cell lines. Actin served as a loading control. The result is representative of two biological replicates.

Site-1 protease activation of the SREBP pathway is required for PDAC cell growth in low-serum conditions. A, PDAC cells (Pa03c and Pa16c) were cultured in either 10% or 1% FBS with indicated concentrations of the Site-1 protease inhibitor PF-429242 for 72 hours, and cell growth was determined using an MTS assay with data normalized to 10% FBS untreated cells. Data are representative of two biological replicates with three technical replicates for each biological replicate. Error bar denotes SD. B, Immunoblot analysis of nuclear extracts from human Pa03c and Pa16c cells cultured for 16 hours in either 10% FBS, 1% FBS, or 1% FBS-containing PF-429242 (10 μmol/L). Blots were probed for either SREBP1-N or SREBP2-N (arrow), and lysine-specific histone demethylase 1A (KDM1A) served as a loading control. The result is representative of two biological replicates. C, Pa03c and Pa16c cells were cultured under the same conditions as in B, and quantitative real-time PCR was performed for target genes of SREBP1 (SCD, FASN, and INSIG1) and SREBP2 (HMGCR, HMGCS1, and LDLR). GAPDH served as a control. Data are representative of two biological replicates with three technical replicates for each biological replicate. Error bar denotes standard deviation. Statistical significance was determined using one-way ANOVA and Tukey honestly significant difference (HSD) test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. D, Immunoblot analysis of whole cell lysates from Pa03c and Pa16c cells cultured as in B for indicated SREBP target protein expression. The asterisk indicates a nonspecific band present in Pa03c and Pa16c cell lines. Actin served as a loading control. The result is representative of two biological replicates.

FIGURE S5 – SCAP is required for PDAC cell growth and survival in low serum conditions.

Inhibition of both SREBP1 and SREBP2 is required to prevent PDAC cell and tumor growth. A, Immunoblot of Pa03c wild-type, SREBF1 KO, SREBF2 KO, SREBF1/2 double KO cells for indicated antibodies. Membrane-enriched extracts (20 μg) were harvested and probed for SREBP1 and SREBP2. Calnexin served as a loading control. B, Wild-type (WT) Pa03c, SREBF1 KO, SREBF2 KO, SREBF1/2 double KO, and MBTPS1 KO cells were cultured in either 10% FBS or 1% FBS for 7 days. Media were changed every 3 days. Plates were stained with crystal violet and quantification is shown (n = 3 per group). For each cell line, growth was normalized to the 10% FBS condition. Statistical significance was determined using two-way ANOVA and Tukey HSD test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ns, not significant. Error bar denotes SD. C, Nude mice were subcutaneously injected with 1 × 106 Pa03c cells of the indicted genotype in both flanks (two tumors per mouse). Once visible, tumors were measured, and volume calculated. Each group contained five mice. Error bar denotes SD. D, Individual tumor volumes at day 25, n = 10 tumors per group. Statistical significance was determined using one-way ANOVA and Dunnett test. Error bar denotes SD.

FIGURE S4 – SCAP is required for human PDAC tumor growth in mouse orthotopic xenograft models

Solid tumors undergo metabolic reprogramming when growth outstrips local nutrient supply. Lipids such as cholesterol and fatty acids are required for continued tumor cell proliferation, and oncogenic mutations stimulate de novo lipogenesis to support tumor growth. Sterol regulatory element-binding protein (SREBP) transcription factors control lipid homeostasis by activating genes required for lipid synthesis and uptake. SREBPs have been implicated in the progression of brain, breast, colon, liver, and prostate cancers. However, the role of the SREBP pathway and its central regulator SREBP cleavage activating protein (SCAP) in pancreatic ductal adenocarcinoma (PDAC) has not been studied in detail. Here, we demonstrated that pancreas-specific knockout of Scap has no effect on mouse pancreas development or function, allowing for examination of the role of Scap in the murine KPC model of PDAC. Notably, heterozygous loss of Scap prolonged survival in KPC mice, and homozygous loss of Scap impaired PDAC tumor progression. Using xenograft models, we showed that SCAP is required for human PDAC tumor growth. Mechanistically, chemical or genetic inhibition of the SREBP pathway prevented PDAC cell growth under low-serum conditions because of a lack of lipid supply. Highlighting its clinical importance, the SREBP pathway is broadly required across cancer cell lines, target genes are upregulated in human PDAC tumors, and increased expression of SREBP targets is associated with poor survival in patients with PDAC. Collectively, these results demonstrate that SCAP and SREBP pathway activity are required for PDAC cell and tumor growth, identifying SCAP as a potential therapeutic target for PDAC. SIGNIFICANCE:Our findings demonstrate that SREBP pathway activation is a critical part of the metabolic reprogramming that occurs in PDAC development and progression. Therefore, targeting the SREBP pathway has significant therapeutic potential.

Table S4. Pa03c cell microarray gene expression data

SCAP is required for human PDAC tumor growth in mouse subcutaneous xenograft models. A, Nude mice were subcutaneously injected with 5 × 105 Pa03c cells in both flanks (two tumors per mouse). Once visible, tumors were measured, and volume calculated. Each group contained six mice. Error bar denotes SD. Statistical significance was determined using the Student t test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. B, Individual tumor volumes at day 22, n = 12 tumors per group. Statistical significance was determined using the Student t test. C, Representative hematoxylin and eosin–stained sections of formalin-fixed tumor tissue from the mice in A showing a low magnification (4×) image (top row) and a higher magnification (20×) image (bottom row) of tumor sections. D, Nude mice were subcutaneously injected with 1 × 106 Pa16c cells in both flanks (two tumors per mouse) as in A–C. Each group contained six mice. Error bar denotes SD. Statistical significance was determined using the Student t test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. E, Individual tumor volumes at day 61, n = 12 tumors per group. Statistical significance was determined using the Student t test. F, Representative hematoxylin and eosin–stained sections of formalin-fixed tumor tissue from the mice in D showing a low magnification (2–4×) image (top row) and a higher magnification (20–40×) image (bottom row) of tumor sections. WT, wild-type.

FIGURE S1 – Scap is not required for development or function of the mouse pancreas.

Causes of death in KPC mouse cohorts

SCAP is required for PDAC cell growth under low-serum and low-lipid conditions. A, Pa03c cells were grown for 16 hours in a medium containing 10% FBS or 0.5% FBS. mRNA expression was determined using microarray, and differentially expressed genes were identified (Supplementary Table S4). Bioinformatic gene set enrichment analysis was performed against four common pathway datasets. Results are shown using a normalized enrichment score (NES) of >2.0. B, Wild-type (WT) and SCAP KO Pa03c cells were grown for 7 days in a medium containing 10% FBS or 1% FBS in the absence or presence of lipid supplements (1 mmol/L mevalonate, 5 μg/mL cholesterol in ethanol, 20 μmol/L sodium oleate, and 50 μg/mL LDL). Media were changed every 3 days. Plates were stained with crystal violet as a measure of cell proliferation. Quantification of crystal violet staining is shown (n = 3 per group). For each cell line, growth was normalized to the 10% FBS condition. Statistical significance was determined using two-way ANOVA and Tukey HSD test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ns, not significant. Error bar denotes SD. C, Cell growth assay of PDAC cell lines. WT, SCAP KO, and SCAP KO rescued cell lines were cultured in either 10% FBS supplemented with cholesterol (5 μg/mL), mevalonate (1 mmol/L) and oleate–albumin (20 μmol/L), or 10% LPDS with no additions for 7 days. Plates were stained with crystal violet. KEGG, Kyoto Encyclopedia of Genes and Genomes.

FIGURE S7 – SREBP pathway activation is required for PDAC cell growth in low serum conditions.

FIGURE S3 – Generation of human PDAC SCAP knockout cell lines