Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 2. KRASG12D increases SLC25A1 expression by activating GLI1. A, Consensus GLI1 binding site in SLC25A1 promoter. The reported consensus GLI1 binding site is GACCACCCA. The putative GLI1 binding site was identified in SLC25A1 promoter. P1 primers (red) are designed to flank the putative GLI1 binding sequence, while P2 primers (green) are designed for detecting negative binding as a control. The length of the detected fragment, including the binding site, is 0.2 kb. B, Correlative expression of GLI1 and SLC25A1 in 179 pancreatic adenocarcinoma tissues based on GEPIA analysis of PAAD dataset. The correlation coefficient was calculated by Spearman rank for R ¼ 0.66. C, PANC1 cells were used for a ChIP assay after GLI1 immunoprecipitation, with primers that flanked the putative GLI1 binding sequence (P1) and non-binding sites (P2). The enrichment was assayed by qRT-PCR. Also, the RT-PCR products at the end of 40 cycles were analyzed by agarose gel electrophoresis (bottom). D, IHC staining of GLI1 on pancreatic tissue sections of fElasCreERT and KrasG12D/þ mice (n ¼ 5; 400). E, Quantitation of GLI1 level in D. F, Western blot analysis of GLI1 in the pancreata of fElasCreERT and KrasG12D/þ mice (n ¼ 3). G, qRT-PCR of GLI1 in the pancreata of fElasCreERT and KrasG12D/þ mice (n ¼ 5). H, Western blot analysis of GLI1 in HPDE cells and pancreatic cancer BxPC3, MIA PaCa-2, AsPC1, PANC1, SW1990, and Su.86.86 cells using the stripped membrane from Fig. 1E. b-Actin was used as loading control as in Fig. 1E. I, Western blot analysis of GLI1 in cytosol and nucleus of HPDE, ASPC1, and PANC1 cells. J, PANC1 cells with or without GANT61 treatment at indicated concentrations for 72 hours and then the cells were harvested to isolate mRNA. The mRNA of SLC25A1 was analyzed by qRT-PCR. K, PANC1 cells were treated with different concentrations of GANT61 for 72 hours and the cell lysates were subjected to Western blot analysis for SLC25A1, GLI1, FASN, and ACSL1. L, PANC1 cells were treated with 20 mmol/L of GANT61 for 4 hours and the cells were collected for GLI1 ChIP assay with primers that flanked the putative GLI binding sequence. The results were analyzed by qPCR. PCR products were analyzed by agarose gel electrophoresis (bottom). M, Western blot analysis of the levels of GLI1 and SLC25A1 in wild-type (WT) and GLI1 knockout AsPC1 cells. N, Representative image (left) and quantification (right) of Transwell migration assays in wild-type and GLI1 knockout AsPC1 cells. O, Representative image (left) and quantification (right) of Transwell migration assaysin AsPC1 cells treated with or without GANT61 (30 mmol/L).Results are expressed as mean SD and were statistically evaluatedwith a t test. ns, not significant; , P < 0.05; , P < 0.01; , P < 0.0001.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Sequencing, Control, Immunoprecipitation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunohistochemistry, Quantitation Assay, Western Blot, Membrane, Knock-Out, Migration

Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 3. HFD challenge stimulates the KRAS-GLI1 axis to induce SLC25A1 expression. A, H&E staining and IHC staining of vimentin in pancreatic tissue sections of KrasG12D/þ mice fed with ND or high HFD (n ¼ 5; 200x). B, Quantitation of vimentin level in A. C, IHC staining of GLI1 (400) and SLC25A1 (200) on pancreatic tissue sections of KrasG12D/þ mice fed with ND or HFD (n ¼ 5). D, Quantitation of GLI1 and SLC25A1 levels in C. E, Western blot analysis for SLC25A1 and GLI1 in the pancreata of KrasG12D/þ mice fed with ND or HFD (n ¼ 3). F, qRT-PCR analysis of Slc25a1 gene expression in the pancreata of KrasG12D/þ mice fed with ND or HFD (n ¼ 5). G, IHC staining of ACSL1 (200) and FASN (200) on pancreatic tissue sections of KrasG12D/þ mice fed with ND or HFD (n ¼ 5). H, Quantitation of ACSL1 and FASN levels in G. I, Western blot analysis of FASN and ACSL1 in the pancreata of fElasCreERT and KrasG12D/þ mice fed with ND or HFD. J and K, Total citrate and FA levels in pancreatic tissues from KrasG12D/þ mice fed with ND or HFD. Results are expressed as mean SD and were statistically evaluated with a t test. , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Expressing, Staining, Immunohistochemistry, Quantitation Assay, Western Blot, Quantitative RT-PCR, Gene Expression

Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 4. Inhibition of GLI1 alleviates pancreatic precancerous lesions by decreasing SLC25A1 expression in KrasG12D/þ mice. A, Experimental scheme for B–H. Sixty-day-old male and female ND-fed KrasG12D/þ mice were treated with TAM to induce KRASG12D expression in pancreatic acinar cells. These mice were fed HFD for 6 weeks and then randomly separated into two groups with one group fed HFD with vehicle (n ¼ 5) and another group of mice fed with HFD plus GANT61 treatment (intraperitoneally twice per week at 50 mg/kg/time; n ¼ 4) for 6 weeks. The mice were analyzed at the age of 150 days. B, Body weight of the KrasG12D/þ mice with ND (n ¼ 5), HFD (n ¼ 5), or HFD plus GANT61 treatment (n ¼ 4). Treatment started at the sixth week of feeding with HFD, as indicated by a red arrow. C, Gross images for pancreatic tissues of HFD-fed KrasG12D/þ mice with or without GANT61 treatment. The pancreatic cysts are indicated by red arrowswith dark edges. D, Representative H&E staining of the pancreata, Alcian blue staining of acidic mucins, Sirius red staining of collagens on pancreatic tissue sections, and coimmunofluorescence of pancreatic amylase and CK19 in HFD-fed KrasG12D/þ mice with or without GANT61 treatment (n ¼ 5; 200). E, Quantitation of Alcian blue and Sirius red staining levels in D. F, IHC staining of SLC25A1 (200), FASN (200), and ACSL1 (200) on pancreatic tissue sections of HFD-fed KrasG12D/þ mice with or without GANT61 treatment (n ¼ 5). G, Quantitation of SLC25A1, FASN, and ACSL1 levels in F. Results are expressed as mean SD and were statistically evaluated with a t test. , P < 0.01; , P < 0.001; , P < 0.0001. H, Western blot analysis of GLI1, SLC25A1, ACSL1, and FASN in the pancreata of HFD-fed KrasG12D/þ mice with or without GANT61 treatment. I, De novo FAS from glucose is decreased in PANC1 cells following GANT61 treatment. U-13C6-glucose metabolic tracing profile for methyl-palmitate (Me-C16:0) and methyl-stearate (Me-C18:0) chain elongation in PANC1 cells after treatment with either 0.13% DMSO (Control; n ¼ 3) or GANT61 (30 mmol/L, also dissolved in 0.13% DMSO; n ¼ 2) for 24 hours. Free FAs were extracted by organic solvents, derivatized as FAMEs and analyzed by GC-MS. The intensity of the endogenous U-12C-Me-C16:0 (Mþ0) and U-12C-Me-C18:0 (Mþ0) and the metabolically labeled 13C-isotopomers of Me-C16:0 (Mþ2 Mþ16) and Me-18:0 (Mþ2 Mþ18) are indicated. The ratio of labeled over the total (unlabeled þ labeled) was calculated. Results are expressed as mean SEM and were statistically analyzed by the multiple unpaired Student t test with Welch correction. , P < 0.05.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Staining, Quantitation Assay, Immunohistochemistry, Western Blot, Control, Gas Chromatography-Mass Spectrometry, Metabolic Labelling, Labeling

Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 6. SLC25A1 inhibitor suppressed PDAC development in KrasG12D/þ mice under a long-term HFD challenge. A, Experimental scheme for B–D. Sixty-day-old ND-fed KrasG12D/þ mice were treated with TAM to induce KRASG12D expression in pancreatic acinar cells. These male and female mice were randomly separated into two groups, with one group of mice (n ¼ 5) fed with HFD for 12 weeks, and pancreatic tissue samples were collected at 150 days of age as indicated by the control group. Another group of mice was fed HFD for 12 weeks and then fed with HFD plus CTPI-2 treatment (intraperitoneally twice per week at 50 mg/kg/time; n ¼ 5) for additional 6 weeks as indicated by the CTPI-2 group. B, Gross images of HFD-fed KrasG12D/þ mice with or without CTPI-2 treatment. C, Representative histology shown by H&E staining, Alcian blue staining, and Sirius red staining on pancreatic tissue sections from the HFD-fed control group and CPTI-2 treatment group (200). D, Quantitation of Alcian blue and Sirius red staining levels in C. E, Hypothetical model of the KRAS-GLI1-SLC25A1 axis that regulates lipid metabolism. Oncogenic KRASG12D upregulates SLC25A1 expression by increasing GLI1 transcription. More SLC25A1 exports more citrate from mitochondria to the cytosol for FAS, leading to severe lipid accumulation. Thus, SLC25A1 acts as a nodal transporter to enhance lipid metabolism. Targeted inhibition of the KRASG12D-GLI1-SLC25A1 axis by GANT61 or CTPI-2 could suppress cytosolic citrate and FA accumulation under HFD, therefore hindering pancreatic tumorigenesis. Results are expressed as mean SD and were statistically evaluated with a t test. , P < 0.01; , P < 0.0001.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Staining, Quantitation Assay, Inhibition