Oncogenic KRAS Reduces Expression of FGF21 in Acinar Cells to Promote Pancreatic Tumorigenesis in Mice on a High-Fat Diet

Abstract

Background & aims: Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis.

Methods: We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (Kras^G12D/+ mice) and fElas^CreERT mice (controls). Kras^G12D/+ mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate.

Results: Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed Kras^G12D/+ had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from Kras^G12D/+ mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed Kras^G12D/+ mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed Kras^G12D/+ mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed Kras^G12D/+ mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle.

Conclusions: Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer.

Keywords: FGFR1; Gene Regulation; KLB; Signaling.

Figure 1.. Pancreatic acinar cells are both a source and a target of FGF21.

All mice in A-H were fed a ND, induced by TM at 70 days of age, and sacrificed one week post-TM. (A) qRT-PCR analysis of Fgf21 expression in the liver (n=11) and pancreas (Panc, n=11) of fElas^CreERT mice. (B) IHC analysis of FGF21 on the liver and pancreas sections. 200x. (C) Western blot analysis for FGF21 levels. (D) Semi-quantitative RT-PCR analysis for Fgfrl in the liver (L) and pancreas (P). (E-F) qRT-PCR analysis for Fgfrl and Klb in the liver (n=9) and pancreas (n=9). (G) Western blot analysis for KLB in the pancreas (p). (H) IHC analysis of KLB in pancreatic sections. 200x. All mice in I-K were fed a ND, induced by TM at 70 days of age, and sacrificed at 7 months of age. Comparative qRT-PCR analysis for Fgf21 (I), Fgfr1 (J) and Klb (K) in the liver (n=7), pancreas (n=7), and adipose tissue (n=7) of fElas^CreERT mice. Open arrow, acinar cells. Closed arrow, islet cells. Data are means ± SEM. *, p<0.05; **, p<0.01; ****, p<0.0001; ns, not significant.

Figure 2.. KRAS^G12D downregulates Fgf21 gene expression in acinar cells.

(A) IHC analysis for FGF21 levels in tissue arrays of human normal pancreas (n=45) and PDAC (n=59) (40x). Inset: 200x. (B) Quantitation of human FGF21 levels as described in 2A. (C) Human FGF21 expression in 39 pairs of normal and tumor tissues resected from pancreatic cancer patients as collected in the GEO database (https://www.ncbi.nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS4103:221433_at). (D) qRT-PCR analysis of FGF21 in human pancreatic cancer cell lines carrying KRAS mutations. Human normal pancreatic ductal epithelial (HPDE) cells served as a control. All mice in E-I were fed a ND. (E) qRT-PCR analysis of pancreatic Fgf21 in 7-month-old Kras^G12D/+ (n=11) or fElas^CreERT mice (n=5) mice five months post-TM. (F) qRT-PCR analysis of pancreatic Fgf21 in 40-day-old Kras^G12D/+ mice one week post-TM (n=7) or without TM (n=9). (G) Representative histology of the pancreata as in 2F. 200x. (H) qRT-PCR analysis of pancreatic Fgf21 of two-month-old Ptf1a^CreERT;Kras^G12D/+ mice two weeks post-TM (n=3) or without TM (n=3). (I) qRT-PCR analysis of pancreatic Pparg in 40-day-old Kras^G12D/+ mice one week post-TM (n=7) or without TM (n=9). (J) qRT-PCR analysis of FGF21 in Panc-1 cells with Rosiglitazone treatment. Data are mean ± SD. *, p<0.05; **, p<0.01; ***, p<0.001; **** p<0.0001.

Figure 3.. Replenishment of FGF21 suppresses KRAS^G12D-induced pancreatic inflammation and PanIN lesions under ND conditions.

(A) Experimental scheme for B-E and G-J: 70-day-old ND-fed Kras^G12D/+;fElas^CreERT mice were treated with TM at 70 days of age to induce Kras^G12D/+ expression in acinar cells, and then treated with rhFGF21 (0.5 mg/kg body weight/day) (n=10) or PBS (n=8) for ten weeks. (B) IHC staining of COX-2 and F4/80, and Sirius Red staining of collagen on pancreatic sections of Kras^G12D/+ mice with indicated treatments. 200x. (C) Co-immunofluorescence (Co-IF) analysis of amylase and CK19 as indicated. 200x. (D-E) Quantitation of amylase+ areas or CK19+ areas on pancreatic sections (n=4) as described in 3C. (F) The explanted acinar cell 3D clusters isolated from 80-day-old Kras^G12D/+ mice one week post-TM were treated daily with rhFGF21 or PBS and then analyzed by brightfield images on day 1, H&E staining on day 4, and Co-IF staining of amylase and CK19 on day 4. (G) Alcian blue staining of acidic mucins and IHC staining of MUC5. (H) IHC analyses of Ki-67 (ND, n=4; ND+rhFGF21, n=4) and Cyclin D1 (ND, n=5; ND+rhFGF21, n=3). (I-J) Quantitation of Ki-67 and Cyclin D1 levels in 3H. Results were expressed as mean ± SD. **, p<0.01.

Figure 4.. Supplementation of FGF21 suppresses HFD and KRAS^G12D induced PanIN lesions and PDAC.

(A) Experimental scheme: 70-day-old ND-fed Kras^G12D/+;fElas^CreERT mice were treated with TM to induce Kras^G12D/+ expression and randomly separated into ND, HFD, or HFD with rhFGF21 treatment groups (n=8 per group) for ten weeks. The age-matched ND-fed fElas^CreERT mice (n=8) served as controls. (B) Gross images of Kras^G12D/+ mice (left) and the pancreata (right) after the indicated treatments. Closed arrows, multiple pancreatic cysts. (C) Histology of representative pancreata. Closed arrows, PanIN lesions. Open arrows, cancerous ductal structure. 100x. (D) Average grades of PanIN1/2 and PanIN3 lesions as in 4C. F21, rhFGF21. (E) Western blot analysis of pancreatic amylase and α-SMA as indicated. (F) Co-IF analysis of pancreatic amylase and CK19 or IHC analysis of MIST1. 200x. (G) Alcian blue+ staining of acidic mucins and IHC staining of MUC5. 200x. Data are mean + SD. *, p<0.05; **, p<0.01.

Figure 5.. Pharmacological FGF21 inhibits HFD and KRAS^G12D induced pancreatic inflammation and ductal cell proliferation.

All the samples were described as in Figure 4A. (A) IHC staining of COX-2, F4/80, and CD3, as well as Sirius Red staining for collagen on pancreatic sections of Kras^G12D/+ mice. 200x. (B-C) qRT-PCR analysis of pancreatic Cxc/5 and Tgfbl expression in Kras^G12D/+ mice with ND (n=4), HFD (n=4) or HFD+rhFGF21 (n=5). The age-matched ND-fed fElas^CreERT mice (n=5) served as a control. (D) Pancreatic RAS activity with the indicated treatments. (E) IHC staining of pancreatic cyclin D1 and Ki-67. 200x. Data are mean ± SD. *, p<0.05; **, p<0.01; ****, p<0.0001.

Figure 6.. Pharmacological FGF21 inhibits HFD-promoted liver and adipose tissue inflammation

All mice were induced by TM at 70 days of age and then treated as indicated for 10 weeks prior to analyses. qRT-PCR analysis of Ccl2, Ccl4, CD68, CD11b, and Tgfbl in adipose tissues (A-E) and Ccl2, Ccl4, CD68, and CD11b in the liver (F-I) of Kras^G12D/+ mice with ND (n=5), HFD (n=6), or HFD+rhFGF21 (n=5). The ND-fed fElas^CreERT mice (n=5) were controls. Data are mean ± SD. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 7.. Pharmacological FGF21 prevents liver metastasis and prolongs KRAS^G12D mice survival.

70-day-old mice were treated with TM to induce fElas^CreERT or Kras^G12D/+ expression, and then fed a ND or a HFD with or without rhFGF21 until they aged. The ND-fed fElas^CreERT mice (n=7) were controls. (A) Body weight of the Kras^G12D/+ mice with ND (n=7), HFD (n=7), or HFD+rhFGF21 treatment (n=7) until the HFD group aged. (B) Survival of the Kras^G12D/+ mice with ND (n=7), ND+rhFGF21 (n=10), HFD (n=7), or HFD+rhFGF21 treatment (n=7). The HFD-fed Kras^G12D/+ mice served as a control for statistical comparison. (C) Histological changes in pancreata of Kras^G12D/+ mice with the indicated treatments upon aging. 200x. (D) Liver metastasis. Upper panel, gross images of the livers. Middle panel, H&E stained sections. Lower panel, IHC analysis of CK19. (E) Schematic summary of the role of pancreatic FGF21 in the HFD and oncogenic KRAS mediated pancreatic tumorigenesis. 200x. Data are mean ± SD. *, p<0.05; ***, p<0.001; ****, p<0.0001.