Increased Bcl-xL Expression in Pancreatic Neoplasia Promotes Carcinogenesis by Inhibiting Senescence and Apoptosis

Abstract

Background & aims: Bcl-xL, an anti-apoptotic Bcl-2 family protein, is overexpressed in 90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, Bcl-xL expression in pancreatic intraepithelial neoplasias (PanINs) and its significance in PDAC carcinogenesis remain unclear. The aim of this study was to elucidate the significance of Bcl-xL expression in PanINs.

Methods: We investigated the expression levels of Bcl-xL in pancreas-specific KrasG12D (P-KrasG12D) mice and human PanINs and PDAC. We examined the impact of Bcl-xL expression on Kras-mutated pancreatic neoplasia using Bcl-xL-overexpressing P-KrasG12D mice and Bcl-xL-knockout P-KrasG12D mice.

Results: In P-KrasG12D mice, the number of PanINs increased and their grades progressed with age. In total, 55.6% of these mice developed PDAC at 12-14 months. According to the immunohistochemistry of mouse pancreas and human resected specimens, Bcl-xL expression was increased significantly in PanIN-1 compared with that in normal pancreatic ducts, and augmented further with the progression of pancreatic neoplasia in PanIN-2/3 and PDAC. Oncogene-induced senescence was observed frequently in PanIN-1, but rarely was detected in PanIN-2/3 and PDAC. Bcl-xL overexpression significantly accelerated the progression to high-grade PanINs and PDAC and reduced the survival of P-KrasG12D mice. Bcl-xL overexpression in P-KrasG12D mice suppressed oncogene-induced senescence in PanIN-1 and inhibited apoptosis in PanIN-3. Bcl-xL deficiency in P-KrasG12D mice induced cellular senescence in PanIN-2/3.

Conclusions: Bcl-xL expression increases with the progression from PanIN-1 to PDAC, whereas oncogene-induced senescence decreases. Bcl-xL overexpression increases PDAC incidence rates by inhibiting oncogene-induced senescence and apoptosis in PanINs. Conversely, Bcl-xL deficiency induced senescence in PanINs. Anti-Bcl-xL treatments may have the potency to suppress the progression from PanINs to PDAC.

Keywords: Bcl-2 Family Protein; IHC, immunohistochemistry; KO, knockout; Kras; P-KrasG12D, Pdx1-Cre LSL-KrasG12D; PDAC, pancreatic ductal adenocarcinoma; PanIN, pancreatic intraepithelial neoplasia; PanINs; SA–β-gal, senescence-associated β-galactosidase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; Tg, transgenic; siRNA, small interfering RNA.

Figure 1

Bcl-xL expression levels are enhanced gradually from PanIN-1 to PDAC in P-KrasG12D mice. (A–C) P-KrasG12D mice (Pdx1-Cre; LSL-KrasG12D) (n = 12 at 2 mo, 10 at 4 mo, 6 at 7 mo, and 9 at 12–14 mo) or wild-type littermates (Pdx1-Cre) (n = 10 at 2 mo, 6 at 4 mo, 4 at 7 mo, and 10 at 12–14 mo) were killed at the indicated months. (A) Representative pictures of H&E staining. Scale bar: 100 μm (original magnification, ×200). (B) The percentage of normal pancreas in defined area. *P < .05. (C) The number of PanINs per defined area in the pancreas sections of P-KrasG12D mice. (D) Representative images of Bcl-xL immunohistochemical staining of pancreas sections in wild-type mice and P-KrasG12D mice at 12–14 months. Scale bar: 100 μm (original magnification, ×200 in the upper row and ×400 in the lower row; width of insets: 50 μm). (E) Total score for the immunostaining of Bcl-xL in normal pancreatic ducts from wild-type mice (n = 5) and PanINs/PDAC from P-KrasG12D mice at 12–14 months (n = 5). (F) Western blot of anti-apoptotic Bcl-2 family proteins in the pancreas of P-KrasG12D mice.

Figure 2

Bcl-xL expression levels are increased in human PanINs and PDAC. Human resected pancreatic tissues of 9 patients with PDAC accompanied by PanIN lesions and 7 patients with other types of pancreatic tumors were stained with H&E and anti–Bcl-xL antibody. Given that the pancreatic tissues of 9 patients with PDAC contained many PanINs in the nontumor area, normal pancreatic ducts were evaluated using nontumor areas of pancreatic tissues from 7 patients with other types of pancreatic tumors. (A) Representative images of H&E and Bcl-xL immunohistochemistry staining of pancreas sections. Scale bar: 100 μm (original magnification, ×200); width of insets: 100 μm. (B) Total score for the immunostaining of Bcl-xL. *P < .05.

Figure 3

Bcl-xL–overexpressing mice do not show any phenotype under physiological conditions. Bcl-xL Tg mice and their control littermates were killed at 2 and 12 months. (A) Western blot analysis of Bcl-xL in the pancreas at 2 months. (B) Representative images of H&E staining of pancreas sections at 2 and 12–14 months. Scale bar: 100 μm (original magnification, ×200). (C) Pancreas/body weight ratio and serum levels of amylase and lipase at 2 months (n = 5 in Bcl-xL Tg mice, n = 4 in control mice).

Figure 4

Bcl-xL overexpression accelerates the incidence rate of PDAC and reduces survival time in P-KrasG12D mice. P-KrasG12D mice and Bcl-xL Tg P-KrasG12D mice were killed at the indicated months. (A) Western blot of anti-apoptotic Bcl-2 family proteins in the pancreas at 4 months. (B) Representative images of pancreas sections at 4 months stained for Bcl-xL. Scale bar: 100 μm (original magnification, ×400); width of insets: 50 μm. (C) Macroscopic pictures of the pancreas at 4 months. Arrows, macroscopically evident tumors. (D) Representative images of H&E staining of pancreas sections. Scale bar: 100 μm (original magnification for A–F, ×40; G–I, ×200). (E) The ratio of microscopic and macroscopic PDAC ∗P < .05. (F) The percentage of normal pancreas at 2 months (n = 12 per each), 4 months (n = 10 or 11), and 7 months (n = 6 per each). *P < .05. (G) The number of PanINs per defined area in the pancreas sections of P-KrasG12D mice and Bcl-xL Tg P-KrasG12D mice (n = 12 per group) at 2 months ∗P < .05. (H) Kaplan–Meier survival analysis of P-KrasG12D (n = 11) and Bcl-xL Tg P-KrasG12D mice (n = 12). *P < .05.

Figure 5

Bcl-xL overexpression suppresses oncogene-induced senescence in low-grade PanINs and apoptosis in high-grade PanINs. (A and B) Pancreatic tissues of P-KrasG12D mice at 12–14 months and Bcl-xL Tg P-KrasG12D mice at 2 months were stained with H&E and TUNEL (n = 5 per group). (A) Representative images of H&E and TUNEL staining and (B) the percentage of TUNEL-positive cells. Scale bar: 100 μm (original magnification, ×400). (C–E) Pancreatic tissues of P-KrasG12D mice at 12–14 months and Bcl-xL Tg P-KrasG12D mice at 2 months were stained for SA–β-gal activity and stained with anti-p21 antibody (n = 5 per group). (C) Representative images of staining for SA–β-gal activity. Scale bar: 100 μm (original magnification, ×400). (D) Representative images of p21 staining of pancreas sections and (E) the percentage of p21-positive cells. Scale bar: 100 μm (original magnification, ×400). *P < .05.

Figure 6

Pancreas-specific Bcl-xL–knockout mice do not show any phenotype under physiological conditions. Bcl-xL KO mice (bcl-x^flox/floxPdx1-Cre) and their control littermates (bcl-x^flox/flox) were killed at the indicated months. (A) Western blot analysis of Bcl-xL in the pancreas at 2 months. (B) Representative images of H&E staining of pancreas sections at 6 months. Scale bar: 100 μm (original magnification, ×200). (C) Pancreas/body weight ratio and serum amylase and lipase levels at 6 months (n = 11 in Bcl-xL KO mice and n = 7 in control mice).

Figure 7

Bcl-xL deficiency increases senescence in high-grade PanINs. Bcl-xL KO P-KrasG12D mice (bcl-x^flox/floxPdx1-Cre LSL-KrasG12D) (n = 14) and their littermate P-KrasG12D mice (bcl-x^+/+Pdx1-Cre LSL-KrasG12D) (n = 6) were killed at 12 months. (A) Western blot of Bcl-xL in the pancreas. (B) Representative images of pancreas sections stained for Bcl-xL. Scale bar: 100 μm (original magnification, ×200). (C) Representative images of staining for SA–β-gal activity. Scale bar: 100 μm (original magnification, ×400). (D) Representative images of p21 staining in pancreas sections and (E) the percentage of p21-positive cells. Scale bar: 50 μm (original magnification, ×1200). *P < .05. (F) Representative images of H&E and TUNEL staining and (G) the percentage of TUNEL-positive cells. Scale bar: 100 μm (original magnification, ×400).

Figure 8

Oncogene-induced senescence is induced by Bcl-xL inhibition in vitro. PANC-1 and MIA Paca-2 cells were transfected with Bcl-xL siRNAs (si1, si2, si3) or negative control siRNA (NC) for 24 hours. After changing the medium, cells were incubated for an additional 24 hours (caspase-3/7 activity) and for 48 hours (WST assay, Western blot analysis, and SA–β-gal activity). (A) Caspase-3/7 activity in culture supernatants and cell viability was determined by the WST assay (n = 4). (B) Western blot of Bcl-xL and p21. (C) Representative images of PDAC cells stained for SA–β-gal activity and the percentage of SA–β-gal–positive cells. Scale bar: 100 μm. *P < .05.