Retinoid signaling in pancreatic cancer, injury and regeneration

Abstract

Background: Activation of embryonic signaling pathways quiescent in the adult pancreas is a feature of pancreatic cancer (PC). These discoveries have led to the development of novel inhibitors of pathways such as Notch and Hedgehog signaling that are currently in early phase clinical trials in the treatment of several cancer types. Retinoid signaling is also essential for pancreatic development, and retinoid therapy is used successfully in other malignancies such as leukemia, but little is known concerning retinoid signaling in PC.

Methodology/principal findings: We investigated the role of retinoid signaling in vitro and in vivo in normal pancreas, pancreatic injury, regeneration and cancer. Retinoid signaling is active in occasional cells in the adult pancreas but is markedly augmented throughout the parenchyma during injury and regeneration. Both chemically induced and genetically engineered mouse models of PC exhibit a lack of retinoid signaling activity compared to normal pancreas. As a consequence, we investigated Cellular Retinoid Binding Protein 1 (CRBP1), a key regulator of retinoid signaling known to play a role in breast cancer development, as a potential therapeutic target. Loss, or significant downregulation of CRBP1 was present in 70% of human PC, and was evident in the very earliest precursor lesions (PanIN-1A). However, in vitro gain and loss of function studies and CRBP1 knockout mice suggested that loss of CRBP1 expression alone was not sufficient to induce carcinogenesis or to alter PC sensitivity to retinoid based therapies.

Conclusions/significance: In conclusion, retinoid signalling appears to play a role in pancreatic regeneration and carcinogenesis, but unlike breast cancer, it is not mediated directly by CRBP1.

Figure 1. RA signalling activity in adult Retinoic Acid Response Element (RARE-LacZ) reporter mouse pancreas showing positive pancreatic islet (A) and rare positive exocrine cells (B), some of which may represent centro-acinar cells (based on location and morphology) (C–D).

Figure 2. H&E staining of pancreata from RARE-LacZ mice treated with caerulein (A), and subsequent recovery for 2 weeks (B), 4 weeks (C) and 6 weeks (D).

RA signalling activity in pancreata from RARE-LacZ mice treated with caerulein (E), and subsequent recovery for 2 weeks (F), 4 weeks (G) and 6 weeks (H). Increased retinoid signaling activity was observed in a significant proportion of acinar cells immediately following cessation of caerulein treatment (E), with retinoid activity peaking at 2 weeks (F), diminishing at 4 weeks (G) and returning to near normal levels following 6 weeks of recovery (H).

Figure 3. RA signalling activity in DMBA-induced tumours (A–B) and in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre;RARE-LacZ pancreatic tumours (C–D) and mPanIN lesions (E–F).

In tumors from DMBA treated RARE-LacZ mice, a distinct absence of any cells exhibiting active retinoid signaling was observed (B). In LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre;RARE-LacZ pancreatic tumours and precursor lesions, there was also a complete absence of retinoid signaling activity (D and F respectively).

Figure 4. CRBP1 expression in (A) normal pancreas, (B) PC positive for CRBP1, (C) PC demonstrating partial loss of CRBP1 expression, (D) PC completely lacking CRBP1 expression, (E) PanIN-1A lesion negative for CRBP1 expression, (F) PanIn-1B (*) lesion negative for CRBP1 expression with positive adjacent normal ductal epithelium.

Figure 5. mRNA expression (A) and protein expression (B) of CRBP1 in PC cell lines.

(C) CRBP1 methylation status in PC cell lines and (D) restoration of CRBP1 expression with combination treatment of MiaPaCa2 cells with 5-Aza and TSA. (E) CRBP1 knockdown in stably transfected HPDE-EcoR cells. (F) HPDE cells grown in 3D demonstrate no change in morphology between siCRBP1 cells and scrambled control.