Molecular determinants of retinoic acid sensitivity in pancreatic cancer

Abstract

Purpose: To identify a predictive molecular "signature" for sensitivity to retinoic acid in pancreatic cancer.

Experimental design: Fourteen patient-derived, low-passage pancreatic ductal adenocarcinoma (PDAC) lines with varied expression of fatty acid-binding protein 5 (FABP5) and cellular retinoic acid-binding protein 2 (CRABP2) were used to evaluate the response to all-trans retinoic acid (ATRA). Cell proliferation, apoptosis, and migration/invasion assays were used to measure the in vitro response. Tumor growth was monitored in subcutaneous xenografts in athymic nude mice for 4 weeks.

Results: Response to ATRA was observed to be dependent upon differential expression of FABP5 versus CRABP2. Thus, elevated FABP5 expression was associated with minimal cytotoxicity and tumor growth inhibition and a paradoxical increase in migration and invasion. Conversely, CRABP2 expression in the absence of FABP5 was associated with significant tumor growth inhibition with ATRA, even in gemcitabine-resistant tumors. The ATRA-resistant phenotype of FABP5(high)CRABP2(null) cells could be circumvented by ectopic expression of CRABP2. Alternatively, reexpression of endogenous CRABP2 could be enabled in FABP5(high)CRABP2(null) PDAC lines by exposure to decitabine and trichostatin A, thereby relieving epigenetic silencing of the CRABP2 gene promoter. Immunohistochemical staining for FABP5 in archival human tissue microarrays identifies a subset of cases (13 of 63, ~20%) which are negative for FABP5 expression and might be candidates for ATRA therapy.

Conclusions: The widely used agent ATRA deserves a "second look" in PDAC, but needs to be targeted to patient subsets with biopsy-proven FABP5-negative tumors, or be combined with a chromatin-modifying agent to reexpress endogenous CRABP2.

Figure 1. Expression Profile of FABP5 and CRABP2 in PDAC cell lines and in vitro responsiveness to ATRA

(A) Real time qRT-PCR with 14 patient-derived, low passage PDAC cell lines showing relative expression levels of FABP5 and CRABP2 normalized to β-actin. Mean of three experiments done in triplicates plotted with error bar denoting ± SEM. (B) Western blot analysis showing protein expression of FABP5 and CRABP2 in a panel of 13 PDAC lines, with actin as protein loading control. Three of 13 (23%) co-expressed both proteins, while 10 of 13 (76%) express one or the other binding protein. (C) ATRA treatment reduces in vitro growth of FABP5^nullCRABP2^high (Pa01C, Pa03C) cells, while FABP5^highCRABP2^null (Pa04C, Pa14C) are completely resistant even at the highest dose. Values are plotted relative to the DMSO treated control and represent mean of three independent experiments, done in triplicates. (D) Apoptosis assay measuring mitochondrial transmembrane depolarization using TMRM dye and measured by flow cytometry, plotted as mean of three independent experiments. Results from two FABP5^highCRABP2^null ATRA-resistant (Pa04C, Pa14C) and two FABP5^nullCRABP2^high ATRA-sensitive (Pa01C, Pa03C) lines are shown. (E) Invasion Assay using modified Boyden chamber and (F) wound healing assay for Pa01C and Pa04C. ATRA exposure significantly blocks invasion (E) and migration (F) in the FABP5^nullCRABP2^high Pa01C cells, while actually enhancing both parameters in the FABP5^highCRABP2^null Pa04C line. Scale bar is 100µm and 250µm in Figs E and F, respectively. Lower panels summarize mean of three independent experiments done in triplicates (after normalizing to growth in MTS assay). Error bar denotes ± SEM. ** P<0.01, *** P<0.001

Figure 2. In vivo therapeutic response to ATRA is dependent on relative expression of FABP5 and CRABP2

Athymic nude mice were injected subcutaneously with either Pa01C (FABP5^nullCRABP2^high) or Pa04C (FABP5^highCRABP2^null) and treated with either ATRA (Red line) or oil as control (Black line). (A) Mean of tumor volumes measured at indicated time intervals plotted for Pa01C (left) and Pa04C (right). Bar denotes mean of tumor weight (in mg) at the end of study. Significant tumor growth inhibition is observed in the FABP5^nullCRABP2^high Pa01C xenografts, while the FABP5^highCRABP2^null Pa04C xenografts are completely resistant. (B) Ki-67 staining on excised xenografts confirms significant reduction in proliferation in FABP5^nullCRABP2^high Pa01C xenografts, and a modest but significant increase in Ki-67 labeling in the FABP5^highCRABP2^null Pa04C xenografts. Representative light microscopic images shown at 40X objective lens magnification. Scale bar is 40µm. Right Bar denotes values plotted as mean of number of positive nuclei from 10 fields per section (5 sections per group). Error bar denotes ± SEM. * P<0.05, ** P<0.001. (C) TUNEL staining on excised xenografts demonstrates pronounced apoptotic nuclei in the ATRA-treated FABP5^nullCRABP2^high Pa01C xenografts, and minimal staining in the ATRA-treated FABP5^highCRABP2^null Pa04C xenografts. Representative images shown at 20X objective. Scale bar is 100µm. (D) Efficacy of single agent ATRA and combination therapy in Pa01C, a gemcitabine-resistant xenograft. Left Mean of tumor volumes measured at various time intervals plotted, treated with indicated drug combinations. Gemcitabine (50mg/kg) in saline was given twice a week. Middle Representative images of tumor-bearing mice from each group. Right Mean of tumor weight (in mg) at the culmination of study. Error bar denotes ± SEM.

Figure 3. CRABP2 overexpression in FABP5^highCRABP2^null Pa20C cell line

(A) Quantitative RT-PCR for FABP5 and CRABP2 expression normalized to β-actin. (B) Western Blotting for FABP5 and CRABP2 in parental Pa20C ("Vector") and CRABP2 over-expressing cell line Pa20C-CRABP2 ("CRABP2"). (C) Pa20C-CRABP2 cells demonstrate significantly reduced growth by MTS assay compared to Pa20C-vector cells (P<0.01) with ATRA treatment. (D) Pa20C-CRABP2 cells demonstrate significantly higher apoptosis by TMRM-based assay, compared to Pa20C-vector cells (P<0.001) with ATRA treatment. (E) Invasion and migration assays using modified Boyden chamber. Ectopic expression of CRABP2 significantly mitigates the paradoxical increase in invasion and migration observed with ATRA treatment in Pa20C cells. Bar denotes mean of three independent experiments done in triplicates (after normalizing to growth in MTS assay). Error bar denotes ± SEM. (F) Wound healing assay. Pa20C with or without CRABP2 overexpression treated with ATRA for 24hrs, before a wound was introduced and monitored for another 24 hrs. Scale bar is 250µm.

Figure 4. Immunohistochemical staining for FABP5 in a human PDAC tissue microarray

(A) Representative images of FABP5 staining representing 63 surgically resected PDAC cases are shown, including negatively-stained normal pancreatic ductal epithelium (left) and pancreatic ductal adenocarcinoma cells (middle, negatively-stained and right, positively-stained). (B) Summary of FABP5 staining pattern in the cases analyzed and the possible predicted outcome to ATRA treatment.