The potential of retinoic acid receptors as prognostic biomarkers and therapeutic targets in gastric cancer

Abstract

Background: Gastric cancer is a heterogeneous collection of tumors characterized by low survival rates. All-trans retinoic acid (retinoic-acid) is a clinically useful therapeutic agent belonging to the chemical family of retinoids, which consists of both natural and synthetic derivatives of vitamin-A. Retinoids are essential components of the normal diet and they regulate different physiological processes. From a therapeutic point of view, retinoic-acid is the first example of clinically useful differentiating agent. Indeed, the differentiating properties of this compound have promoted the use of retinoic-acid as a standard of care in Acute-Promyelocytic-Leukemia, a rare form of acute myeloid leukemia. In this study, we determine the RNA expression of the six isoforms of Retinoic-Acid-Receptors (RARα/RARβ/RARγ/RXRα/RXRβ/RXRγ) in view of their potential use as gastric cancer progression markers and/or therapeutic targets. In addition, we evaluate associations between the expression of these receptors and a simplified molecular classification of stomach tumors as well as the clinical characteristics of the cohort of patients analyzed. Finally, we define the prognostic value of the various Retinoic-Acid-Receptors in gastric cancer.

Methods: In this single institution and retrospective RAR-GASTRIC study, we consider 55 consecutive gastric cancer patients. We extract total RNA from the pathological specimens and we perform a NanoString Assay using a customized panel of genes. This allows us to determine the expression levels of the RAR and RXR mRNAs as well as other transcripts of interest.

Results: Our data demonstrate ubiquitous expression of the RAR and RXR mRNAs in gastric cancers. High levels of RARα, RARβ, RXRα and RXRβ show a significant association with stage IV tumors, "de novo" metastatic disease, microsatellite-stable-status, epithelial-to-mesenchymal-transition, as well as PIK3CA and TP53 expression. Finally, we observe a worse overall-survival in gastric cancer patients characterized by high RARα/RARβ/RARγ/RXRβ mRNA levels.

Conclusions: In gastric cancer, high expression levels of RARα/RARβ/RARγ/RXRβ transcripts are associated with poor clinical and molecular characteristics as well as with reduced overall-survival. Our data are consistent with the idea that RARα, RARβ, RARγ and RXRβ represent potential prognostic markers and therapeutic targets of gastric cancer.

Keywords: gastric cancer; prognosticbiomarkers; retinoic-acid-receptors; retrospective-clinical-study; therapeutic-targets.

Figure 1

Expression levels of the mRNAs coding for different marker proteins in gastric cancer specimens. The bar-graphs illustrate the expression levels of the indicated mRNAs in the tumor samples of each cohort patient, which were determined with the use of the NanoString nCounter Analysis System. The genes considered are involved in EBV (Epstein-Barr-Virus) positivity (EBER1), in EMT (Epithelial to Mesenchymal Transition; CDH1/ZEB1), in the control of the TP53 pathway (TP53/CDKN1A/MDM2), in the molecular landscape of gastric cancer (PIK3CA) and in the control of the MMR (Mutation-Mismatch-Repair; MLH1/MSH2/MSH6/PMS2) process. The number and percentage of patients (black values) presenting with the indicated expression quartiles (black blue and red values) of the various mRNAs considered. *We determined the normalized expression-levels (see Materials and Methods) of the indicated mRNAs in tumor tissues and we calculated the expression-level percentiles. We divided the calculated expression-level percentiles in tertiles, as indicated. Normalization mRNAs = B2M (Beta-2-Microglobulin); GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase); HPRT1 (Hypoxanthine-Phosphoribosyltransferase-1); RPL19 (Ribosomal-Protein-L19). Acronyms of the mRNAs considered: CDH1, Cadherin-1; ZEB1, Zinc-Finger-E-Box-Binding Homeobox1; TP53 = Tumor-Protein-P53, CDKN1A, Cyclin-Dependent-Kinase-Inhibitor-1A; EBER1, Epstein–Barr-Virus–Encoded-small-RNA1; MDM2, MDM2-Proto-Oncogene; MLH1, MutL-Homolog-1; MSH2, MutS-Homolog-2; MSH6, MutS-Homolog-6; PIK3CA, Phosphatidylinositol-4,5-Bisphosphate-3-Kinase-Catalytic-Subunit-Alpha; PMS2, PMS1-Homolog-2, Mismatch-Repair-System-Component; MSI, MicroSatellite-Instability; MSS, MicroSatellite-Stability.

Figure 2

RAR/RXR mRNA levels in gastric cancer specimens. The bar-graphs illustrate the expression levels of the indicated RAR/RXR mRNAs in the tumor samples of each cohort patient, which were determined with the use of the NanoString nCounter Analysis System. The number and percentage of patients (black values) presenting with the indicated quartile (black blue and red values) of RARα, RARβ, RARγ, RXRα, RXRβ and RXRγ mRNA levels.

Figure 3

Associations between RARs levels and the clinical/molecular characteristics of gastric cancers. The box plots show the relative levels of the RARα (A), RARβ (B) and RARγ (C) mRNAs in the indicated subgroups of gastric cancers. The relative levels of the 3 transcripts are grouped into expression quartiles, as indicated. The statistical-significance p-values of the correlations between the calculated RARα/RARβ/RARγ mRNA levels and the indicated clinical/molecular characteristics of the gastric cancer samples are shown in red above the corresponding box plots. Each panel illustrates only the p-values of the comparisons reaching statistical significance. PIK3CA, Phosphatidylinositol-4,5-Bisphosphate-3-Kinase-Catalytic-Subunit-Alpha; CDKN1A, Cyclin-Dependent-Kinase-Inhibitor-1A; EMT, Epithelial-to-Mesenchymal-Transition; MMR, DNA-Mismatch-Repair; MSI, MicroSatellite-Instability; MSS, MicroSatellite-Stability; TP53, Tumor-Protein-P53.

Figure 4

Associations between the expression of RXRs and the clinical/molecular characteristics of gastric cancers. The box plots show the relative levels of the RXRα (A), RXRβ (B) and RXRγ (C) mRNAs in the indicated subgroups of gastric cancers. The statistical-significance p-values of the correlations between the calculated RXRα/RXRβ/RXRγ mRNA levels and the indicated clinical/molecular characteristics of the gastric cancer samples are shown in red above the corresponding box plots. Each panel illustrates only the p-values of the comparisons reaching statistical significance. PIK3CA, Phosphatidylinositol-4,5-Bisphosphate-3-Kinase-Catalytic-Subunit-Alpha; CDKN1A, Cyclin-Dependent-Kinase-Inhibitor-1A; EMT, Epithelial-to-Mesenchymal-Transition; MMR, DNA-Mismatch-Repair; MSI, MicroSatellite-Instability; MSS, MicroSatellite-Stability; TP53, Tumor-Protein-P53.

Figure 5

Associations between the overall-survival of gastric cancer patients and the expression levels of RARα/RARβ/RARγ/RXRβ mRNAs. The associations of the RARα (A), RARβ (B), RARγ (C) and RXRβ (D) mRNA expression levels and overall-survival values are illustrated by the curves. The expression levels of the various RAR and RXR isoforms are gathered binomially using the indicated expression ranges (IQ). The red curves illustrate the overall-survival rates of patients whose gastric cancer tumors are characterized by high levels of the indicated Retinoic-Acid-Receptor. The blue curves illustrate the overall-survival rates of patients whose gastric cancer tumors are characterized by low levels of the indicated Retinoic-Acid-Receptor. The associations were explored with the use of the COX proportional Hazard-Ratios (HRs). The analysis results are expressed as HRs and 95% Confidence-Intervals (95%-CIs). Survival curves are estimated according to the Kaplan-Meier method and compared using the log-rank test. Statistical significance is set at p<0.05 for bilateral tests.