Genotyping and expression analysis of IDO2 in human pancreatic cancer: a novel, active target

Abstract

Background: The recently discovered indoleamine 2,3-dioxygenase-2 (IDO2) gene has 2 functional polymorphisms that abolish its enzymatic activity. We hypothesize that expression of the IDO2 enzyme in primary pancreatic ductal adenocarcinomas (PDA) can help cancer cells evade immune detection.

Study design: Because the IDO2 enzyme might be the preferential target of d-1-methyl-tryptophan, a clinical lead inhibitor of IDO currently being evaluated in phase I trials, we sequenced IDO2 in 36 pancreatic specimens and evaluated its expression.

Results: We found that 58% (21 of 36) of cases were heterozygous for the R248W polymorphism; 28% (10 of 36) were homozygous wild-type; and only 14% (5 of 36) were homozygous for the functionally inactive polymorphism. As for the Y359STOP polymorphism, we found that 27% (10 of 36) of cases were heterozygous, 62% (22 of 36) were homozygous wild-type, and only 11% (4 of 36) were homozygous for this functionally inactive allele. Ruling out the possibility of compound polymorphic variants, we estimated 75% of our resected patient cohort had an active IDO2 enzyme, with a conservative estimate that 58% of the patients had at least 1 functional allele. IDO2 was expressed in PDA tissue from each genetically polymorphic subgroup. We also detected IDO2 protein expression in the genetically distinct pancreatic cancer cell lines after exposure with interferon-gamma.

Conclusions: This is the first study to report IDO2 expression in PDA and related cancers indicating that IDO2 genetic polymorphisms do not negate interferon-gamma-inducible protein expression. Taken together, our data strongly suggest that the clinical lead compound d-1-methyl-tryptophan might be useful in treatment of PDA.

Figure 1

Representative chromatograms of direct sequencing of patient constitutional genomic DNA showing the three possible sequences of homozygous, heterozygous, or wild-type sequence: R235W polymorphism (left) and Y359STOP (right).

Figure 2

Functionally active IDO2 alleles are frequently found in PDA patients. A) Percent distribution of alleles for the R235W and the Y359STOP polymorphisms in our patient cohort. B) Combined distribution of amino acid changes when surveying both polymorphisms in our patient cohort. In the corresponding legend: both active is the presence of wild type sequence present on both alleles (R/R - Y / Y); both inactive is the presence of a functional polymorphism on both alleles (i.e., R /W -stop / stop, R /R– stop/stop, W / W - Y / Y, W/W - Y / stop); heterozygous is the presence of at least one functional allele; het/Inactive ? indicates that direct sequencing could not determine if the polymorphism was located on the same alleles. The bar on the right indicates that the majority of the alleles likely translate into active functional IDO2 enzyme.

Figure 2

Functionally active IDO2 alleles are frequently found in PDA patients. A) Percent distribution of alleles for the R235W and the Y359STOP polymorphisms in our patient cohort. B) Combined distribution of amino acid changes when surveying both polymorphisms in our patient cohort. In the corresponding legend: both active is the presence of wild type sequence present on both alleles (R/R - Y / Y); both inactive is the presence of a functional polymorphism on both alleles (i.e., R /W -stop / stop, R /R– stop/stop, W / W - Y / Y, W/W - Y / stop); heterozygous is the presence of at least one functional allele; het/Inactive ? indicates that direct sequencing could not determine if the polymorphism was located on the same alleles. The bar on the right indicates that the majority of the alleles likely translate into active functional IDO2 enzyme.

Figure 3

IDO2 protein expression in pancreatic ductal adenocarcinoma ex vivo. A) Weak cytoplasmic IDO2 staining in well differentiated pancreatic ductal adenocarcinoma heterozygous for the R235W polymorphism. B) Moderate cytoplasmic IDO2 staining in well differentiated pancreatic adenocarcinoma homozygous for the R235W polymorphism. Both panels A and B were wild type at Y359. C) Moderate cytoplasmic staining in poorly differentiated pancreatic ductal adenocarcinoma heterozygous for the STOP polymorphism (Y359STOP). D) Cytoplasmic IDO2 staining in moderately differentiated PDA homozygous for the STOP polymorphism (Y359STOP). Both panels C and D were wild type at R235.

Figure 4

IDO2 expression in pancreatic cancer cell lines (Hs766T, CAPAN1, and MiaPaCa2 cells) is expressed only after IFN-γ exposure (750U/ml) for 48 h. A) IDO protein expression assessed by immunoblot analysis. Equal protein loading was confirmed by Fast Green staining of the membrane. Amino acid polymorphic sequences of the cell lines are: Hs766T (wild type at codon R235, harbored a homozygous premature stop codon at 346), CAPAN1 (wild type at codon R235, wild type at codon 359), and MiaPaCa2 (homozygous at codon R235W, wild type at codon 359). Two additional pancreatic cancer cell lines were sequenced (PL-5 and BxPC3) and both were heterozygous at codon R235W and wild type at codon 359. B) CAPAN1 cells exposed to IFN-γ (750U/ml) for 48 h. IDO2 protein detected in the cytoplasm via immunoflouresence (Alexa Fluor 488 staining, green). DAPI, blue stain, represents nuclear staining.