Functional analysis of the Aurora Kinase A Ile31 allelic variant in human prostate

Abstract

Overexpression of the centrosome-associated serine/threonine kinase Aurora Kinase A (AURKA) has been demonstrated in both advanced prostate cancer and high-grade prostatic intraepithelial neoplasia lesions. The single-nucleotide polymorphism T91A (Phe31Ile) has been implicated in AURKA overexpression and has been suggested as a low-penetrance susceptibility allele in multiple human cancers, including prostate cancer. We studied the transcriptional consequences of the AURKA Ile31 allele in 28 commercial normal prostate tissue RNA samples (median age, 27 years). Significant overexpression of AURKA was demonstrated in homozygous and heterozygous AURKA Ile31 prostate RNA (2.07-fold and 1.93-fold, respectively; P < .05). Expression levels of 1509 genes differentiated between samples homozygous for Phe31 alleles and samples homozygous for Ile31 alleles (P = .05). Gene Ontology classification revealed overrepresentation of cell cycle arrest, ubiquitin cycle, antiapoptosis, and angiogenesis-related genes. When these hypothesis-generating results were subjected to more stringent statistical criteria, overexpression of a novel transcript of the natural killer tumor recognition sequence (NKTR) gene was revealed and validated in homozygous Ile31 samples (2.6-fold; P < .05). In summary, our data suggest an association between the AURKA Ile31 allele and an altered transcriptome in normal non-neoplastic prostates.

Keywords: Aurora Kinase A; NKTR; cancer; overexpression; prostate.

Figures

Figure 1

Quantitative RT-PCR analysis of the AURKA gene in normal prostate RNA samples homozygous for AURKA Phe31, heterozygous for Phe31/Ile31, and homozygous for Ile31 alleles. Values represent mRNA expression levels that were obtained from 10 ng of total RNA divided by NPC1 mRNA values (cDNA obtained from 1 µg of total RNA). Relative expression units were calculated by a standard curve using LightCycler 5.1 software (Roche Applied Bioscience). n, number of RNA samples tested.

Figure 2

Significant overexpression of the novel NKTR transcript in prostate RNA samples homozygous for the AURKA Ile31 compared to prostate RNA samples homozygous for AURKA Phe31. (A) Affymetrix-normalized expression values of probe set 231235_at following multiple correction restriction analysis using the Benjamini and Hochberg FDR algorithm (P = .05). (B) Relative expression of the novel NKTR transcript using quantitative RT-PCR with primer pair Ex6F-Int6aR to generate a 138-bp fragment from NKTR exon 6 to intron 6. Values represent mRNA levels of expression that were obtained from 10 ng of total RNA divided by the values of the expression levels of the NPC1 gene (P < .005). Relative expression units were calculated by standard curves using LightCycler 5.1 software (Roche Applied Bioscience). Pr, prostate RNA sample number.

Figure 3

Expression analysis and gene structure of the novel NKTR transcript that is overexpressed in normal homozygous AURKA Ile31 prostates. (A) Expression of the NKTR transcript in a variety of human tissues. RT-PCR amplification of the 658-bp full ORF transcript spanning from exon 1 (upstream of the ATG codon) to intron 6 (downstream of the putative stop codon; upper panel). The expression pattern of the 138-bp-short fragment spanning from exon 6 to intron 6, which is presented on the microarray (middle panel) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (lower panel), is also demonstrated. RTPCR was performed using human commercial whole brain RNA (lane 1 from left), placenta (lane 2), muscle (lane 3), skeletal muscle (lane 4), lung (lane 5), kidney (lane 6), colon (lane 7), adipose (lane 8), white blood cells (lane 9), and prostate (lane 10). RT-PCR without cDNA was used as a negative control (lane 11). Lane 12 contains a 100-bp ladder. (B) The structure of the NKTR gene (upper row, according to Ensembl gene report for ENSG00000114857) and the two alternative splice variants that contain sequences from intron 6. The novel alternative splice site variant (lower row) shares the (5′) 28-bp sequence of intron 6 with the transcript previously reported by Rinfret and Anderson [21], which contains only the 28 bp of intron 6 (middle row). The coding sequence in intron 6 of both alternative splice variants start at the same 5′ base. Lines and boxes represent introns and exons, respectively. Full box, coding sequences; empty box, noncoding sequences; ex, exon; int, intron. (C) Sequence of the novel splice site NKTR variant, including an ORF of 193 amino acids. The ATG and putative stop codon are in boldface. Intronic sequences are in lower case. The first 28 bp that originated from intron 6, identical to those previously published [21], are underlined.

Figure 3

Expression analysis and gene structure of the novel NKTR transcript that is overexpressed in normal homozygous AURKA Ile31 prostates. (A) Expression of the NKTR transcript in a variety of human tissues. RT-PCR amplification of the 658-bp full ORF transcript spanning from exon 1 (upstream of the ATG codon) to intron 6 (downstream of the putative stop codon; upper panel). The expression pattern of the 138-bp-short fragment spanning from exon 6 to intron 6, which is presented on the microarray (middle panel) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (lower panel), is also demonstrated. RTPCR was performed using human commercial whole brain RNA (lane 1 from left), placenta (lane 2), muscle (lane 3), skeletal muscle (lane 4), lung (lane 5), kidney (lane 6), colon (lane 7), adipose (lane 8), white blood cells (lane 9), and prostate (lane 10). RT-PCR without cDNA was used as a negative control (lane 11). Lane 12 contains a 100-bp ladder. (B) The structure of the NKTR gene (upper row, according to Ensembl gene report for ENSG00000114857) and the two alternative splice variants that contain sequences from intron 6. The novel alternative splice site variant (lower row) shares the (5′) 28-bp sequence of intron 6 with the transcript previously reported by Rinfret and Anderson [21], which contains only the 28 bp of intron 6 (middle row). The coding sequence in intron 6 of both alternative splice variants start at the same 5′ base. Lines and boxes represent introns and exons, respectively. Full box, coding sequences; empty box, noncoding sequences; ex, exon; int, intron. (C) Sequence of the novel splice site NKTR variant, including an ORF of 193 amino acids. The ATG and putative stop codon are in boldface. Intronic sequences are in lower case. The first 28 bp that originated from intron 6, identical to those previously published [21], are underlined.

Figure 4

Comparison of normalized gene expression values that are differentially expressed in normal prostates homozygous for either AURKA Phe31 or Ile31. The expression values of two molecular annotations are presented: cell cycle arrest (A) and antiapoptosis (B). Values on the two left bars represent the average of normalized mRNA expression obtained from each group homozygous for the AURKA Ile31 allele (n = 5; middle bar) and for the Phe31 allele (n = 3; left bar). The ratio between groups (right bar) was calculated as the mean in AURKA Ile31 samples divided by the mean in AURKA Phe31 samples. *Genes whose expression levels were significantly changed between the two AURKA genotype groups of normal prostate RNA samples (t-test, P < .05).