Point mutations and genomic deletions in CCND1 create stable truncated cyclin D1 mRNAs that are associated with increased proliferation rate and shorter survival

Abstract

A gene expression signature of tumor proliferation rate in mantle cell lymphoma (MCL) is an overriding molecular predictor of the length of survival following diagnosis. Many strongly proliferative MCL tumors have exceptionally high cyclin D1 mRNA levels and preferentially express short cyclin D1 mRNA isoforms. We demonstrate here that these short mRNAs are cyclin D1a isoforms with truncated 3'UTRs, not alternatively spliced cyclin D1b mRNA isoforms. Among 15 MCL tumors with truncated cyclin D1 mRNAs, 7 had genomic deletions in the CCND1 3'UTR region. In 3 others, CCND1 contained point mutations that created premature polyadenylation signals, giving rise to 1.5-kb mRNAs lacking most of the 3'UTR. Both types of genomic alteration created transcripts lacking mRNA destabilization elements present in the wild-type cyclin D1a mRNA. Premature polyadenylation due to a 3'UTR mutation also was present in the Z-138 MCL cell line, which expressed both truncated and full-length cyclin D1a mRNAs. In these cells, the half-life of the short cyclin D1a mRNA was much longer than that of the full-length mRNA. We conclude that alterations of CCND1 3'UTR structure can significantly increase its oncogenic effect and worsen the clinical course of MCL patients.

Figure 1

CCND1 locus and expression of cyclin D1 mRNA isoforms in MCL. (A) Schematic representation of the CCND1 locus and alternatively spliced cyclin D1 mRNA transcripts cyclin D1a and D1b. Shaded boxes represent coding sequences, open boxes represent noncoding exon sequences, and filled boxes on the dotted line indicate the location of qPCR probes. (B) The relative amount of cyclin D1 mRNA containing full-length 3′UTR in relation to all cyclin D1 mRNA transcripts measured by the exon 1-2 probe is shown on a log2 scale. The dotted line indicates the cutoff, below which more than 50% of transcripts lack full-length 3′UTR. Quartiles of proliferation as determined in a prior study are given on the x-axis with tumors in quartile 1 having the lowest and those in quartile 4 having the highest proliferation (P < .001 for any difference between quartiles). Number of samples analyzed were for Q1 n = 22, Q2 n = 20, Q3 n = 22, Q4 n = 20. (C) The A870G polymorphism at the last nucleotide of exon 4 (codon 241) was determined using an NciI RFLP. PCR primers are indicated by arrows, the NciI restriction site is underlined, and a representative analysis of cDNA samples separated on a 3% agarose gel with a size marker (m) in the first lane is shown. (D) The frequency of cyclin D1 G870 alleles (white columns) or A870 alleles (black columns) involved in the t(11;14) translocation is summarized for all patients as well as for each proliferation quartile. There was no significant difference in genotype of the translocated alleles between quartiles (P = .84). Number of samples analyzed were for Q1 n = 22, Q2 n = 23, Q3 n = 21, Q4 n = 22.

Figure 2

Cyclin D1a transcripts with a truncated 3′UTR are associated with inferior survival. (A) The relative amount of cyclin D1a transcripts with a truncated 3′UTR compared to full-length form was determined with probes in the proximal UTR and the distal UTR (see map in Figure 1). The mean and standard deviation for this ratio is given for tumors expressing full-length transcripts (square; a representative subset of 32 samples from Figure 1B was analyzed) as compared to transcripts with truncated 3′UTR (triangle; n = 15). (B) Kaplan-Meier estimates of overall survival according to predominant type of cyclin D1a transcript. The median survival for patients whose tumors expressed full-length cyclin D1 mRNA (n = 69) was 3.28 years, compared to 1.38 years for tumors expressing truncated cyclin D1a mRNAs (n = 15).

Figure 3

Genomic deletions cause truncation of cyclin D1a mRNA. Proximal and distal 3′UTR probes were used to amplify genomic DNA, and the ratio of the respective amplification strength was determined. The black square indicates the mean in tumors with normal cyclin D1a mRNA transcripts (n = 69), and the bars represent the standard deviation of this mean estimate. Individual tumor samples expressing truncated cyclin D1a mRNA transcripts are represented by triangles (n = 14, for one patient no DNA was available). Samples with a ratio of distal/proximal UTR below the 5th percentile of the samples with normal cyclin D1 locus (n = 69, black squares) were considered to have a genomic deletion of the 3′UTR.

Figure 4

Mutations in the 3′UTR generate premature polyadenylation signals. Sequencing chromatograms showing premature polyadenylation signals (boxed). (A) Wild-type and mutated cyclin D1a alleles from MCL-tumors 942 and 1176 are shown. The yellow bar highlights the 3 nucleotides at position 1344-46 of wild-type cyclin D1a mRNA that were deleted in MCL-942. In MCL-1176 and Z-138, there was an A insertion before position 1250. (B) The mutated allele of MCL-970 contained a partial duplication (yellow bars), starting at position 1233.

Figure 5

Cyclin D1a transcripts with full-length and truncated 3′UTRs are expressed in Z-138, but the transcript with a truncated 3′UTR is more stable. (A) Schematic representation of the 2 cyclin D1a transcripts found in Z-138. Lines represent the mRNA, with shaded boxes indicating the coding sequence. Ovals indicate the position of the polyadenylation signal (AATAAA), while the light gray square and the black box represent mRNA destabilizing elements, including an AU-rich element (ARE). PCR probes are represented by filled boxes. (B) Expression of cyclin D1 transcripts in Z-138 cells in relation to beta-2 microglobulin (B2M). (C-E) Relative expression levels of cyclin D1 and B2M transcripts in Z-138 cells after inhibition of transcription with 10 μg/mL actinomycin D.

Figure 6

Cyclin D1 protein expression is higher and persists longer after inhibition with actinomycin D in cells expressing a truncated cyclin D1a transcript than in cells expressing a normal cyclin D1a transcript. Western blots of cyclin D1 protein expression in KMS-12 (normal cyclin D1a transcript) and Z-138 cells (truncated cyclin D1a transcript) treated with actinomycin D for the indicated time points. Antibodies against alpha tubulin (loading control at 55 kDa) and cyclin D1 (38 kDa) were used simultaneously. Vehicle (dimethyl sulfoxide [DMSO]) only treated cells at 12 hours are included as control (far right lane).