Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer

Abstract

Background: The claudin 1 tight junction protein, solely responsible for the barrier function of epithelial cells, is frequently down regulated in invasive human breast cancer. The underlying mechanism is largely unknown, and no obvious mutations in the claudin 1 gene (CLDN1) have been identified to date in breast cancer. Since many genes have been shown to undergo deregulation through splicing and mis-splicing events in cancer, the current study was undertaken to investigate the occurrence of transcript variants for CLDN1 in human invasive breast cancer.

Methods: RT-PCR analysis of CLDN1 transcripts was conducted on RNA isolated from 12 human invasive breast tumors. The PCR products from each tumor were resolved by agarose gel electrophoresis, cloned and sequenced. Genomic DNA was also isolated from each of the 12 tumors and amplified using PCR CLDN1 specific primers. Sanger sequencing and single nucleotide polymorphism (SNP) analyses were conducted.

Results: A number of CLDN1 transcript variants were identified in these breast tumors. All variants were shorter than the classical CLDN1 transcript. Sequence analysis of the PCR products revealed several splice variants, primarily in exon 1 of CLDN1; resulting in truncated proteins. One variant, V1, resulted in a premature stop codon and thus likely led to nonsense mediated decay. Interestingly, another transcript variant, V2, was not detected in normal breast tissue samples. Further, sequence analysis of the tumor genomic DNA revealed SNPs in 3 of the 4 coding exons, including a rare missense SNP (rs140846629) in exon 2 which represents an Ala124Thr substitution. To our knowledge this is the first report of CLDN1 transcript variants in human invasive breast cancer. These studies suggest that alternate splicing may also be a mechanism by which claudin 1 is down regulated at both the mRNA and protein levels in invasive breast cancer and may provide novel insights into how CLDN1 is reduced or silenced in human breast cancer.

Fig 1. Identification of CLDN1 transcript variants in invasive human breast cancer.

PCR analysis was carried out on reverse transcribed RNA from 12 breast tumors using primers (Table 1) flanking the coding regions of CLDN1, 3, and 4. The expected full length cDNA products (representing the classical transcripts) for CLDN1 and CLDN4 (700 bases and 706 bases respectively) was evident in all tumors. However, no CLDN3 transcript was detected in some of the breast tumors (middle panel; lanes 4, 10, 11). Colored arrows indicate CLDN1 PCR products which were verified by Sanger sequencing: yellow arrow, transcript variant 1 (V1) is 615 bp; red arrow, transcript variant 2 (V2) is 440bp; blue arrow, transcript variant 3 (V3) is 362 bp; and green arrow, transcript variant 4 (V4) is 217 bp. In the lower panel, the product indicated by the black arrow, was a non-specific band and had no sequence homology to CLDN4.

Fig 2. CLDN1 transcript variants reveal alternate splicing within exons.

The shorter CLDN1 transcript variants were aligned with the full length coding region (636 bp). Translation of the putative protein of each variant resulted in alignment only in the N-terminal of claudin 1 (exon 1). Remaining sequence was out of frame and translated into a non-functional claudin 1 protein. * = location of stop codon.

Fig 3. CLDN1 transcript variants in human breast tumors versus normal breast.

A. Primer Design. Forward primers were designed to specifically amplify V1 and V2 CLDN1 transcripts. The reverse primer flanked the coding region of CLDN1 mRNA and was identical to the one used for the PCR amplification shown in Fig 1. A reverse primer was specifically designed to amplify the V3 transcript variant and used in conjunction with the CLDN1 forward primer (Table 1). B. Differential expression of transcript variants. V1 was amplified in both breast cancer (2/6 tumors) and normal tissue samples (3/6). However, V2 was only evident in the breast tumors (3/6). V3 was detected in both tumors (3/6 tumors) and normal tissue (4/6). Gapdh was used as a control for the RT-PCR reaction. (blk = no template).

Fig 4. Identification of SNPs in human breast cancers within CLDN1 exons.

Several SNPs were identified by sequencing of the PCR products (see Table 1). The cDNA position of the SNPs are shown and designated by their corresponding Reference SNP (rs) numbers. The frequency of the missense SNP detected in exon 2, was significantly higher in the breast tumors than predicted for the normal population (p< 0.5; two-tailed Fisher’s Exact Test).

Fig 5. Representative immunostaining of breast tumors with claudin 1 antibody.

A human breast tumor exhibiting high (A; tumor #11) and one exhibiting low (B; Tumor #3) levels of the claudin 1 protein. Scale bars = 60 μm.