DMP1β, a splice isoform of the tumour suppressor DMP1 locus, induces proliferation and progression of breast cancer
Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo‐insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functi...
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| Published in | The Journal of pathology Vol. 236; no. 1; pp. 90 - 102 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Chichester, UK
John Wiley & Sons, Ltd
01.05.2015
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| Subjects | |
| Online Access | Get full text |
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| Summary: | Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo‐insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1β and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1β expression. RNA‐seq analyses of a publicly available database showed significantly increased DMP1β mRNA in 43–55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1β protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1β overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1β may have a biological function. Indeed, DMP1β increased proliferation of non‐tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1β's role in vivo, we established MMTV‐DMP1β transgenic mouse lines. DMP1β overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7–18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up‐regulating the tumour‐promoting DMP1β isoform. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. |
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| Bibliography: | ArticleID:PATH4504 istex:FC87F33A9647C5F9344C0AAC0724A09883C92A16 ark:/67375/WNG-N8LWHTQ0-R Appendix S1. Supplementary materials and methodsThe domain structure of DMP1 splice isoforms. A. Protein domain structures of DMP1 splice isoforms indicating lack of a DNA-binding domain and C-terminal transactivation domain in DMP1β and DMP1γ isoforms. The green box shows a unique amino acid sequence found in DMP1 β and DMP1 γ. The DMP1 isoform specific antibody (RAB) was developed using an epitope sequence (NH2-LWTPKKGHTFKLWLSKYC-COOH) as shown. B. The sequence of DMP1 Exon 10 showing splice donor and acceptor sites that generate DMP1α, DMP1β, and DMP1γ proteins. DMP1β- and DMP1γ- unique portions of Exon 10 contain the TAA stop codon.Alteration of DMP1 splicing in human breast cancer and mouse mammary tumours. A. Quantitative RT-PCR analyses for alteration of DMP1β/a splicing in 20 human breast tumours versus matched normal breast tissue showing infrequent increase in DMP1β mRNA. The error bars for normal and tumour tissues of each patient represent experimental variations in the real-time PCR analyses. The arrows indicate patients with aberrant DMP1β/α splicing in tumours compared to their matched normal tissues. DMP1 LOH (loss of heterozygosity) below each patient indicates breast tumours with one copy deletion of DMP1. The patient IDs are the same as those for Figure 1A. B. Quantitative RT-PCR of mammary tumours from MMTV-neu mice to detect alteration of Dmp1β/α ratios in tumour tissue compared to the normal mammary glands. Tumours were separated into those that retained wild-type Dmp1 (ND) and those that had naturally deleted one allele of Dmp1 (HD). NMMG = normal mammary glands; ND = non-deleted Dmp1 tumours; HD = hemizygous Dmp1 deleted tumours. The red bars indicate mouse tumours with aberrant Dmp1β/α ratios when compared to the normal mammary glands. The error bars indicated for normal and tumour tissues represent experimental variations in the real-time PCR analyses.DMP1β expression in human breast cancers from the publically available RNA-seq data. A, B. Hits mean alignments for the RNA-seq readings within the NCBI database. The data were obtained from analysis of RNA-seq data of 42 estrogen receptor-positive (ER+) and HER2 negative (ER+/HER2-) breast cancer primary tumours, 30 uninvolved breast tissue samples that were adjacent to ER+/HER2- primary tumours, 42 different triple-negative breast cancer (TNBC) primary tumours, and 21 uninvolved breast tissue samples that were adjacent to TNBC primary tumours http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58135. The 172 base blue-coloured sequence shown in Suppl. Fig 1B was used to detect the DMP1β + γ, and the 39 base green-colored sequence was used to detect the DMP1γ mRΝΑ. Then the DMP1γ reading was subtracted from the DMP1β + γ reading for each sample to get the DMP1β-specific value, which was subjected to statistical analyses. C. Statistical analyses of the number of DMP1β readings (i.e., blast alignment). The DMP1β readings are higher in BC than in uninvolved tissues as shown by the Student's t analyses (p = 0.0058 in all breast cancer and p = 0.013 in ER+/HER2- BC). The mode for DMP1β is higher in breast cancer than uninvolved neighbouring tissue (95 vs. 45 in ER+/HER2- BC; 85 vs. 60 in TNBC). Significantly increased expression of human DMP1β mRNA (i.e. 80 hits or higher) was found in 23/42 (54.8%) of ER+/HER2- BC, and 18/42 (42.9%) of TNBC, which is consistent with the percentage of high DMP1β protein expression in immunohistochemistry. The second peak (at 95) in uninvolved tissue for TNBC is possibly from pre-cancerous breast tissues where tumour cells are emerging.Detection of the endogenous DMP1α/β proteins in breast cancer cells. A. MDA-MB-231 cells with mutant p53 were infected with a retrovirus encoding DMP1β shRNA, and puromycin resistant cells were expanded to study endogenous DMP1β protein by Western blotting with the RAB antibody. pSR-293 and −465 were directed to the 3' noncoding region of hDMP1 while pSR-1131 was directed to the coding region . The filter was probed with an Actin antibody (sc-1615) to verify equal loading of proteins. B. Human breast epithelial (MCF-10A) or breast cancer (MCF7 and others) cells were studied for endogenous DMP1α/β expression by Western blotting with RAX and RAB antibodies, respectively. The filter was probed with an Actin antibody (sc-1615) to verify equal loading of proteins.Evaluation of a newly generated DMP1 splice isoform-specific antibody (RAB). A. Immunofluorescence analysis of endogenous DMP1β in MDA-MB-175VII cells expressing DMP1 shRNA. The DMP1β-specific antibody (RAB) shows reduced protein in DMP1-465 shRNA-expressing cells compared to those expressing a control shRNA targeting Luciferase. The signal intensity for DMP β (green) was quantified to be 3.25 ms exposure for control cells compared to 11.1 ms for DMP1-465 shRNA-expressing cells. B. NIH 3 T3 cells were transfected with vector, DMP1α, DMP1β, or DMP1 γ cDNA constructs. Western blot analysis shows that the pan-DMP1 antibody (RAX) detects all three isoforms, including the endogenous Dmp1 in the vector transfected cells, while RAB detects only DMP1β protein. C. Representative RAB immunohistochemistry from two patients showing strong staining that was abolished when the RAB antibody was pre-incubated with 5 µg of the immunization peptide used for raising the antibody. D. Quantitative RT-PCR analysis of DMP1β mRNA expression in breast tumours compared to matched normal tissues indicates positive correlation between DMP1β mRNA and RAB antibody IHC staining. E. The whole Western blots for Suppl. Fig. 5B. Two filters with equal loading for each sample were generated, and were probed with RAX (left, pan-DMP1), or RAB (right, to DMP1β). Finally, the lower half of the left filter was probed with the Actin antibody (sc-1615) to verify equal loading of proteins.Intensity grading of human breast cancers with the RAB antibody. The numbers indicate the intensity of DMP1β staining with RAB in 3-0 grades. This intensity (3: strong 40% [02-135], 2, medium 50% [08-1030], 1: weak 90% [05-958], 0: none 5% [04-1001]) together with percentage of positive cells within the field (3: 60-100% of the cells stained, 2: 30-59%, 1: 10-29%, 0: 0-10%) have been counted on each slide and Kaplan-Meier analysis has been performed between the high (4-6) and the low (0-3) and groups in Figure 2B. The scale shows 500 µm.Influences of DMP1β overexpression and knockdown on breast epithelial cell growth. A. SK-BR-3 cells with mutant p53 were infected with a retrovirus encoding DMP1β-puromycin, and puromycin resistant cells were cultured for 7 days. DMP1β accelerated the growth of this cell line indicating the growth-promoting effect of DMP1β was p53-independent. B. MDA-MB-175VII cells expressing DMP1 shRNA (pSR-DMP1-465; targeting all three DMP1 isoforms) compared to the cells expressing non-targeting control shRNA proliferate significantly slower in 2D culture. The qRT-PCR analysis confirms reduction of DMP1α and DMP1β mRNA in the cells expressing pSR-DMP1-465 shRNA (* p < 0.01). C. MCF10A cells were infected with pSR-DMP1-1131 shRNA retrovirus and puromycin-resistant cells were cultured for 4 days. The growth of non-transformed breast epithelial cells was less influenced by DMP1 shRNA in comparison to transformed BT474, MDA-MB-231 (Fig. 4A, B), and MDA-MB-175VII (Suppl. Fig. 7B) cells, indicating that the growth inhibitory effect of DMP1β depletion was specific to breast cancer cells.Whole mammary gland mounts from MMTV-DMP1β female mice. Representative whole mammary gland mounts from two 18-month old MMTV-DMP1β (upper 2 images) female mice and a non-transgenic (lower image) female mouse. Mammary glands from MMTV-DMP1β mice show diffuse hyperplasia and multifocal lesions compared to non-transgenic mammary glands. White arrows indicate tumour lesions.Mammary gland phenotype of 18-month-old MMTV-DMP1β female mice. H&E stain of mammary glands from 4 different MMTV-DMP1β mouse strains showing adenosquamous carcinoma phenotype. The arrows indicate areas of keratin deposition.High-magnification images of mammary tumours found in MMTV-DMP1β transgenic mice. A. Immunohistochemical staining of mammary tumours from #8124 with CD3 antibody. Arrows show membrane-positive cells. B. A high-magnification image of the mammary tumour found in mouse #8124 doubly stained with cytokeratin 8 (red) and 14 (blue) antibodies. Mononuclear cell infiltration is also evident. C. A high-magnification image of the mammary tumour found in mouse #8127 doubly stained with cytokeratin 8 (red) and 14 (blue) antibodies.Double staining immunohistochemistry of mammary tumours found in an MMTV-DMP1β transgenic mouse. Mammary tumour tissue from mouse #8124 (A, 20X magnification; B, 40X magnification) doubly stained for DMP1β (peroxidase, red) and cytokeratin 14 (alkaline phosphatase, blue). The majority of tumour cells were positive for both proteins, suggesting transdifferentiation of mammary tumour cells to adenosquamous carcinoma.Correlation of DMP1bexpression with current histological and molecular classifications of human breast cancer. Currently used prognostic indicators, clinical stage and sub-classification, for breast cancer were correlated to DMP1bprotein (IHC) expression and aberrant DMP1b/asplicing. High DMP1bprotein staining in IHC was found associated with stage I of breast cancer (p = 0.0218, χ2 = 5.26). Aberrant DMP1b/a(high) ratios were found to trend with stage I, but the data were not statistically significant. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Current address: North Carolina Wesleyan College; Rocky Mt, NC, USA. Current address: Boston Children's Hospital, Harvard University, Boston. MA, USA. Former affiliation Current address: Department of Biotechnology Sharda University India. |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4504 |