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PubMed articles by:
Lott, S.
Chen, N.
Chandler, D.
Killary, A.
PLoS Med. 2009 May; 6(5): e1000068.
Published online 2009 May 5. doi: 10.1371/journal.pmed.1000068.
PMCID: PMC2673042
Copyright This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
DEAR1 Is a Dominant Regulator of Acinar Morphogenesis and an Independent Predictor of Local Recurrence-Free Survival in Early-Onset Breast Cancer
Steven T. Lott,1 Nanyue Chen,1 Dawn S. Chandler,1 Qifeng Yang,2 Luo Wang,1 Marivonne Rodriguez,1 Hongyan Xie,1 Seetharaman Balasenthil,1 Thomas A. Buchholz,3 Aysegul A. Sahin,4 Katrina Chaung,1 Baili Zhang,1 Shodimu-Emmanu Olufemi,1 Jinyun Chen,5 Henry Adams,1 Vimla Band,6 Adel K. El-Naggar,4 Marsha L. Frazier,5 Khandan Keyomarsi,7 Kelly K. Hunt,8 Subrata Sen,4 Bruce Haffty,2 Stephen M. Hewitt,9 Ralf Krahe,1 and Ann McNeill Killary1*
1Department of Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
2Department of Radiation Oncology, University of Medicine & Dentistry of New Jersey–Robert Wood Johnson Medical School, New Brunswick, New Jersey, United States of America
3Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
4Division of Pathology and Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
5Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America
6Department of Genetics, Cell Biology and Anatomy, The University of Nebraska Medical Center, Eppley Cancer Center, Omaha, Nebraska, United States of America
7Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
8Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
9Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America
John D. Minna, Editor
Univesity of Texas Southwestern Medical Center at Dallas, United States of America
* E-mail: akillary/at/mdanderson.org
ICMJE criteria for authorship read and met: STL NC DSC QY LW MR HX SB TB AS KC BZ SEO JC HA VB AKEN MLF KK KKH SS BH SMH RK AK. Agree with the manuscript's results and conclusions: STL NC DSC QY LW MR HX SB TB AS KC BZ SEO JC HA VB AKEN MLF KK KKH SS BH SMH RK AK. Designed the experiments/the study: STL NC DSC BZ RK AK. Analyzed the data: STL NC DSC LW MR HX SB TB AS KC BZ HA MLF KKH SS BH SMH RK AK. Collected data/did experiments for the study: STL NC DSC QY LW BZ SEO JC KKH SMH RK. Enrolled patients: KKH. Wrote the first draft of the paper: DSC AK. Contributed to the writing of the paper: STL NC DSC QY LW VB MLF KK SS BH SMH RK. Conceived of and/or performed many of the experiments in the paper: STL. Designed some experiments, DEAR1 knock-down assay and DEAR1 antibody confirmation by Western: NC. Performed experiments, collected data, and analyzed experiments as well as made the revised figures: NC. Contributed to the identification of the homozygous deletion: LW. Collected sequencing, gene expression, and acinar morphogenesis data: MR. Helped in the identification of a missense mutation in DEAR1 by direct sequencing: KC. Designed and analyzed results from methylation assays: BZ. Analyzed and interpreted data: JC. Performed imaging of 3D cultures and helped with interpretation of the imaging data: HA. Provided 21T series cells and protocols and consultation in culture and 3D acinar structure of these cells: VB. Provided materials: AKEN. Assisted in the evaluation of results: AKEN. Helped design some of the experiments: MLF. Provided some of the sequencing data: MLF. Helped in experiments analyzing expression of DEAR1 gene in breast cancer cells: SS. Gave comments and suggestions on the draft manuscript: SS. Interpreted and photographed immunohistochemistry in some figures: SMH. Collected pyrosequencing data and supervised BZ and SEO in experimentation, data generation, and analysis: RK. Conceived of most of the experiments in the paper, including the experiments that led to the cloning, identification, and characterization of DEAR1 and its role as a regulator of acinar morphogenesis and independent predictor of recurrence-free survival in early onset breast cancer: AK. Supervised or initiated the collaboration for most of the experiments in the manuscript: AK.
Received September 11, 2008; Accepted March 17, 2009.
Small right arrow pointing to: See "A New Tumor Suppressor That Regulates Tissue Architecture" on page e1000073.
Small right arrow pointing to: See commentary "A New Tumor Suppressor That Regulates Tissue Architecture" on page e1000073.
 
Abstract
Background
Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium–associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer.
Methods and Findings
Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER−, PR−, HER-2−) of breast cancers with poor prognosis.
Conclusions
Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.
 
Editors' Summary
Background
Each year, more than one million women discover that they have breast cancer. This type of cancer begins when cells in the breast that line the milk-producing glands or the tubes that take the milk to the nipples (glandular and ductal epithelial cells, respectively) acquire genetic changes that allow them to grow uncontrollably and to move around the body (metastasize). The uncontrolled division leads to the formation of a lump that can be detected by mammography (a breast X-ray) or by manual breast examination. Breast cancer is treated by surgical removal of the lump or, if the cancer has started to spread, by removal of the whole breast (mastectomy). Surgery is usually followed by radiotherapy or chemotherapy. These “adjuvant” therapies are designed to kill any remaining cancer cells but can make patients very ill. Generally speaking, the outlook for women with breast cancer is good. In the US, for example, nearly 90% of affected women are still alive five years after their diagnosis.
Why Was This Study Done?
Although breast cancer is usually diagnosed in women in their 50s or 60s, some women develop breast cancer much earlier. In these women, the disease is often very aggressive. Compared to older women, young women with breast cancer have a lower overall survival rate and their cancer is more likely to recur locally or to metastasize. It would be useful to be able to recognize those younger women at the greatest risk of cancer recurrence so that they could be offered intensive surveillance and adjuvant therapy; those women at a lower risk could have gentler treatments. To achieve this type of “stratification,” the genetic changes that underlie breast cancer in young women need to be identified. In this study, the researchers discover a gene that is genetically altered (by mutations or deletion) in early-onset breast cancer and then investigate whether its expression can predict outcomes in women with this disease.
What Did the Researchers Do and Find?
The researchers used “suppression subtractive hybridization” to identify a new gene in a region of human Chromosome 1 where loss of heterozygosity (LOH; a genetic alteration associated with cancer development) frequently occurs. They called the gene DEAR1 (ductal epithelium-associated RING Chromosome 1) to indicate that it is expressed in ductal and glandular epithelial cells and encodes a “RING finger” protein (specifically, a subtype called a TRIM protein; RING finger proteins such as BRCA1 and BRCA2 have been implicated in early cancer development and in a large fraction of inherited breast cancers). DEAR1 expression was reduced or lost in several ductal carcinomas in situ (a local abnormality that can develop into breast cancer) and advanced breast cancers, the researchers report. Furthermore, many breast tumors carried DEAR1 missense mutations (genetic changes that interfere with the normal function of the DEAR1 protein) or had lost both copies of DEAR1 (the human genome contains two copies of most genes). To determine the function of DEAR1, the researchers replaced a normal copy of DEAR1 into a breast cancer cell that had a mutation in DEAR1. They then examined the growth of these genetically manipulated cells in special three-dimensional cultures. The breast cancer cells without DEAR1 grew rapidly without an organized structure while the breast cancer cells containing the introduced copy of DEAR1 formed structures that resembled normal breast acini (sac-like structures that secrete milk). In normal human mammary epithelial cells, the researchers silenced DEAR1 expression and also showed that without DEAR1, the normal mammary cells lost their ability to form proper acini. Finally, the researchers report that DEAR1 expression (detected “immunohistochemically”) was frequently lost in women who had had early-onset breast cancer and that the loss of DEAR1 expression correlated with reduced local recurrence-free survival, a strong family history of breast cancer and with a breast cancer subtype that has a poor outcome.
What Do These Findings Mean?
These findings indicate that genetic alteration and loss of expression of DEAR1 are common in breast cancer. Although laboratory experiments may not necessarily reflect what happens in people, the results from the three-dimensional culture of breast epithelial cells suggest that DEAR1 may regulate the normal acinar structure of the breast. Consequently, loss of DEAR1 expression could be an early event in breast cancer development. Most importantly, the correlation between DEAR1 expression and both local recurrence in early-onset breast cancer and a breast cancer subtype with a poor outcome suggests that it might be possible to use DEAR1 expression to identify women with early-onset breast cancer who have an increased risk of local recurrence so that they get the most appropriate treatment for their cancer.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000068.
  • This study is further discussed in a PLoS Medicine Perspective by Senthil Muthuswamy
  • The US National Cancer Institute provides detailed information for patients and health professionals on all aspects of breast cancer, including information on genetic alterations in breast cancer (in English and Spanish)
  • The MedlinePlus Encyclopedia provides information for patients about breast cancer; MedlinePlus also provides links to many other breast cancer resources (in English and Spanish)
  • The UK charities Cancerbackup (now merged with MacMillan Cancer Support) and Cancer Research UK also provide detailed information about breast cancer
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