DEAR1 Is a Chromosome 1p35 Tumor Suppressor and Master Regulator of TGF-β–Driven Epithelial–Mesenchymal Transition

Abstract

Deletion of chromosome 1p35 is a common event in epithelial malignancies. We report that DEAR1 (annotated as TRIM62) is a chromosome 1p35 tumor suppressor that undergoes mutation, copy number variation and loss of expression in human tumors. Targeted disruption in the mouse recapitulates this human tumor spectrum with both Dear1−/− and Dear1+/− mice developing primarily epithelial adenocarcinomas and lymphoma with evidence of metastasis in a subset of mice. DEAR1 loss of function in the presence of TGFβ results in failure of acinar morphogenesis, upregulation of EMT markers, anoikis resistance, migration and invasion. Furthermore, DEAR1 blocks TGFβ-SMAD3 signaling resulting in decreased nuclear phosphorylated SMAD3 by binding to and promoting the ubiquitination of SMAD3, the major effector of TGFβ-induced EMT. Moreover, DEAR1 loss increases levels of SMAD3 downstream effectors, SNAI1 and SNAI2, with genetic alteration of DEAR1/SNAI2 serving as prognostic markers of overall poor survival in an 889 invasive breast cancer cohort.

DEAR1, a Novel Tumor Suppressor That Regulates Cell Polarity and Epithelial Plasticity

“…LOH within chromosome 1p has been shown to predict poor prognosis in node-negative breast cancers, and allelic deletions in the 1p36 and 1p32 region correlate with poor survival (20). Our laboratory discovered a novel gene DEAR1 ( Ductal Epithelium Associated Ring Chromosome 1 ) by suppression subtractive hybridization cloning of genes differentially expressed between cells with and without functional complementation of the tumor suppressor locus NRC-1 within 3p12 (21-25). DEAR1 was identified as a differentially expressed cDNA obtained from the suppression library that mapped into chromosome 1p35.1, a genomic interval downstream of chromosome 3p loss in a cytogenetic pathway for the evolution of smoking related cancers including lung, kidney, and breast cancer (24).…”

“…DEAR1 is expressed in normal tissues but limited to the ductal and glandular epithelium, and undergoes loss or downregulation of expression in breast cancer cell lines as well as in DCIS (71%), one of the earliest histologic precursors of invasive breast cancer (24, 25) DEAR1 is also mutated (13%) and homozygously deleted in breast cancer, including early onset cases (24). Targeted disruption of Dear1 in the mouse determined that Dear1 was a bona fide tumor suppressor, the knockout of which resulted in late onset epithelial adenocarcinomas in multiple organs, including mammary gland, kidney, pancreas and lung as well as sarcomas and lymphomas (25). Interestingly, Dear1 heterozygous as well as homozygous null animals formed tumors with approximately equal frequency, suggesting that Dear1 might be a haploinsufficient tumor suppressor.…”

“…Immunohistochemical staining of representative tumors from the mouse model indicated that in the majority of adenocarcinomas, expression was absent which would be expected for loss of function. However, in the majority of lymphomas, Dear1 was expressed in tumors (30%-80% of cells staining positively), indicating that Dear1 is a haploinsufficient tumor suppressor in certain tumor types (25). Unlike classical tumor suppressors that regulate cellular proliferation, however, DEAR1 loss to date has not been shown to affect growth rates of human mammary epithelial cells (HMECs) in both two dimensional culture (2D) (unpublished observations) and 3D culture (24).…”

“…The importance of DEAR1 in the regulation of TGFβ signaling from the ECM was discovered when immortal HMECs for which DEAR1 expression had been stably knocked down (DEAR1-KD), failed to undergo acinar morphogenesis when TGFβ was added to the 3D culture media as compared to wild type DEAR1 controls which formed proper acini (25). DEAR1-KD clones failed to upregulate Caspase 3, indicative that the cells were not undergoing apoptosis, but rather the cells upregulated Vimentin, an immediate early EMT marker, indicative that loss of DEAR1 resulted in a phenotype shift to a more mesenchymal phenotype in the presence of TGFβ and furthermore, in wild type (WT) DEAR1 expressing HMECs, DEAR1 restricted TGFβ signaling from the extracellular milieu.…”

“…DEAR1-KD clones failed to upregulate Caspase 3, indicative that the cells were not undergoing apoptosis, but rather the cells upregulated Vimentin, an immediate early EMT marker, indicative that loss of DEAR1 resulted in a phenotype shift to a more mesenchymal phenotype in the presence of TGFβ and furthermore, in wild type (WT) DEAR1 expressing HMECs, DEAR1 restricted TGFβ signaling from the extracellular milieu. In addition, compared to HMEC clones that express DEAR1, TGFβ-treated DEAR1-KD clones exhibited pronounced migration across the wound in 2D culture as well as prolonged migration and invasion on top of and throughout the matrix in 3D culture (25). TGFβ-treated DEAR1-KD clones also displayed elevated levels of mesenchymal markers β-integrin and N-Cadherin, as well as acquired markedly enhanced anoikis resistance, another important marker of EMT, in which increased survival of epithelial tumor cells occurs in the presence of inadequate or inappropriate contact with the extracellular matrix (7, 32).…”