Tumor suppressor DEAR1 regulates mammary epithelial cell fate and predicts early onset and metastasis in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a disease of poor prognosis, with the majority classified as the basal-like subtype associated with epithelial-mesenchymal transition and metastasis. Because basal breast cancers originate from proliferative luminal progenitor-like cells upon dysregulation of proper luminal differentiation, genes regulating luminal-basal transition are critical to elucidate novel therapeutic targets to improve TNBC outcomes. Herein we demonstrate that the tumor suppressor DEAR1/TRIM62 is a critical regulator of luminal cell fate. DEAR1 loss in human mammary epithelial cells results in significantly enhanced mammosphere formation that is accelerated in the presence of TGF-ß/SMAD3 signaling. Mammospheres formed following DEAR1 loss are enriched for ALDH1A1 and CK5 expression, EpCAM-/CD49f+ and CD44high/24low basal-like epithelial cells, indicating that DEAR1 regulates stem/progenitor cell properties and luminal-basal progenitor transition. We show that DEAR1 maintains luminal differentiation as a novel ubiquitin ligase for SNAI2/SLUG, a master regulator driving stemness and generation of basal-like progenitor populations. We also identify a significant inverse correlation between DEAR1 and SNAI2 expression in a 103 TNBC case cohort and show that low DEAR1 expression significantly correlates with young age of onset and shorter time to metastasis, suggesting DEAR1 could serve as a biomarker to stratify early onset TNBCs for targeted stem cell therapies.

DEAR1, a novel tumor suppressor that regulates cell polarity and epithelial plasticity.

Elucidation of the regulatory controls on epithelial plasticity is pivotal not only to better understand the nature of metastasis but also for the design of targeted therapies to prevent the earliest steps in migration and invasion from the primary tumor. This review will highlight the role of the novel TRIM protein DEAR1 (annotated as TRIM62) in the regulation of apical-basal polarity and acinar morphogenesis as well as its function as a chromosome 1p35 tumor suppressor and negative regulator of TGFß-driven epithelial-mesenchymal transition (EMT). DEAR1 binds to and promotes the ubiquitination of SMAD3, the major effector of TGFß-mediated EMT, as well as downregulates SMAD3 targets SNAIL1/2, master transcriptional regulators of EMT. Cumulative results suggest a novel paradigm for DEAR1 in the regulation of the breast tumor microenvironment, polarity, and EMT. Because DEAR1 undergoes loss-of-function mutations, homozygous deletion, as well as copy-number losses in multiple epithelial cancers, including breast cancer, DEAR1 has clinical use as a predictive and prognostic biomarker as well as for stratifying breast cancers and potentially other epithelial tumor types for targeted therapies aimed at the pathways regulated by DEAR1.

DEAR1 is a chromosome 1p35 tumor suppressor and master regulator of TGF-ß-driven epithelial-mesenchymal transition.

UNLABELLED: 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 TGF-ß results in failure of acinar morphogenesis, upregulation of epithelial-mesenchymal transition (EMT) markers, anoikis resistance, migration, and invasion. Furthermore, DEAR1 blocks TGF-ß-SMAD3 signaling, resulting in decreased nuclear phosphorylated SMAD3 by binding to and promoting the ubiquitination of SMAD3, the major effector of TGF-ß-induced EMT. Moreover, DEAR1 loss increases levels of SMAD3 downstream effectors SNAIL1 and SNAIL2, with genetic alteration of DEAR1/SNAIL2 serving as prognostic markers of overall poor survival in a cohort of 889 cases of invasive breast cancer. SIGNIFICANCE: Cumulative results provide compelling evidence that DEAR1 is a critical tumor suppressor involved in multiple human cancers and provide a novel paradigm for regulation of TGF-ß-induced EMT through DEAR1's regulation of SMAD3 protein levels. DEAR1 loss of function has important therapeutic implications for targeted therapies aimed at the TGF-ß-SMAD3 pathway.

A migration signature and plasma biomarker panel for pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma is a disease of extremely poor prognosis for which there are no reliable markers of asymptomatic disease. To identify pancreatic cancer biomarkers, we focused on a genomic interval proximal to the most common fragile site in the human genome, chromosome 3p12, which undergoes smoking-related breakage, loss of heterozygosity, and homozygous deletion as an early event in many epithelial tumors, including pancreatic cancers. Using a functional genomic approach, we identified a seven-gene panel (TNC, TFPI, TGFBI, SEL-1L, L1CAM, WWTR1, and CDC42BPA) that was differentially expressed across three different expression platforms, including pancreatic tumor/normal samples. In addition, Ingenuity Pathways Analysis (IPA) and literature searches indicated that this seven-gene panel functions in one network associated with cellular movement/morphology/development, indicative of a "migration signature" of the 3p pathway. We tested whether two secreted proteins from this panel, tenascin C (TNC) and tissue factor pathway inhibitor (TFPI), could serve as plasma biomarkers. Plasma ELISA assays for TFPI/TNC resulted in a combined area under the curve (AUC) of 0.88 and, with addition of CA19-9, a combined AUC for the three-gene panel (TNC/TFPI/CA19-9), of 0.99 with 100% specificity at 90% sensitivity and 97.22% sensitivity at 90% specificity. Validation studies using TFPI only in a blinded sample set increased the performance of CA19-9 from an AUC of 0.84 to 0.94 with the two-gene panel. Results identify a novel 3p pathway-associated migration signature and plasma biomarker panel that has utility for discrimination of pancreatic cancer from normal controls and promise for clinical application.

DEAR1 is a dominant regulator of acinar morphogenesis and an independent predictor of local recurrence-free survival in early-onset breast cancer.

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.

Fine mapping of the NRC-1 tumor suppressor locus within chromosome 3p12.

Identification of tumor suppressor genes based on physical mapping exercises has proven to be a challenging endeavor, due to the difficulty of narrowing regions of loss of heterozygosity (LOH), infrequency of homozygous deletions, and the labor-intensive characterization process for screening candidates in a given genomic interval. We previously defined a chromosome 3p12 tumor suppressor locus NRC-1 (Nonpapillary Renal Carcinoma-1) by functional complementation experiments in which renal cell carcinoma microcell hybrids containing introduced normal chromosome 3p fragments were either suppressed or unsuppressed for tumorigenicity following injection into athymic nude mice. We now present the fine-scale physical mapping of NRC-1 using a QPCR-based approach for measuring copy number at sequence tagged sites (STS) which allowed a sub-exon mapping resolution. Using STS-QPCR and a novel statistical algorithm, the NRC-1 locus was narrowed to 4.615-Mb with the distal boundary mapping within a 38-Kb interval between exon 3 and exon 4 of the DUTT1/Robo1 gene, currently the only candidate tumor suppressor gene in the interval. Further mutational screening and gene expression analyses indicate that DUTT1/ROBO1 is not involved in the tumor suppressor activity of NRC-1, suggesting that there are at least two important tumor suppressor genes within the chromosome 3p12 interval.

High frequency loss of heterozygosity in von Hippel-Lindau (VHL)-associated and sporadic pancreatic islet cell tumors: evidence for a stepwise mechanism for malignant conversion in VHL tumorigenesis.

Germ-line mutation of the von Hippel-Lindau (VHL) gene predisposes to the development of multifocal, benign lesions, including retinal and central nervous system hemangioblastomas, pheochromocytomas, and renal and pancreatic cysts. Progression to malignancy in VHL disease is associated primarily with the development of renal cell carcinoma (RCC) and pancreatic islet cell tumors (PICT). Although many reports have documented the multiple functions of the VHL protein, few have investigated the intriguing question related to the tissue-specificity of malignant conversion in VHL disease, a problem not easily explained by strict genotype-phenotype correlations. We investigated a novel VHL kindred with a preponderance of PICTs to determine whether loss of additional genetic loci associated with the sporadic forms of RCC and PICTs might play a role in malignant conversion in this disease. We report the high frequency loss of heterozygosity (LOH) of genetic loci distinct from and mapping proximal to VHL within human chromosome 3p in the VHL kindred under study. Furthermore, chromosome 3p LOH occurs subsequent to VHL mutation and cyst formation, and correlates with malignant progression in VHL-associated PICTs. High frequency LOH was also observed in sporadic PICTs in regions of 3p associated with LOH in sporadic clear cell RCC as well as homozygous deletion in lung cancer. A stepwise model for malignant conversion in VHL disease is herein proposed.