Loss of the novel tumour suppressor and polarity gene Trim62 (Dear1) synergizes with oncogenic Ras in invasive lung cancer.

Deregulation of cell polarity proteins has been linked to the processes of invasion and metastasis. TRIM62 is a regulator of cell polarity and a tumour suppressor in breast cancer. Here, we demonstrate that human non-small cell lung cancer lesions show a step-wise loss of TRIM62 levels during disease progression, which was associated with poor clinical outcomes. To directly examine the role of Trim62 in development of lung cancer, we deleted Trim62 in a mutant K-Ras mouse model of lung cancer. In this context, haploinsufficiency of Trim62 synergized with a K-RasG12D mutation to promote invasiveness and disrupt three-dimensional morphogenesis, both of which are associated with epithelial-mesenchymal transitions. Re-expression of Trim62 reverted these phenotypes in tumour cell lines. Thus, Trim62 loss cooperates with K-Ras mutation in tumourigenesis and metastasis in vivo, indicating that decreased levels of TRIM62 may play an important role in the evolution of lung cancer.

Blood-based Migration Signature Biomarker Panel Discriminates Early Stage New Onset Diabetes related Pancreatic Ductal Adenocarcinoma from Type 2 Diabetes.

BACKGROUND AND AIMS: While type 2 diabetes is a well-known risk factor for pancreatic ductal adenocarcinoma (PDAC), PDAC-induced new-onset diabetes (PDAC-NOD) is a manifestation of underlying PDAC. In this study, we sought to identify potential blood-based biomarkers for distinguishing PDAC-NOD from type 2 diabetes (T2DM) without PDAC. MATERIALS AND METHODS: By ELISA analysis, a migration signature biomarker panel comprising tissue factor pathway inhibitor (TFPI), tenascin C (TNC-FNIII-C) and CA 19-9 was analyzed in plasma samples from 50 PDAC-NOD and 50 T2DM controls. RESULTS: Both TFPI (area under the curve (AUC) 0.71) and TNC-FNIII-C (AUC 0.69) outperformed CA 19-9 (AUC 0.60) in distinguishing all stages of PDAC-NOD from T2DM controls. The combined panel showed an AUC of 0.82 (95% CI = 0.73-0.90) (p = 0.002). In the PDAC-NOD early stage II samples, the three biomarkers had an AUC of 0.84 (95% CI = 0.73-0.93) vs CA 19-9, AUC = 0.60, (95% CI = 0.45-0.73), which also improved significance (p = 0.0123). CONCLUSION: The migration signature panel adds significantly to CA 19-9 to discriminate PDAC-NOD from T2DM controls and warrants further validation for high-risk group stratification.

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 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.

A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells.

The propensity for prostate cancer to metastasize to bone led us and others to propose that bidirectional interactions between prostate cancer cells and bone are critical for the preferential metastasis of prostate cancer to bone. We identified previously a secreted isoform of ErbB3 (p45-sErbB3) in bone marrow supernatant samples from men with prostate cancer and bone metastasis and showed by immunohistochemical analysis of human tissue specimens that p45-sErbB3 was highly expressed in metastatic prostate cancer cells in bone. Here, we show that p45-sErbB3 stimulated mouse calvaria to secrete factors that increased the invasiveness of prostate cancer cells in a Boyden chamber invasion assay. Using gene array analysis to identify p45-sErbB3-responsive genes, we found that p45-sErbB3 up-regulated the expression of osteonectin/SPARC, biglycan, and type I collagen in calvaria. We further show that recombinant osteonectin increased the invasiveness of PC-3 cells, whereas osteonectin-neutralizing antibodies blocked this p45-sErbB3-induced invasiveness. These results indicate that p45-sErbB3 enhances the invasiveness of PC-3 cells in part by stimulating the secretion of osteonectin by bone. Thus, p45-sErbB3 may mediate the bidirectional interactions between prostate cancer cells and bone via osteonectin.

A Plasma Biomarker Panel to Identify Surgically Resectable Early-Stage Pancreatic Cancer.

Background: Blood-based biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC) are urgently needed. Current biomarkers lack high sensitivity and specificity for population screening. The gold-standard biomarker, CA 19-9, also fails to demonstrate the predictive value necessary for early detection. Methods: To validate a functional genomics-based plasma migration signature biomarker panel, plasma tissue factor pathway inhibitor (TFPI), tenascin C (TNC-FN III-C), and CA 19-9 levels were measured by enzyme-linked immunosorbent assays in three early-stage PDAC plasma cohorts, including two independent blinded validation cohorts containing a total of 43 stage I, 163 stage II, 86 chronic pancreatitis, 31 acute biliary obstruction, and 108 controls. Logistic regression models developed classification rules combining TFPI and/or TNC-FN III-C with CA 19-9 for patient cases and control subjects, with or without adjustment for age and diabetes status. Model classification performance was evaluated and analyses repeated among subpopulations without diabetes and pancreatitis history. Two-sided P values were calculated using bootstrap method. Results: The TFPI/TNC-FN III-C/CA 19-9 panel improved CA 19-9 performance in all early-stage cohorts, including discriminating stage IA/IB/IIA, stage IIB, and all early-stage cancer from healthy controls. Statistical significance was reached for a number of subcohorts, including for all early-stage cancer vs healthy controls (cohort 1 AUC = 0.92, 95% CI = 0.86 to 0.96, P = .04; cohort 3 AUC = 0.83, 95% CI = 0.76 to 0.89, P = .045). Among subcohorts without diabetes and pancreatitis history, the panel approaches potential clinical utility for early detection to discriminate early-stage PDAC from healthy controls including an area under the curve (AUC) of 0.87 (95% CI = 0.77 to 0.95) for stage I/IIA, an AUC of 0.93 (95% CI = 0.87 to 0.98) for stage IIB, and a statistically significant AUC of 0.89 (95% CI = 0.82 to 0.95) for all early-stage cancer ( P = .03). Conclusions: TFPI/TNC-FN III-C migration signature adds statistically significantly to CA 19-9's predictive power to detect early-stage PDAC and may have clinical utility for early detection of surgically resectable PDAC, as well as for enhanced survival from this routinely lethal cancer.

Differential gene expression profiles of Jnk1- and Jnk2-deficient murine fibroblast cells.

c-Jun NH(2)-terminal kinase (JNK) 1 and JNK2 have been assumed to complement each other and mediate the same or similar biological functions. However, our recent reports indicated that 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced tumor development is suppressed in Jnk2 knockout mice but enhanced in Jnk1 knockout mice. In the present work, primary embryo cells were isolated from wild-type, Jnk1(-/-) and Jnk2(-/-) mice and used for cDNA microarray analysis. The patterns of gene expression in Jnk1(-/-), Jnk2(-/-), and wild-type cells are different. After 12-O-tetradecanoylphorbol-13-acetate treatment, the changes in the gene expression profiles in three different kinds of cells appear to agree with the differences in susceptibility to tumorigenesis of each respective animal model. These results suggest that JNK1 and JNK2 proteins have different roles in modulating cell function.

Deficiency of c-Jun-NH(2)-terminal kinase-1 in mice enhances skin tumor development by 12-O-tetradecanoylphorbol-13-acetate.

The c-Jun NH(2)-terminal kinase (JNK) has been implicated in regulating cell survival, apoptosis, and transformation. However, the distinct role of JNK isoforms in regulating tumor development is not yet clear. We have found previously that skin tumor formation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is suppressed in JNK2-deficient (Jnk2(-/-)) mice. Here, we show that JNK1-deficient (Jnk1(-/-)) mice are more susceptible to TPA-induced skin tumor development than wild-type mice. The rate of tumor development in Jnk1(-/-) mice was significantly more rapid than that observed in wild-type mice (P < 0.0001). At the end of 33 weeks of TPA promotion, the number of skin tumors and tumors >1.5 mm in diameter per mouse in Jnk1(-/-) mice was significantly increased by 71% (P < 0.03) and 82% (P < 0.03), respectively, relative to the wild-type mice. Furthermore, the carcinoma incidence and the number of carcinomas per mouse were also higher in Jnk1(-/-) mice. Strikingly, Jnk1(-/-) mouse skin was more sensitive to TPA-induced AP-1 DNA binding activity and phosphorylation of extracellular signal-regulated kinases and Akt, which are two important survival signaling components. These results suggest that JNK1 is a crucial suppressor of skin tumor development.

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.

Human prolyl-4-hydroxylase alpha(I) transcription is mediated by upstream stimulatory factors.

Prolyl-4-hydroxylase alpha(I) (P4Halpha(I)) is the rate-limiting subunit for P4H enzyme activity, which is essential for procollagen hydroxylation and secretion. In the current study, we have characterized the human P4Halpha(I) promoter for transcription factors and DNA elements regulating P4Halpha(I) expression. Using a progressive deletion cloning approach, we have constructed pGL3-P4Halpha(I) recombinant plasmids. We have identified a positive regulatory region at the positions of bp -184 to -97 responsible for approximately 80% of the P4Halpha(I) promoter efficiency. Three E-boxes were located within this region, and the E-box at position bp -135 explains most of the regulatory capacity. Upstream stimulatory factors (USF1/USF2) were shown to bind on the E-box using chromatin immunoprecipitation assay. Suppression of USF1 and/or USF2 using specific short interference RNA resulted in a significant reduction in P4Halpha(I) promoter activity, and overexpressed USF1 or USF2 increased P4Halpha(I) promoter activity significantly. Although transforming growth factor beta1 increased the USF1/USF2-E-box binding and P4Halpha(I) promoter activity, this up-regulatory effect can be largely prevented by USF1/USF2-specific short interference RNA. On the other hand, cigarette smoking extracts, which have been shown to suppress P4Halpha(I) expression, inhibited the binding between the USF1/USF2 and E-box, resulting in a reduced P4Halpha(I) promoter activity. Furthermore, the E-box on the P4Halpha(I) promoter appeared to indiscriminately bind with either USF1 or USF2, with a similar outcome on the promoter efficiency. In conclusion, our study shows that USF1/USF2 plays a critical role in basal P4Halpha(I) expression, and both positive (transforming growth factor beta1) and negative (cigarette smoking extract) regulators appear to influence the USF-E-box interaction and affect P4Halpha(I) expression.

Decreased tumorigenicity of c-Myc-transformed fibroblasts expressing active USF2.

USF is a small family of basic helix-loop-helix leucine zipper (bHLH-zip) transcription factors with DNA binding specificities similar to that of the c-Myc oncoprotein. Evidence for a role of USF in growth control includes inhibition of c-Myc-dependent cellular transformation in vitro and loss of USF transcriptional activity in many cancer cell lines. However, a direct effect of USF on the tumorigenicity of an established cell line has never been demonstrated. Here, cell lines derived from rat embryo fibroblasts transformed by c-Ha-Ras and either c-Myc or E1A were used as model system to investigate the tumor suppression ability of USF. Overexpression of USF2 stimulated transcription and inhibited colony formation in c-Myc-transformed, but not E1A-transformed, fibroblasts. Stable clones expressing high USF2 levels were constructed from c-Myc-transformed fibroblasts. In two of these clones, overexpressed USF2 did not activate transcription, and there was no significant change in the transformed phenotype. In contrast, a clone that expressed transcriptionally active USF2 exhibited altered morphology and a strongly decreased ability to proliferate in semisolid medium. The ability of these cells to form tumors in nude mice was also decreased by a factor of more than 30 as compared to the parental cell line or cells overexpressing transcriptionally inactive USF2. Cotransfection assays with USF- or Myc-specific dominant-negative mutants indicated that active USF2 inhibited cellular transformation by preventing transcriptional repression by c-Myc.

2-Arachidonoylglycerol stimulates activator protein-1-dependent transcriptional activity and enhances epidermal growth factor-induced cell transformation in JB6 P+ cells.

2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and it plays a critical role in cannabinoid receptor-mediated cell signaling. Although 2-AG was shown to induce ERK activation via the cannabinoid receptor 1 (CB1), only a nonspecific CB receptor agonist and antagonist was used in those studies. Whether cannabinoid receptor 2 (CB2) is involved in 2-AG-induced ERK activation is still unclear. Moreover, whether 2-AG is involved in mediation of AP-1 activity and cell transformation is also not known. In the present study, we show that 2-AG stimulates AP-1-dependent transcriptional activity and enhances epidermal growth factor-induced cell transformation in mouse epidermal JB6 P+ Cl41 cells. Using JB6 P+ C141 cells, stably transfected with an AP-1 luciferase reporter, we found that 10 microm 2-AG induced up to a 3-fold stimulation of AP-1 transcriptional activity. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 kinase. PD98059, a specific inhibitor of MEK1, almost completely blocked 2-AG-induced ERK phosphorylation and AP-1 activation. Using CB1/2-/- murine embryonic fibroblasts, we present the first direct evidence that both cannabinoid receptors 1 and 2 (CB1/2) are involved in 2-AG-induced ERK activation. 2-AG could not stimulate ERK phosphorylation or Fyn kinase activity in dominant negative Fyn. In addition, the Fyn inhibitor PP2 blocked 2-AG-induced Fyn kinase activity and ERK phosphorylation and activity. Small interfering RNA Fyn also suppressed 2-AG-induced ERK phosphorylation. Interestingly, 2-AG enhanced epidermal growth factor-induced AP-1 DNA binding and cell transformation. Taken together, our data provide direct evidence suggesting that 2-AG may have a novel role in cell transformation and carcinogenesis in a signaling pathway involving CB1/2 and activation of Fyn, ERKs, and AP-1.