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The WWOX gene encodes a tumor suppressor spanning the second most common human fragile site, FRA16D. Targeted deletion of the Wwox gene in mice led to an increased incidence of spontaneous and ethyl nitrosourea–induced tumors. In humans, loss of heterozygosity and reduced or loss of WWOX expression has been reported in esophageal squamous cell cancers (SCC). In the present study, we examined whether inactivation of the Wwox gene might lead to enhanced esophageal/forestomach tumorigenesis induced by N-nitrosomethylbenzylamine. Wwox+/− and Wwox+/+ mice were treated with six intragastric doses of N-nitrosomethylbenzylamine and observed for 15 subsequent weeks. Ninety-six percent (25 of 26) of Wwox+/− mice versus 29% (10 of 34) of Wwox+/+ mice developed forestomach tumors (P = 1.3 × 10−7). The number of tumors per forestomach was significantly greater in Wwox+/− than in Wwox+/+ mice (3.2 ± 0.34 versus 0.47 ± 0.17; P < 0.0001). In addition, 27% of Wwox+/− mice had invasive SCC in the forestomach, as compared with 0% of wild-type controls (P = 0.002). Intriguingly, forestomachs from Wwox+/− mice displayed moderately strong Wwox protein staining in the near-normal epithelium, but weak and diffuse staining in SCC in the same tissue section, a result suggesting that Wwox was haploinsufficient for the initiation of tumor development. Our findings provide the first in vivo evidence of the tumor suppressor function of WWOX in forestomach/esophageal carcinogenesis and suggest that inactivation of one allele of WWOX accelerates the predisposition of normal cells to malignant transformation. [Cancer Res 2007;67(12):5606– 10]

Onco-fetal LIN28, present in two isoforms A and B and expressed in cancer cells, promotes tumorigenesis by suppressing the biogenesis of the tumor suppressor microRNA, let-7, thereby activating an array of oncogenes including RAS, MYC, HMGA2 and Cyclin D1. Studies have demonstrated a positive correlation between LIN28 A/B expression and advanced epithelial ovarian cancers (Histological grade 2 or 3). Correlation of LIN28 expression to a poor progression-free survival and resistance to platinum-based therapies makes LIN28-let-7 pathway an attractive target for ovarian cancer chemotherapy. The overall goal of this study was to identify small molecules that upregulate let-7 levels in LIN28A or LIN28B positive ovarian cancer cells. We hypothesize that induction of let-7 levels would potentially improve prognosis for ovarian cancer patients bearing LIN28 positive tumors. To test this hypothesis, we developed a dual luciferase let-7 reporter assay in which let-7 binding sites found in the 3’ UTR of the HMGA2 gene were cloned downstream of the nanoluciferase reporter gene. In this assay, elevated let-7 levels would result in repression of the nanoluciferase signal. Data obtained was normalized to Firefly luciferase activity. We tested efficacy of the assay system using let-7 mimics and inhibitors in LIN28A (OVK-18) and LIN28B (TOV-112D) positive ovarian cancer cell lines stably expressing the reporter cassette that were generated in house. Transfection of the let-7 mimic resulted in a 0.5 fold decrease in both cell lines relative to the negative controls, whereas transfection of the let-7 inhibitor resulted in a two-fold increase in relative luminescence. We miniaturized the assay to 384-well format for small molecule screening. The Z prime (Z’) scores for both cell lines was 0.58 and 0.84 respectively, indicating that the assay was robust to carry out the screens. We performed a primary screen using the Prestwick and LOPAC1280 libraries for each cell line in duplicate as well as a counter screen using a reporter cassette that lacked let-7 binding sites. A compound was identified as a hit if it ranked within the top 100 compounds in the primary screen and outside the top 250 compounds in the counter screen. The hit rate for LIN28A positive cell line was 80 % for the Prestwick and 74 % for the LOPAC1280 libraries. For the LIN28 B positive cell line, the hit rate was 73 % and 53 % for the Prestwick and LOPAC libraries. The hits were validated by assaying for let-7 levels using qRTPCR. The hits identified included compounds that inhibited PI3K-mTOR, nFkB, c-MYC, Cyclin D1, BET1 as well as aurora kinase. These targets have been reported in the literature to downregulate let-7 levels. In conclusion, our assay-system is the first to utilize a biological assay of let-7 levels for small molecule library screening thus serving as a valuable tool for cancer drug discovery. Citation Format: Miriam-Rose Menezes, Goeun Bae, Yong Sung Park, Julien Balzeau, Clifford C. Stephan, John P. Hagan. An in vitro biological assay to identify small molecule upregulators of let-7 miRNA in LIN28- positive ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1244. doi:10.1158/1538-7445.AM2017-1244

Inactivation of tumor suppressors is among the rate-limiting steps in carcinogenesis that occur during the tumor promotion stage. The translation inhibitor programmed cell death 4 (Pdcd4) suppresses tumorigenesis and invasion. Although Pdcd4 is not mutationally inactivated in human cancer, the mechanisms controlling Pdcd4 inactivation during tumorigenesis remain elusive. We report that tumor promoter 12-O-tetradecanoylphorbol-13-acetate exposure decreases protein levels of Pdcd4 in mouse skin papillomas and keratinocytes as well as in human HEK293 cells. This decrease is attributable to increased proteasomal degradation of Pdcd4 and is mediated by protein kinase C–dependent activation of phosphatidylinositol 3-kinase–Akt–mammalian target of rapamycin–p70S6K and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)–ERK signaling. Both Akt and p70S6K phosphorylate Pdcd4, allowing for binding of the E3-ubiquitin ligase β-TrCP and consequently ubiquitylation. MEK-ERK signaling on the other hand facilitates the subsequent proteasomal degradation. We further show that Pdcd4 protein levels in vivo are limiting for tumor formation, establishing Pdcd4 as a haploinsufficient tumor suppressor in Pdcd4-deficient mice. Thus, because endogenous Pdcd4 levels are limiting for tumorigenesis, inhibiting signaling to Pdcd4 degradation may prove a valid strategy for cancer prevention and intervention. [Cancer Res 2008;68(5):1254–60]

TAL1 oncogene encodes a helix-loop-helix transcription factor, Tal1, which is required for blood cell development, and its activation is a frequent event in T-cell acute lymphoblastic leukemia. Tal1 interacts and inhibits other helix-loop-helix factors such as E47 and HEB. To investigate the function of Tal1 in B cells, we generated Eμ-TAL1 transgenic mouse line, expressing Tal1 in mouse B-cell lineage. Fluorescence-activated cell sorting (FACS) analysis of lymphocytes isolated from spleens of five out of five founders reveals complete absence of IgM- or CD19-expressing cells. Only 2% to 3% of these cells were B220+ and 100% of B220+ cells were CD43+, indicating that these mice were able to make pro-B cells. Similarly, FACS analysis of bone marrow cells in Eμ-TAL1 mice revealed complete absence of B220+IgM+ and B220+CD19+ cells. Analysis of the recombination status of IgH genes revealed the presence of D-J but absence or drastic reduction of V-D-J rearrangements. Our results suggest that Tal1 overexpression in B cells results in a phenotype similar to that of B cells of E47/E2A knockout animals. This represents first in vivo evidence that Tal1 can completely inhibit E47/E2A function. (Cancer Res 2006; 66(12): 6014-7)

The gene for protein tyrosine phosphatase receptor-type O encodes two functional isoforms in a tissue-specific manner. The full-length form is expressed in epithelial cells, particularly in the brain and kidney, whereas the truncated form (PTPROt) is primarily expressed in hematopoietic cells. We have previously demonstrated that PTPROt is a bona fide tumor suppressor (J Biol Chem. 2009 Jan 2;284(1):455-64) and is down-regulated in primary CLL samples (Clin Cancer Res. 2007 Jun 1;13(11):3174-81). Further, studies from two laboratories including ours have shown that Syk and Lyn, two key protein tyrosine kinases involved in B-cell receptor signaling, are substrates of PTPROt, igniting interest in understanding deregulation of this tyrosine phosphatase in CLL. While the PTPRO promoter is methylated, the mechanism of its silencing in the pathogenesis of CLL is unexplored. Here, we utilized the Tcl1 transgenic (Tg) mouse model of CLL and a cell line with TPA-inducible expression of PTPROt to elucidate the mechanism of PTPROt silencing. This study demonstrates that PTPROt is transcriptionally silenced in CD19+ spleen B cells from Tcl1 transgenic (Tg) mouse when compared to wild type mice. As observed with primary human CLL, the methylation of PTPRO CGI (CpG island) is significantly higher in Tcl1 Tg CLL B-lymphocytes than in normal B lymphocytes. Significant suppression of PTPROt is also observed at very early time-point (∼7 wks) in CD19+ spleen B-cells from Tcl1 Tg mice compared to normal spleen B-lymphocytes from age-matched WT mice in the absence of detectable methylation at CGI. This data suggests that transcriptional silencing occurs prior to methylation. To understand further the potential transcriptional mechanism of PTPROt suppression in CLL, the role of AP1 in the expression of this gene was explored. We first demonstrate that AP-1 activation by TPA in U937 cells induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. PTPROt promoter is also responsive to over- and under-expression of AP-1 confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that Tcl1 can repress PTPROt promoter by altering c-fos expression and c-jun activation state. These findings further substantiate the importance of TCL1 in the pathogenesis of CLL. [Supported, in part, by grants CA101956 and CA086978]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 276. doi:10.1158/1538-7445.AM2011-276

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the western world. Human B-CLL occurs in two forms: aggressive (showing high ZAP-70 expression and unmutated IgH VH) and indolent (showing low ZAP-70 expression and mutated IgH VH). We found that miR-29a is upregulated in indolent human B-CLL compared to aggressive B-CLL and normal CD19+ B-cells. To study the role of miR-29 in B-CLL, we generated Eµ-miR-29 transgenic mice overexpressing miR-29 in mouse B-cells. Flow cytometric analysis revealed a markedly expanded CD5+ population in the spleen of these mice starting at 2 months of age. Over 80% of miR-29 transgenic mice exhibited an expanded population of CD5+ B-cells, a characteristic of the B-CLL phenotype. An average of 56% of the B-cell population in these transgenics were CD5 positive. At the age of 2 years these mice showed significantly enlarged spleens and an increase in CD5+ B-cell population of up to 100% of B-cells. Over 8% (3/36) of Eµ-miR-29 transgenic mice developed frank leukemia and prematurely died from the disease at the age of 24-26 months. The expanded CD5+ B-cell population was found to be proliferative, with an increased number of cells in the S-phase of the cell cycle, compared to wild type CD19+ B-cells. These results suggest that deregulation of miR-29 can cause, or at least significantly contribute to the pathogenesis of indolent B-CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-352.