Citations

In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1‐Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1‐Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild‐type mice. Lung metastases were found in 75% of Sulf1‐Tg mice but not in wild‐type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor‐β (TGF‐β)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo . This effect of SULF1 on the TGF‐β/SMAD pathway is functional; overexpression of SULF1 promotes TGF‐β‐induced gene expression and epithelial–mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF‐β from the cell surface. We also show that SULF1 expression decreases the interaction between TGF‐β1 and its heparan sulfate proteoglycan sequestration receptor, TGFβR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence‐free survival (hazard ratio 4.1, 95% confidence interval 1.9‐8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF‐β expression and with several TGF‐β‐related epithelial–mesenchymal transition genes in human HCC. Conclusion : Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF‐β pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC. (H epatology 2015;61:1269–1283)

Abstract Signaling lymphocyte activation molecule (SLAM) family receptors are critically involved in modulating innate and adaptive immune responses. Several SLAM family receptors have been shown to interact with the adaptor molecule SAP; however, subsequent intracellular signaling is poorly defined. Notably, mutations in SLAM-associated protein (SAP) lead to X-linked lymphoproliferative disease, a rare but fatal immunodeficiency. Although the SLAM family member Ly9 (CD229) is known to interact with SAP, the functions of this receptor have remained elusive. Therefore, we have generated Ly9−/− mice and compared their phenotype with that of SLAM−/− and SAP−/− mice. We report that Ly9−/− T cells exhibit a mild Th2 defect associated with reduced IL-4 production after stimulation with anti-TCR and anti-CD28 in vitro. This defect is similar in magnitude to the previously reported Th2 defect in SLAM−/− mice but is more subtle than that observed in SAP−/− mice. In contrast to SLAM−/− and SAP−/− mice, T cells from Ly9−/− mice proliferate poorly and produce little IL-2 after suboptimal stimulation with anti-CD3 in vitro. We have also found that Ly9−/− macrophages exhibit no defects in cytokine production or bacterial killing as was observed in SLAM−/− macrophages. Additionally, Ly9−/− mice differ from SAP−/− mice in that they foster normal development of NKT cells and mount appropriate T and B cell responses to lymphocytic choriomeningitis virus. We have identified significant phenotypic differences between Ly-9−/− mice as compared with both SLAM−/− and SAP−/− mice. Although Ly9, SLAM, and SAP play a common role in promoting Th2 polarization, Ly-9 is uniquely involved in enhancing T cell activation.

Abstract Interactions of LIGHT and its receptors, herpesvirus entry mediator on T cells and lymphotoxin β receptor on stromal cells, are implicated in the regulation of lymphoid organogenesis, costimulation of T cells, and activation of dendritic cells. In this work we report that LIGHT-deficient mice had normal lymphoid organs with T cells and APCs that normally responded to Ag stimulation and normally stimulated T cells. Although the number of Vβ8+ T cells in naive LIGHT+/+ and LIGHT−/− mice was identical, Vβ8+CD8+ T cell proliferation in response to staphylococcal enterotoxin B was significantly lower in LIGHT−/− mice. Consistently, induction and cytokine secretion of CD8+ CTL to MHC class I-restricted peptide was also reduced in LIGHT−/− mice. However, the proliferative response of Vβ8+CD4+ T cells to staphylococcal enterotoxin B was comparable in LIGHT−/− and LIGHT+/+ mice. Our results suggest that LIGHT is required for activation of normal CD8+ T cells but not CD4+ T cells.

Abstract Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbppc−/−;Ptenpc+/− mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp−/−; Pten+/− and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27KIP1, and p21CIP1. Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbppc−/−;Ptenpc+/− mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic-targeted therapy in patients with prostate cancer. Cancer Res; 74(7); 2050–61. ©2014 AACR.

Abstract Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion–induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNA interference–mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these findings, in PTEN-deficient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss. Cancer Res; 74(6); 1870–80. ©2014 AACR.

Aneuploidy, an aberrant number of chromosomes, has been recognized as a feature of human malignancies for over a century, but compelling evidence for causality was largely lacking until mouse models for chromosome number instability were used. These in vivo studies have not only uncovered important new insights into the extremely complex aneuploidy–cancer relationship but also into the molecular mechanisms underlying proper and aberrant chromosome segregation. A series of diverse mouse models for the mitotic checkpoint protein BubR1 has provided evidence for a provocative novel link between aneuploidization and the development of age-related pathologies.

The mitotic checkpoint protein Bub1 is essential for embryogenesis and survival of proliferating cells, and bidirectional deviations from its normal level of expression cause chromosome missegregation, aneuploidy, and cancer predisposition in mice. To provide insight into the physiological significance of this critical mitotic regulator at a modular level, we generated Bub1 mutant mice that lack kinase activity using a knockin gene-targeting approach that preserves normal protein abundance. In this paper, we uncover that Bub1 kinase activity integrates attachment error correction and mitotic checkpoint signaling by controlling the localization and activity of Aurora B kinase through phosphorylation of histone H2A at threonine 121. Strikingly, despite substantial chromosome segregation errors and aneuploidization, mice deficient for Bub1 kinase activity do not exhibit increased susceptibility to spontaneous or carcinogen-induced tumorigenesis. These findings provide a unique example of a modular mitotic activity orchestrating two distinct networks that safeguard against whole chromosome instability and reveal the differential importance of distinct aneuploidy-causing Bub1 defects in tumor suppression.

Abstract Recent molecular and pathologic evidence indicate that high-grade serous carcinomas of the ovary, which are the most common type of epithelial carcinomas, may arise from the distal part of fallopian tubes or tubal carcinoma implants. Oviductal glycoprotein 1 (Ovgp1), a glycosylated protein found in the epithelial cells in the oviduct, is the only specific tubal marker so far. Ovgp1 promoter-driven SV40 large T-antigen transgenic mice develop tumors in the female reproductive organs. To characterize these tumors, we performed immunohistochemical analysis in paraffin-embedded sections. Mice were sacrificed at 8 or 13 weeks old. Macroscopically we observed large tumors in the uterus and cervix and markedly enlarged region in the oviduct. Due to the large tumor in the uterus and cervix, we could not keep the mice longer than 13 weeks. Histologically, there were no major differences in these affected regions between 8 weeks old and 13 weeks old mice. We observed, (i) relatively small ovaries and ovarian follicular hyperplasia, (ii) intact ovarian surface epithelium showing no atypical histology, (iii) atypical epithelial hyperplasia within the oviduct (with endometrioid architectural and cytologic features), (iv) leiomyosarcomatous proliferation in the myometrium, and (v) squamous carcinoma in situ in the cervix or early invasive cervical cancer with extensive papillary architecture. We did not detect metastasis from the tumor to other organs. The atypical abnormalities of the oviduct were more significant in the proximal site to the uterus than the distal site. The proximal oviduct demonstrated weak and spotty p53 expression, beneath the epithelium, similar to p53 signature in the human oviduct. We hypothesized that stem cell components may contribute to the tumor development and that putative cancer stem cells could be identified by stem cell markers. In the oviduct epithelium, Bmi1 expression was not detectable. Cytoplasmic expression of Sox2 was observed in all epithelial cells of the oviduct. There was little correlation between Sox2/Bmi1 staining and p53 staining in the oviduct. Interestingly, Sox2 and Bmi1 staining in the wild-type control mice showed immunoreactive cells interspersed between glandular structures, a pattern similar to previously described label-retaining cells in the endometrium. The expression pattern in Ovgp1-SV40 mice, Bmi1 and Sox2 expression were more obvious in the endometrial stromal area but not in the leiomyosarcoma region, which implies that stem cell components in the endometrial region may support the leiomyosarcoma progression in this mouse model. In conclusion, Ovgp1-SV40 transgenic mouse is a useful model for studies involving fallopian and lower genital tract carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3272. doi:1538-7445.AM2012-3272

Abstract During mitosis, sister chromatids are separated and equally divided over two daughter cells. If missegregation of whole chromosomes occurs, progeny cells with an abnormal number of chromosomes will arise. This is referred to as aneuploidy, a hallmark of most human cancers. We found that inactivation of a single Pten allele in mouse splenocytes and mouse embryonic fibroblasts (MEFs) is sufficient to cause aneuploidy, suggesting a role for Pten in the chromosome segregation process. Upon further study by live cell imaging of haploinsufficient and null Pten MEFs, we indeed found many missegregation errors (59% and 79% respectively, compared to 15% in wildtype MEFs). These abnormalities were independent from the AKT pathway since overexpression of a myristoylated, constitutively active form of AKT in wildtype MEFs resembled a wildtype instead of a Pten heterozygous phenotype. In addition, overexpression of a catalytic mutant Pten (C124S) in Pten+/- MEFs corrected the missegregation errors. We therefore propose a novel tumor-suppressive function of Pten to prevent whole chromosome instability, independent of the increased cell proliferation and survival caused by the activation of AKT. BubR1 is a regulator of kinetochore-microtubule attachment and a potent inhibitor of Cdc20, a key co-activator of the anaphase promoting complex/cyclosome (APC/C) that drives cells into anaphase by targeting the anaphase inhibitors cyclin B1 and securin for degradation by the 26S proteosome. We found that overexpression of BubR1 completely restores accurate chromosome segregation in Pten+/- MEFs, alluding to a specific role for Pten in mitosis. As a first step to test the hypothesis that Pten's mitotic role in preventing aneuploidization represents a key tumor suppressive function of this phosphatase, we bred a Flag-tagged BubR1 transgene into Pten+/- mice. We are currently in the process of studying the impact of the transgene on the development of prostatic intraepithelial neoplasia (PIN). Consistent with our hypothesis, preliminary analysis of two mice shows that BubR1 overexpression considerably decreases PIN lesion multiplicity. Data from the complete analysis of our double transgenic mouse cohort will be presented at the meeting. 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 2992. doi:10.1158/1538-7445.AM2011-2992

Abstract Notch is activated by somatic mutation in more than 50% of cases of T-cell acute lymphoblastic lymphoma/leukemia (T-ALL). Exuberant notch signaling drives several oncogenic phenotypes, including enhanced proliferation, improved survival, and impaired differentiation. Inhibition of Notch is likely to yield improved treatment strategies for this disease and certain other malignancies, as well. However, because global inhibition of Notch may have intolerable toxicities, efforts are underway to identify the key downstream mediators of its neoplastic effects. We find that the clathrin-mediated receptor uptake adapter Hrb (HIV-rev binding protein) is a downstream target of Notch1. Hrb protein was induced to high levels by retroviral-mediated expression of constitutively active Notch (ICN1) in bone marrow stem cells and T cell precursors in vitro. To explore a potential role for Hrb downstream of Notch, we have examined the effects of ICN1 on bone marrow progenitor cells (BMPCs) from Hrb-deficient mice. Wild type BMPCs transduced with ICN1 grew to higher density in culture than did Hrbdeficient cells. There was no difference in distribution in cell cycle phases, and proliferative rates appeared to be the same. However, we found a significant increase in apoptosis in Hrb-deficient BMPCs in comparison to cells from littermate control mice. This increase in apoptosis could be reversed by reexpression of Hrb in the mutant cells, thus verifying that Hrb provides an enhanced survival signal to ICN1-expressing bone marrow cells. Potential downstream mediators of this survival signaling cascade are currently being identified. Because resistance to apoptosis is a key feature of leukemic cells, we hypothesize that the pro-survival effect of Hrb provides a portion of the oncogenic properties of Notch. Thus, because Hrb-knockout mice have no evident phenotype, other than male sterility, Hrb may be a useful molecular target for therapy of Notch-dependent malignancies, such as T-ALL. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B23.