Almada, Luciana L.

Abstract Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

Abstract Pancreatic cancer, one of the most lethal forms of human cancer, is largely resistant to many conventional chemotherapeutic agents. Although many therapeutic approaches focus on tumor growth, metastasis is a primary factor contributing to lethality. Therefore, novel therapies to target metastatic invasion could prevent tumor spread and recurrence resulting from local and distant metastasis. The protein Vav1 is aberrantly expressed in more than half of pancreatic cancers. Its expression promotes activation of Rac and Cdc42 and leads to enhanced invasion and migration, as well as increased tumor cell survival and proliferation, suggesting that Vav1 could be a potent therapeutic target for pancreatic cancer. The purine analogue azathioprine, well known for its function as an anti-inflammatory compound, was recently shown to function by inhibiting Vav1 signaling in immune cells. We therefore hypothesized that azathioprine could also inhibit Vav1 in pancreatic tumor cells to reduce its proinvasive functions. Indeed, we have found that treatment of cultured pancreatic tumor cells with azathioprine inhibited Vav1-dependent invasive cell migration and matrix degradation, through inhibition of Rac and Cdc42 signaling. Furthermore, azathioprine treatment decreased metastasis in both xenograft and genetic mouse models of pancreatic cancer. Strikingly, metastasis was dramatically reduced in Vav1-expressing tumors arising from p48Cre/+, KrasG12D/+, p53F/+ mice. These inhibitory effects were mediated through Vav1, as Vav1-negative cell lines and tumors were largely resistant to azathioprine treatment. These findings demonstrate that azathioprine and related compounds could be potent antimetastatic agents for Vav1-positive pancreatic tumors. Cancer Res; 75(14); 2907–15. ©2015 AACR.

Abstract Interferon regulatory factor-4 (IRF4) is a tightly-regulated transcription factor involved in growth and differentiation of normal lymphocytes. IRF4 is expressed in multiple lymphoid malignancies and its expression drives tumor growth. Thus, characterization of the molecular events regulating IRF4 levels is key to design any targeting strategy. Here, we define a novel regulatory mechanism controlling IRF4 expression in peripheral T-cell lymphoma (PTCL), an aggressive non-Hodgkin lymphoma with poor overall survival rates and high levels of this transcription factor. In normal T cells, IRF4 was induced upon external activation of protein kinase C (PKC) by phorbol myristate acetate and ionomycin, an effect reversed by the PKC inhibitor, enzastaurin. Unlike normal T cells, PTCL cells constitutively expressed IRF4 in the absence of external stimulation, and this expression was resistant to enzastaurin. To identify regulators of IRF4 expression in PTCL, we performed a pharmacological screen using inhibitors that targeted PKC-associated pathways, including mitogen-activated protein kinases, PI3K/AKT/mTOR, and NF-κB. Of the inhibitors tested, only the proteasome inhibitor, bortezomib, showed a decrease in IRF4 expression in a dose- and time-dependent manner. To confirm this result we used a more specific NF-κB inhibitor blocking IκB kinase, BMS-345541. This inhibitor dramatically reduced IRF4 expression by 95.6% (P<0.0001). We used cell-based assays to investigate further the role of NF-κB in the transcriptional regulation of IRF4. A reporter assay for the IRF4 promoter demonstrated reduced promoter activity by siRNA knockdown of RelB and p52 NF-κB subunits to 31.4% and 36.9% of control (P=0.009). IRF4 promoter activity also was inhibited 67.9% by IκBαΔN, a dominant negative inhibitor of NF-κB activity (P=0.002), and was increased 71% by siRNA knockdown of IκBα (P<0.05). Chromatin immunoprecipitation assays confirmed the occupancy of NF-κB subunits in the IRF4 promoter, and this interaction was diminished in the presence of BMS-345541. Finally, knockdown of RelB and p52 subunits had a tendency to decrease IRF4 gene expression by 24.4% and 27.3% (P=0.1). Taken together, these data demonstrate transcriptional regulation of IRF4 by the NF-κB family in PTCL, and suggest the NF-κB pathway may represent a therapeutic target for PTCLs with concurrent IRF4 and NF-κB expression. Citation Format: Rebecca L. Boddicker, N. Sertac Kip, Luciana L. Almada, Julie C. Porcher, Deanna M. Grote, Stephen M. Ansell, Martin E. Fernandez-Zapico, Andrew L. Feldman. Interferon regulatory factor-4 is transcriptionally regulated by NF-κB in peripheral T-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2014-2323

Key PointsThe NF-κB subunits p52 and RelB increase IRF4 promoter activity and expression in PTCL cells. A positive feedback loop involving CD30, NF-κB, and IRF4 drives PTCL cell proliferation and can be blocked by NF-κB inhibitors.

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

Abstract Background/Aim: Glioma-associated oncogene 1 (GLI1) is one of the most important transcription factors in the hedgehog (Hh) signaling pathway and has been shown to be involved in embryonic development and tumor progression. The epithelial-to-mesenchymal transition (EMT) is an important cellular program which contributes to invasion and metastasis of cancer. We have previously shown that GLI1 may promote HCC metastasis via EMT. Here, we demonstrate that GLI1 upregulates SNAI1 expression, induces EMT, and promotes HCC recurrence in vivo and in vitro. Methods: Gli1 mRNA was assessed in 139 HCCs by microarray and its correlation with HCC recurrence and SNAI1 mRNA expression was analyzed. In vitro, the mRNA and protein expression of GLI1 was measured in Gli1-negative Huh7 cells transfected with GU1 and GU1-expressing SNU398 cells transfected with antisense oligonucleotides targeting GU1 (GLI1 ASO). GLI1-dependent transcriptional activity was assessed with GU reporter luciferase assay and electrophoretic mobility shift assay (EMSA). The interaction between Gli1, SNAI1 and EMT was assessed by real-time PCR, Western immunoblotting and immunofluorescence. Effects of GLI1 on proliferation, viability, migration, and invasion of tumor cells were also assessed. Results: GLI1 mRNA was detected in 74 HCC tumors and found to be upregulated in 43 (58.1 %) HCCs. Increased Gli1 mRNA in HCCs was associated with tumor recurrence after resection. There was a positive correlation between GLI1 and SNAI1 mRNA expression. In HCC cells, GLI1 overexpression increased Gli1-dependent transcriptional activity, proliferation, viability, migration, and invasion of tumor cells. GU1 also increased SNAI1 expression and induced the EMT. Opposite effects were observed in GU1-knockdown models. Conclusion: Together, these findings identify a novel mechanism mediating HCC recurrence that includes GLI1-mediated activation of EMT via upregulation of SNAI1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B20.

Abstract The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here, we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility, and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC. Cancer Res; 72(1); 88–99. ©2011 AACR.

AbstractAlthough proinflammatory and chemotactic cytokines can profoundly affect the tumor microenvironment, and many of them have been shown to have therapeutic efficacy in preclinical models, the role of these molecules in Waldenström macroglobulinemia (WM) remains poorly understood. In this study, simultaneous analysis of WM patient sera and bone marrow biopsies identified a set of dysregulated cytokines including CCL5, G-CSF, and soluble IL-2 receptor, that were significantly elevated in WM patients whereas IL-8 and EGF levels were significantly lower in these patients compared with healthy controls. Interestingly, CCL5 levels positively correlated with features of disease aggressiveness such as elevated IgM levels and bone marrow involvement. Functional analysis of tumor microenvironment revealed a functional correlation between CCL5 levels and IL-6 levels, a proinflammatory cytokine with an important role in normal and malignant B-cell biology. Furthermore, CCL5 stimulated IL-6 secretion in WM stromal cells resulting in increased IgM secretion by WM malignant cells via the JAK/STAT signaling pathway. Thus, together these results define a novel signaling network in the WM tumor microenvironment controlling IgM secretion and suggest CCL5 as a potential target for the treatment of this disease.

The chromosomal translocation t (11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1–driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1–expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G 1 –S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2’s antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1–expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1–expressing tumors.