RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing.

RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.

Constitutively Activated DAP12 Induces Functional Anti-Tumor Activation and Maturation of Human Monocyte-Derived DC.

Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.

Data on SVCT2 transporter expression and localization in cancer cell lines and tissues.

The data presented in this article are related to the research paper entitled "Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer", available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.

Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer.

The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines. We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.

Data supporting the functional role of Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) in B cell lymphoma cell line cells.

The data presented here are related to the research article entitled "Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival" (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented.

Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival.

B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt's lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma's with a germinal center origin.

PRDM1/Blimp1 downregulates expression of germinal center genes LMO2 and HGAL.

Human germinal center-associated lymphoma (HGAL) and LIM domain only-2 (LMO2) are proteins highly expressed in germinal center (GC) B lymphocytes. HGAL and LMO2 are also expressed in GC-derived lymphomas and distinguish biologically distinct subgroups of diffuse large B-cell lymphomas (DLBCL) associated with improved survival. However, little is known about their regulation. PRDM1/Blimp1 is a master regulator of terminal B cell differentiation and may also function as a tumor suppressor in the pathogenesis of DLBCL, where it is frequently inactivated by mutations and deletions. We now demonstrate that both HGAL and LMO2 are directly regulated by the transcription repressor PRDM1. In vivo studies demonstrate that PRDM1 directly binds to the recognition sites within the upstream promoters of both HGAL and LMO2. PRDM1 binding suppresses endogenous protein and mRNA levels of HGAL and LMO2. In addition, promoter analysis reveals that site-specific binding of PRDM1 to the promoters is capable of repressing transcriptional activity. This inhibitory effect of PRDM1 suggests that it has a key role in the loss of HGAL and LMO2 expression upon differentiation of GC B cells to plasma cells and may also contribute to absence of HGAL and LMO2 expression in post-GC lymphoid tumors.

PRDM1/Blimp-1 controls effector cytokine production in human NK cells.

NK cells are major effectors of the innate immune response through cytolysis and bridge to the adaptive immune response through cytokine release. The mediators of activation are well studied; however, little is known about the mechanisms that restrain activation. In this report, we demonstrate that the transcriptional repressor PRDM1 (also known as Blimp-1 or PRDI-BF1) is a critical negative regulator of NK function. Three distinct PRDM1 isoforms are selectively induced in the CD56(dim) NK population in response to activation. PRDM1 coordinately suppresses the release of IFN-γ, TNF-α, and TNF-ß through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFN-γ and TNF-α but does not alter cytotoxicity, whereas overexpression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. Collectively, these data demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response.

PRDM1 is required for mantle cell lymphoma response to bortezomib.

Mantle cell lymphoma (MCL) is an aggressive form of B-cell lymphoma with a poor disease-free survival rate. The proteasome inhibitor bortezomib is approved for the treatment of relapsed and refractory MCL and has efficacy in about 30% of patients. However, the precise mechanism of action of bortezomib is not well understood. This report establishes a requirement for the transcription repressor PR domain zinc finger protein 1 (PRDM1, Blimp1) in the response to bortezomib. Bortezomib rapidly induces transcription of PRDM1 as part of the apoptotic response in both cell lines and primary MCL tumor cells. Knockdown of PRDM1 blocks activation of NOXA and inhibits apoptosis, whereas ectopic expression of PRDM1 alone leads to apoptosis in MCL. Two novel direct targets of PRDM1 were identified in MCL cells: MKI67 (Ki67) and proliferating cell nuclear antigen (PCNA). Both MKI67 and PCNA are required for proliferation and survival. Chromatin immunoprecipitation and knockdown studies reveal that specific repression of MKI67 and PCNA is mediated by PRDM1 in response to bortezomib. Furthermore, promoter studies and mutation/deletion analysis show that PRDM1 functions through specific sites in the PCNA proximal promoter and an MKI67 distal upstream repression domain. Together, these findings establish PRDM1 as a key mediator of bortezomib activity in MCL.

PU.1 regulates positive regulatory domain I-binding factor 1/Blimp-1 transcription in lymphoma cells.

The human positive regulatory domain I-binding factor 1 (PRDI-BF1) and its murine homolog Blimp-1 promote differentiation of mature B cells into Ab-secreting plasma cells. In contrast, ectopic expression of PRDI-BF1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1, the gene encoding PRDI-BF1. This report establishes that in lymphoma cells stimulation through the BCR rapidly induces endogenous PRDM1 at the level of transcription with minor changes in mRNA stability. The induced PRDM1-encoded protein localizes to its target genes in vivo and suppresses their expression. In vivo genomic footprinting of the PRDM1 promoter in unstimulated lymphoma and myeloma cells reveals multiple common in vivo occupied elements throughout the promoter. Further functional and structural analysis of the promoter reveals that the promoter is preloaded and poised for activation in the B cell lines. The transcription factor PU.1 is shown to be required for the BCR-induced expression of PRDM1 in lymphoma cells and in PU.1-positive myeloma cells. Activation of PRDM1 is associated with loss of the corepressor transducin-like enhancer of split 4 from the PU.1 complex. These findings indicate that PRDM1 is poised for activation in lymphoma cells and therefore may be a potential therapeutic target to inhibit lymphoma cell proliferation and survival.

The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance.

Antigen-presenting cells (APCs) induce T cell activation as well as T cell tolerance. The molecular basis of the regulation of this critical 'decision' is not well understood. Here we show that HDAC11, a member of the HDAC histone deacetylase family with no prior defined physiological function, negatively regulated expression of the gene encoding interleukin 10 (IL-10) in APCs. Overexpression of HDAC11 inhibited IL-10 expression and induced inflammatory APCs that were able to prime naive T cells and restore the responsiveness of tolerant CD4+ T cells. Conversely, disruption of HDAC11 in APCs led to upregulation of expression of the gene encoding IL-10 and impairment of antigen-specific T cell responses. Thus, HDAC11 represents a molecular target that influences immune activation versus immune tolerance, a critical 'decision' with substantial implications in autoimmunity, transplantation and cancer immunotherapy.

Enhancement of sonochemical degradation of phenol using hydrogen atom scavengers.

Sonochemical degradation of phenol was found to be enhanced in the presence of the volatile hydrogen atom scavengers CCl4 and perfluorohexane. The non-volatile hydrogen atom scavenger iodate did not enhance phenol degradation. The first order rate constant for aqueous phenol degradation in separate experiments using different sonochemical probes increased in the presence of 150 microM CCl4 from 0.014 to 0.031 min(-1) (probe 1) and from 0.022 to 0.061 min(-1) (probe 2). In the presence of <1.5 microM C6H14, the first order rate constant increased from 0.014 to 0.032 min(-1) (probe 1). Hydroquinone was the major observed reaction intermediate both in the presence and absence of hydrogen atom scavengers. Hydroquinone yields were substantially higher in the presence of hydrogen atom scavengers, suggesting that hydroxyl radical pathways for phenol degradation were enhanced by the hydrogen atom scavengers. These additives may be useful in improving pollutant degradation efficiency or improving synthetic processes that rely on hydroxyl radical as a key intermediate.