Knockdown of core binding factorß alters sphingolipid metabolism.

Core binding factor (CBF) is a heterodimeric transcription factor containing one of three DNA-binding proteins of the Runt-related transcription factor family (RUNX1-3) and the non-DNA-binding protein, CBFß. RUNX1 and CBFß are the most common targets of chromosomal rearrangements in leukemia. CBF has been implicated in other cancer types; for example RUNX1 and RUNX2 are implicated in cancers of epithelial origin, including prostate, breast, and ovarian cancers. In these tumors, CBF is involved in maintaining the malignant phenotype and, when highly over-expressed, contributes to metastatic growth in bone. Herein, lentiviral delivery of CBFß-specific shRNAs was used to achieve a 95% reduction of CBFß in an ovarian cancer cell line. This drastic reduction in CBFß expression resulted in growth inhibition that was not associated with a cell cycle block or an increase in apoptosis. However, CBFß silencing resulted in increased autophagy and production of reactive oxygen species (ROS). Since sphingolipid and ceramide metabolism regulates non-apoptotic cell death, autophagy, and ROS production, fumonsin B1 (FB1), an inhibitor of ceramide synthase, was used to alter ceramide production in the CBFß-silenced cells. FB1 treatment inhibited the CBFß-dependent increase in autophagy and provided a modest increase in cell survival. To document alterations to sphingolipids in the CBFß-silenced cells, ceramide, and lactosylceramide levels were directly examined by mass spectrometry. Substantial increases in ceramide species and decreases in lactosylceramides were identified. Altogether, this report provides evidence that CBF transcriptional pathways control cellular survival, at least in part, through sphingolipid metabolism.

The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.

Down regulation of CSL activity inhibits cell proliferation in prostate and breast cancer cells.

The Notch receptor pathway provides a paradigm for juxtacrine signaling pathways and controls stem cell function, developmental cell fate decisions, and cellular differentiation. The Notch pathway is constitutively activated in human cancers by chromosomal rearrangements, activating point mutations, or altered expression patterns. Therefore, the Notch pathway is the subject of chemotherapeutic intervention in a variety of human cancers. Notch receptor activation results in the gamma-secretase dependent proteolytic cleavage of the receptor to liberate the Notch intracellular domain that acts to mediate co-activator recruitment to the DNA binding transcription factor, CSL (CBF-1/RBP-Jκ, Su(H), Lag-1). Therapeutic targeting of the Notch pathway by gamma-secretase inhibitors prevents NICD production and regulates CSL-dependent transcriptional activity. To interrogate the loss of CSL activity in breast and prostate cancer cells, we used lentiviral-based shRNA knockdown of CSL. Knockdown of CSL expression was assessed by decreased DNA binding activity and resulted in decreased cell proliferation. In contrast, gamma-secretase inhibitor (GSI) treatment of these prostate and breast cancer cell lines resulted in minimal growth effects. PCR profiling of Notch pathway genes identified expression changes in few genes (Delta-like-1, Deltex-1, LMO2, and SH2D1A) after CSL knockdown. Consistent with differential effects of GSI on cell survival, GSI treatment failed to recapitulate the gene expression changes observed after CSL knockdown. Thus, CSL inhibition may provide a more effective mechanism to inhibit Notch-pathway dependent cancer cell proliferation as compared to GSI treatment.

Association of core-binding factor ß with the malignant phenotype of prostate and ovarian cancer cells.

Core binding factor (CBF) is a transcription factor complex that plays roles in development, stem-cell homeostasis, and human disease. CBF is a heterodimer composed of one of three DNA-binding RUNX proteins plus the non-DNA-binding protein, CBFß. Recent studies have showed that the RUNX factors exhibit complex expression patterns in prostate, breast, and ovarian cancers, and CBF has been implicated in the control of cancer-related genes. However, the biologic roles of CBF in solid tumors have not been fully elucidated. To test whether CBF is required for the malignant phenotype of various epithelial cancers, we used lentiviral delivery of CBFß-specific shRNA to significantly decrease CBFß expression in two prostate cancer cell lines (PPC1 and PC-3) and the SKOV-3 ovarian cancer cell line. We found that knockdown of CBFß significantly inhibited anchorage independent growth of each cell line. Further, CBFß knockdown in PPC1 cells suppressed xenograft tumor growth compared to controls. Mice injected with SKOV-3 ovarian cancer cells knocked-down for CBFß exhibited a survival time similar to control mice. However, human cells recovered from the ascites fluid of these mice showed CBFß expression levels similar to those from mice injected with control SKOV-3 cells, suggesting that CBFß knockdown is incompatible with tumor cell growth. Gene expression profiling of CBFß knockdown cells revealed significant changes in expression in genes involved in various developmental and cell signaling pathways. These data collectively suggest that CBFß is required for malignancy in some human cancers.

Solution structure of Gfi-1 zinc domain bound to consensus DNA.

Gfi-1 is a crucial transcriptional repressor for the precise regulation of cell proliferation and differentiation in hematopoiesis. Recently, this protein has also been demonstrated to be capable of restricting the proliferation of hematopoietic stem cells, a process that appears to be vital for the long-term competency of hematopoietic stem cells. These two seemingly opposite outcomes of regulation are likely to arise from its interactions with a variety of cellular partners. Such interactions can directly affect the genes that Gfi-1 recognizes through its DNA binding zinc-finger domain. In this work, we report the determination of the solution structure of Gfi-1 zinc fingers 3-5 in complex with a 16-mer consensus DNA using multidimensional NMR method. Unlike a proposed minor-groove binding model based on methylation interference experiments, our structure clearly shows that Gfi-1 zinc fingers 3-5 bind into the major groove of the target DNA reminiscent of canonical C(2)H(2) zinc-finger domains. The fourth and fifth zinc fingers recognize the AATC core sequence by forming base-specific hydrogen bonds between the side chains of Asn382, Gln379, and Asp354 and the bases of the invariant adenines and cytosine. Overall, the current work provides valuable insight into the structural determinants for DNA binding specificity, in particular for the TCA triplet that has not been observed in any other structures of zinc finger-DNA complexes, as well as molecular rationales for a naturally occurring mutation that causes acute myeloid leukemia.

RUNX1 (AML-1) and RUNX2 (AML-3) cooperate with prostate-derived Ets factor to activate transcription from the PSA upstream regulatory region.

The RUNX transcription factors (RUNX1, RUNX2, and RUNX3) play essential roles in hematopoiesis and skeletal development. Consistent with these roles in differentiation and cell cycle, the activity of both RUNX1 and RUNX3 is perturbed in cancer. To determine a role for the RUNX factors in prostate biology, we investigated the expression of RUNX factors in prostate epithelial cell lines and normal prostate tissue. RUNX1, RUNX2, and RUNX3 were expressed in both normal prostate tissue and an immortalized, non-transformed cell line. We found that prostate cancer-derived cell lines expressed RUNX1 and RUNX2, but not RUNX3. Next, we sought to identify prostate-specific genes whose expression could be regulated by RUNX proteins. Four consensus RUNX sites are located within the prostate-specific antigen (PSA) regulatory region. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 is specifically bound to the PSA regulatory region in LNCaP cells. RUNX1 and RUNX2 activated the PSA regulatory region alone or cooperatively with prostate-derived ETS factor (PDEF) and RUNX1 physically associated with PDEF. Taken together, our results suggest that RUNX factors participate in prostate epithelial cell function and cooperate with an Ets transcription factor to regulate PSA gene expression.

Mtgr1 is a transcriptional corepressor that is required for maintenance of the secretory cell lineage in the small intestine.

Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.

Gfi-1 attaches to the nuclear matrix, associates with ETO (MTG8) and histone deacetylase proteins, and represses transcription using a TSA-sensitive mechanism.

Gfi-1 and Gfi-1B can repress transcription and play important roles in hematopoietic cell survival and differentiation. Although these proteins are known to bind DNA through a C-terminal zinc-finger domain and may require an N-terminal SNAG domain (SNAIL/Gfi-1) to repress transcription, the mechanism by which Gfi-1 and Gfi-1B act is unknown. A first step towards understanding the mechanism by which these proteins repress transcription is to identify interacting proteins that could contribute to transcriptional repression. ETO (also termed MTG8), was first identified through its involvement in the (8;21) translocation associated with acute myelogenous leukemia. It attaches to the nuclear matrix and associates with histone deacetylases and the co-repressors N-CoR, SMRT, and mSin3A, and may act as a co-repressor for site-specific transcriptions factors. In this report we demonstrate that Gfi-1 interacts with ETO and related proteins both in vitro and in vivo and with histone deacetylase proteins in vivo. We observed that a portion of Gfi-1 and Gfi-1B associated with the nuclear matrix, as is the case with ETO. Moreover, Gfi-1 and ETO co-localize to punctate subnuclear structures. When co-expressed in mammalian cells, Gfi-1 associates with histone deacetylse-1 (HDAC-1), HDAC-2, and HDAC-3. These data identify ETO as a partner for Gfi-1 and Gfi-1B, and suggest that Gfi-1 proteins repress transcription through recruitment of histone deacetylase-containing complexes.

The ETO (MTG8) gene family.

Cloning and characterization of the 8;21 chromosomal breakpoint identified AML1 on chromosome 21 and ETO (MTG8) on chromosome 8, and the resultant chimeric gene product, AML-1/ETO. The ETO gene family now includes three human members encoding proteins composed of four evolutionarily conserved domains termed nervy homology regions (NHR) 1-4. ETO associates with N-CoR/Sin3a/HDAC complexes in vivo and acts as a corepressor for the promyelocytic zinc finger protein. Moreover, ETO is nuclear matrix attached at sites coincident with histone deacetylase enzymes and mSin3a. These data suggest that ETO proteins function as transcriptional corepressors. This review focuses on the ETO gene family in terms of expression and function. Specifically, the role of ETO as a co-repressor will be detailed. Additionally, the impact of this recent discovery on treatment of t(8;21)-containing leukemia will be discussed.