Cholesterol and CDON Regulate Sonic Hedgehog Release from Pancreatic Cancer Cells.

Background: Sonic Hedgehog (Shh) is a tightly regulated membrane-associated morphogen and a known driver of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). After processing, Shh remains at the plasma membrane of Shh producing cells, thereby limiting its distribution and signal strength. In PDAC, the release of Shh from tumor cells is necessary to promote a tumor-permissive microenvironment. Mechanisms regulating Shh sequestration and/or release from tumor cells to signal distant stromal cells are not well known. Previously, our laboratory demonstrated that the Drosophila transmembrane protein Boi, sequesters Hh at the membrane of Hh-producing cells. In response to dietary cholesterol or in the absence of boi, Hh is constitutively released to promote proliferation in distant cells. In this study, we investigated the conservation of this mechanism in mammals by exploring the role of the human boi homolog, CDON, in PDAC. Methods: Using PDAC cell-lines BxPC-3, Capan-2, and MIA PaCa-2, along with normal pancreatic epithelial cells (PDEC), we investigated Shh expression via Immunoblot and real-time, quantitative polymerase chain reaction in addition to Shh release via enzyme-linked immunoassay following cholesterol treatment and/or transfection with either RNA interference to reduce CDON expression or with human CDON to increase expression. Results: Consistent with our Boi model, CDON suppresses Shh release, which is alleviated in response to dietary cholesterol. However, over-expressing CDON suppresses cholesterol-mediated Shh release in some PDAC contexts, which may be relative to the mutational burden of the cells. Conclusion: Identifying mechanisms that either sequester or stimulate Shh release from the tumor cell membrane may provide new avenues to reduce signaling between the tumor and its surrounding environment, which may restrain tumor development.

Functional proteomics interrogation of the kinome identifies MRCKA as a therapeutic target in high-grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological cancer with few effective, targeted therapies. HGSOC tumors exhibit genomic instability with frequent alterations in the protein kinome; however, only a small fraction of the kinome has been therapeutically targeted in HGSOC. Using multiplexed inhibitor beads and mass spectrometry, we mapped the kinome landscape of HGSOC tumors from patients and patient-derived xenograft models. The data revealed a prevalent signature consisting of established HGSOC driver kinases, as well as several kinases previously unexplored in HGSOC. Loss-of-function analysis of these kinases in HGSOC cells indicated MRCKA (also known as CDC42BPA) as a putative therapeutic target. Characterization of the effects of MRCKA knockdown in established HGSOC cell lines demonstrated that MRCKA was integral to signaling that regulated the cell cycle checkpoint, focal adhesion, and actin remodeling, as well as cell migration, proliferation, and survival. Moreover, inhibition of MRCKA using the small-molecule BDP9066 decreased cell proliferation and spheroid formation and induced apoptosis in HGSOC cells, suggesting that MRCKA may be a promising therapeutic target for the treatment of HGSOC.

O-GlcNAc Transferase Regulates Cancer Stem-like Potential of Breast Cancer Cells.

Breast tumors are heterogeneous and composed of different subpopulation of cells, each with dynamic roles that can change with stage, site, and microenvironment. Cellular heterogeneity is, in part, due to cancer stem-like cells (CSC) that share properties with stem cells and are associated with treatment resistance. CSCs rewire metabolism to meet energy demands of increased growth and biosynthesis. O-GlcNAc transferase enzyme (OGT) uses UDP-GlcNAc as a substrate for adding O-GlcNAc moieties to nuclear and cytoplasmic proteins. OGT/O-GlcNAc levels are elevated in multiple cancers and reducing OGT in cancer cells blocks tumor growth. Here, we report that breast CSCs enriched in mammosphere cultures contain elevated OGT/O-GlcNAcylation. Inhibition of OGT genetically or pharmacologically reduced mammosphere forming efficiency, the CD44H/CD24L, NANOG+, and ALDH+ CSC population in breast cancer cells. Conversely, breast cancer cells overexpressing OGT increased mammosphere formation, CSC populations in vitro, and also increased tumor initiation and CSC frequency in vivo. Furthermore, OGT regulates expression of a number of epithelial-to-mesenchymal transition and CSC markers including CD44, NANOG, and c-Myc. In addition, we identify Krüppel-like factor 8 (KLF8) as a novel regulator of breast cancer mammosphere formation and a critical target of OGT in regulating CSCs. IMPLICATIONS: These findings demonstrate that OGT plays a key role in the regulation of breast CSCs in vitro and tumor initiation in vivo, in part, via regulation of KLF8, and thus inhibition of OGT may serve as a therapeutic strategy to regulate tumor-initiating activity.

O-GlcNAcylation in Cancer Biology: Linking Metabolism and Signaling.

The hexosamine biosynthetic pathway (HBP) is highly dependent on multiple metabolic nutrients including glucose, glutamine, and acetyl-CoA. Increased flux through HBP leads to elevated post-translational addition of ß-D-N-acetylglucosamine sugars to nuclear and cytoplasmic proteins. Increased total O-GlcNAcylation is emerging as a general characteristic of cancer cells, and recent studies suggest that O-GlcNAcylation is a central communicator of nutritional status to control key signaling and metabolic pathways that regulate multiple cancer cell phenotypes. This review summarizes our current understanding of changes of O-GlcNAc cycling enzymes in cancer, the role of O-GlcNAcylation in tumorigenesis, and the current challenges in targeting this pathway therapeutically.

mTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer.

UNLABELLED: Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-ß-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K-mTOR-MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. IMPLICATIONS: Evidence indicates OGT as a therapeutic target in c-MYC-amplified cancers.

O-GlcNAcylation regulates cancer metabolism and survival stress signaling via regulation of the HIF-1 pathway.

The hexosamine biosynthetic pathway elevates posttranslational addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. Cancer cells elevate total O-GlcNAcylation by increasing O-GlcNAc transferase (OGT) and/or decreasing O-GlcNAcase (OGA) levels. Reducing O-GlcNAcylation inhibits oncogenesis. Here, we demonstrate that O-GlcNAcylation regulates glycolysis in cancer cells via hypoxia-inducible factor 1 (HIF-1α) and its transcriptional target GLUT1. Reducing O-GlcNAcylation increases α-ketoglutarate, HIF-1 hydroxylation, and interaction with von Hippel-Lindau protein (pVHL), resulting in HIF-1α degradation. Reducing O-GlcNAcylation in cancer cells results in activation of endoplasmic reticulum (ER) stress and cancer cell apoptosis mediated through C/EBP homologous protein (CHOP). HIF-1α and GLUT1 are critical for OGT-mediated regulation of metabolic stress, as overexpression of stable HIF-1 or GLUT1 rescues metabolic defects. Human breast cancers with high levels of HIF-1α contain elevated OGT, and lower OGA levels correlate independently with poor patient outcome. Thus, O-GlcNAcylation regulates cancer cell metabolic reprograming and survival stress signaling via regulation of HIF-1α.

Protein phosphatase 2A (PP2A) regulates low density lipoprotein uptake through regulating sterol response element-binding protein-2 (SREBP-2) DNA binding.

LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake.

Light-dependent expression of four cryptic archaeal circadian gene homologs.

Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man, yet it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. As an initial investigation of this question, we examined the regulation of four cyanobacterial-like circadian gene homologs present in the genome of the haloarchaeon Haloferax volcanii. These genes, designated cirA, cirB, cirC, and cirD, display similarity to the KaiC-family of cyanobacterial clock proteins, which act to regulate rhythmic gene expression and to control the timing of cell division. Quantitative RT-PCR analysis was used to examine the expression of each of the four cir genes in response to 12 h light/12 h dark cycles (LD 12:12) in H. volcanii during balanced growth. Our data reveal that there is an approximately two to sixteen-fold increase in cir gene expression when cells are shifted from light to constant darkness, and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii cir genes result in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing, in trans, a wild-type copy of individual cir genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kaiC homologs found among archaeal genomes.

Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) has been identified as a proto-oncogene that is overexpressed in various types of human cancers. CIP2A acts by inhibiting protein phosphatase 2A-dependent destabilization of c-Myc, resulting in increased cell proliferation. Here, we have characterized the proximal promoter region of the human CIP2A gene in cervical, endometrial and liver carcinoma cells. The 5' flanking minimal proximal promoter of the CIP2A gene consists of putative binding sites for Ets1 and Elk1 in forward and reverse orientations. Here, we show that Ets1 and Elk1 binding is essential for CIP2A basal expression in several urogenital cancer cell lines. Interestingly, both Ets1 and Elk1 are required together for CIP2A expression, as siRNA knockdown of Ets1 and Elk1 together decreased CIP2A gene transcription, whereas knockdown of Ets1 or Elk1 alone had no effect. Moreover, ectopic expression of Ets1 and Elk1 together increased CIP2A expression. To gain physiological significance of the Ets1 and Elk1 regulation we observed, a panel of matched human cervical carcinoma samples was analyzed for the expression of CIP2A and Ets1 and/or Elk1. We found a direct correlation between the levels of CIP2A and the levels of Ets1 and Elk1. Our results suggest that the binding of Ets1 and Elk1 together to the proximal CIP2A promoter is absolutely required for CIP2A expression in cervical, endometrial and liver carcinoma cell lines. Thus, different factors regulate CIP2A expression in a cell-type specific manner. As previous work has shown a requirement for only Ets1 in prostate and gastric carcinomas, our results now indicate that CIP2A regulation is more complex than previously determined.