Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice

Lithocholic acid (LCA) is an endogenous compound associated with hepatic toxicity during cholestasis. LCA exposure in mice resulted in decreased serum lysophosphatidylcholine (LPC) and sphingomyelin levels due to elevated lysophosphatidylcholine acyltransferase (LPCAT) and sphingomyelin phosphodiesterase (SMPD) expression. Global metabolome analysis indicated significant decreases in serum palmitoyl-, stearoyl-, oleoyl-, and linoleoyl-LPC levels after LCA exposure. LCA treatment also resulted in decreased serum sphingomyelin levels and increased hepatic ceramide levels, and induction of LPCAT and SMPD messenger RNAs (mRNAs). Transforming growth factor-β (TGF-β) induced Lpcat2/4 and Smpd3 gene expression in primary hepatocytes and the induction was diminished by pretreatment with the SMAD3 inhibitor SIS3. Furthermore, alteration of the LPCs and Lpcat1/2/4 and Smpd3 expression was attenuated in LCA-treated farnesoid X receptor-null mice that are resistant to LCA-induced intrahepatic cholestasis. CONCLUSION: This study revealed that LCA induced disruption of phospholipid/sphingolipid homeostasis through TGF-β signaling and that serum LPC is a biomarker for biliary injury.     

Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3(-/-) and smpd2(-/-)smpd3(-/-) double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3(-/-) mouse might mimic a form of human combined pituitary hormone deficiency.     

Mutation of C/EBPalpha predisposes to the development of myeloid leukemia in a retroviral insertional mutagenesis screen

The CCAAT enhancer binding protein (C/EBP) is an important myeloid tumor suppressor that is frequently mutated in human acute myeloid leukemia (AML). We have previously shown that mice homozygous for the E2F repression–deficient Cebpa^BRM2 allele develop nonfatal AML with long latency and incomplete penetrance, suggesting that accumulation of secondary mutations is necessary for disease progression. Here, we use SRS19-6–driven retroviral insertional mutagenesis to compare the phenotypes of leukemias arising in Cebpa^+/+, Cebpa^+/BRM2, and Cebpa^BRM2/BRM2 mice, with respect to disease type, latency of tumor development, and identity of the retroviral insertion sites (RISs). Both Cebpa^+/BRM2 and Cebpa^BRM2/BRM2 mice preferentially develop myeloid leukemias, but with differing latencies, thereby demonstrating the importance of gene dosage. Determination of RISs led to the identification of several novel candidate oncogenes, some of which may collaborate specifically with the E2F repression–deficient allele of Cebpa. Finally, we used an in silico pathway analysis approach to extract additional information from single RISs, leading to the identification of signaling pathways which were preferentially deregulated in a disease- and/or genotype-specific manner.     

Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

We sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed a high frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.     

Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia

Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples ("germline") from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1^V623A, JAK1^T478S, DDR1^A803V, and NTRK1^S677N, once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis.     

Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma

Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell–derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.     

The enigma of ectopic expression of FGFR3 in multiple myeloma: a critical initiating event or just a target for mutational activation during tumor progression

The t(4;14)(p16.3;q32) translocation that occurs uniquely in a subset of multiple myeloma tumors results in ectopic expression of wild-type FGFR3 and enhanced expression of MMSET, a gene that is homologous to the MLL gene that is involved in acute myeloid leukemias. Wild-type FGFR3 appears to be weakly transforming in a hematopoietic murine model, whereas FGFR3 that contains kinase-activating mutations is strongly transforming in NIH3T3 cells and the hematopoietic model. The subsequent acquisition of FGFR3 kinase-activating mutations in some tumors with t(4;14) translocations confirms a role for FGFR3 in tumor progression. However, it remains to be proven if and how dysregulation of FGFR3 or MMSET mediates an early oncogenic process in multiple myeloma.     

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18⁺ cells but failed to further differentiate into K14⁺ cells (epidermis/limbus) or K3/K12⁺ cells (corneal epithelium). APR-246 (PRIMA-1^MET), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.     

Expression of the Wnt antagonist DKK3 is frequently suppressed in sporadic epithelial ovarian cancer

Purpose  

The Wnt pathway plays an important role in embryonic development, and defects in this pathway have been implicated in the tumorigenesis. The Dickkopf 3 (DKK3) is a putative Wnt signaling inhibitor that is frequently inactivated in human cancers. However, the expression of DKK3 in ovarian cancer remains unknown.     

Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis

The Hedgehog (Hh) pathway has been implicated in pancreatic cancer but its role remains controversial. To delineate the cell populations able to respond to Hh ligand stimulation, we expressed an oncogenic allele of Smoothened (SmoM2) to cell autonomously activate Hh signaling in the mouse pancreas. Surprisingly, we found that expression of SmoM2 in epithelial cells was not able to activate the pathway and had no impact on pancreatic development or neoplasia. In contrast, activation of Smo in the mesenchyme led to Hh pathway activation, indicating that only the tumor stroma is competent to transduce the Hh signal. Using a Ptc-LacZ reporter mouse, we show that Hh signaling is active in stromal cells surrounding Hh-expressing tumor epithelium in various mouse pancreatic cancer models. Activation of the Hh pathway in the tumor stroma of human pancreatic and metastatic cancer specimens was confirmed by quantitative RT-PCR of microdissected tissue samples. These data support a paracrine model of Hh-mediated tumorigenesis, in which tumor cells secrete Hh ligand to induce tumor-promoting Hh target genes in adjacent stroma.     

Inactivating germ-line and somatic mutations in polypeptide N-acetylgalactosaminyltransferase 12 in human colon cancers

Aberrant glycosylation is a pathological alteration that is widespread in colon cancer, and usually accompanies the onset and progression of the disease. To date, the molecular mechanisms underlying aberrant glycosylation remain largely unknown. In this study, we identify somatic and germ-line mutations in the gene encoding for polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) in individuals with colon cancer. Biochemical analyses demonstrate that each of the 8 GALNT12 mutations identified inactivates the normal function of the GALNT enzyme in initiating mucin type O-linked protein glycosylation. Two of these inactivating GALNT12 mutations were identified as acquired somatic mutations in a set of 30 microsatellite stable colon tumors. Relative to background gene mutation rates, finding these somatic GALNT12 mutations was statistically significant at P < 0.001. Six additional inactivating GALNT12 mutations were detected as germ-line changes carried by patients with colon cancer; however, no inactivating variants were detected among cancer-free controls (P = 0.005). Notably, in 3 of the 6 individuals harboring inactivating germ-line GALNT12 mutations, both a colon cancer and a second independent epithelial cancer had developed. These findings suggest that genetic defects in the O-glycosylation pathway in part underlie aberrant glycosylation in colon cancers, and they contribute to the development of a subset of these malignancies.     

Hedgehog signaling in the murine melanoma microenvironment

The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function. L. Geng and K.C. Cuneo contributed equally to this work.     

p16INK4A tumor suppressor gene expression and CD3epsilon deficiency but not pre-TCR deficiency inhibit TAL1-linked T-lineage leukemogenesis

Inactivation of the CDKN2 genes that encode the p16^INK4A and p14^ARF proteins occurs in the majority of human T-cell acute lymphoblastic leukemias (T-ALLs). Ectopic expression of TAL1 and LMO1 genes is linked to the development of T-ALL in humans. In TAL1xLMO1 mice, leukemia develops in 100% of mice at 5 months. To identify the molecular events crucial to leukemic transformation, we produced several mouse models. We report here that expression of P16^INK4A in developing TAL1xLMO1 thymocytes blocks leukemogenesis in the majority of the mice, and the leukemias that eventually develop show P16^INK4A loss of expression. Events related to the T-cell receptor ß selection process are thought to be important for leukemic transformation. We show here that the absence of the pT chain only slightly delays the appearance of TAL1xLMO1-induced T-ALL, which indicates a minor role of the pT chain. We also show that the CD3-mediated signal transduction pathway is essential for this transformation process, since the TAL1xLMO1xCD3-deficient mice do not develop T-ALL for up to 1 year.      

Notch signalling suppresses apoptosis in adult human and mouse pancreatic islet cells

Aims/hypothesis The pathogenesis of diabetes and the success of islet transplantation depend on the control of pancreatic beta cell fate. The Notch signalling pathway is essential for normal prenatal pancreatic development, but the presence and function of this gene network in adult islets has received much less attention. Methods The presence of Notch signalling components was assessed in vitro using RT-PCR, western blotting and immunofluorescence. The functional consequences of altering Notch signalling on insulin secretion and programmed cell death were examined. Results Adult mouse islets, human islets and mouse insulinoma MIN6 cells possess key components of the Notch pathway. RT-PCR, western blotting and immunofluorescence indicated that the Notch target gene, neurogenin3 (Ngn3, also known as Neurog3), is also present in adult islet cells. Inhibiting Notch signalling with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) increased Ngn3 mRNA expression and protein levels in adult islets. The activated notch homologue 1 (NOTCH1) protein level was decreased upon serum withdrawal, as well as after treatment with a phosphatidylinositol 3-kinase inhibitor, or hydroxy-2-naphthalenylmethylphosphonic acid, an insulin receptor inhibitor. While islets cultured in DAPT did not exhibit defects in insulin secretion, indicating that differentiation is unaltered, inhibiting gamma-secretase-dependent Notch activation led to a dose-dependent increase in caspase-3-dependent apoptosis in both MIN6 cells and human islets. Conversely, gamma-secretase overactivity resulted in an accumulation of cleaved NOTCH1 and protection from apoptosis. Conclusions/interpretation Together these results show that the Notch/Ngn3 signalling network is intact and functional in adult islets. This pathway represents an attractive target for modulating beta cell fate in diabetes, islet transplantation and efforts to derive beta cell surrogates in vitro.     

The pathogenesis and treatment of acid sphingomyelinase-deficient Niemann–Pick disease

Summary Patients with types A and B Niemann–Pick disease (NPD) have an inherited deficiency of acid sphingomyelinase (ASM) activity. The clinical spectrum of this disorder ranges from the infantile, neurological form that results in death by 3 years of age (type A NPD) to the non-neurological form (type B NPD) that is compatible with survival into adulthood. Intermediate cases also have been reported, and the disease is best thought of as a single entity with a spectrum of phenotypes. ASM deficiency is panethnic, but appears to be more frequent in individuals of Middle Eastern and North African descent. Current estimates of the disease incidence range from ∼0.5 to 1 per 100 000 births. However, these approximations likely under estimate the true frequency of the disorder since they are based solely on cases referred to biochemical testing laboratories for enzymatic confirmation. The gene encoding ASM (SMPD1) has been studied extensively; it resides within an imprinted region on chromosome 11, and is preferentially expressed from the maternal chromosome. Over 100 SMPD1 mutations causing ASM-deficient NPD have been described, and some useful genotype–phenotype correlations have been made. Based on these findings, DNA-based carrier screening has been implemented in the Ashkenazi Jewish community. ASM ‘knockout’ mouse models also have been constructed and used to investigate disease pathogenesis and treatment. Based on these studies in the mouse model, an enzyme replacement therapy clinical trial has recently begun in adult patients with non-neurological ASM-deficient NPD. Competing interests: None declared References to electronic databases: OMIM accession numbers: 257200, 607616, 607608.