Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Cognitive control in social situations: a role for the dorsolateral prefrontal cortex.

Using functional magnetic resonance imaging (fMRI), we investigated brain activity elicited by a computer-animated child's actions that appeared consistent and inconsistent with a computer-animated adult's instructions. Participants observed a computer-animated adult verbally instructing a computer-animated child to touch one of two objects. The child performed correctly in half of the trials and incorrectly in the other half. We observed significantly greater activity when the child performed incorrectly compared to correctly in regions of the dorsolateral prefrontal cortex (DLPFC) that have been implicated in maintaining our intentions in working memory and implementing cognitive control. However, no such effects were found in regions of the posterior superior temporal sulcus (posterior STS) that have been posited to interpret other people's behavior. These findings extend the role of the DLPFC in cognitive control to evaluating the social outcomes of other people's behavior and provide important new constraints for theories of how the posterior STS contributes to social cognition.

Formation of Neu/ErbB2-induced mammary tumors is unaffected by loss of ErbB4.

The four members of the ErbB family of receptor tyrosine kinases are involved in development and tumorigenesis of the mammary gland. Whereas the epidermal growth factor receptor, ErbB2 and ErbB3 are positively associated with various cancers, clinical studies of ErbB4 in breast cancer are contradictory. Results from tissue culture analyses and some clinical studies suggested that ErbB4 is either a tumor suppressor or is a negative regulator of ErbB2-driven tumors. Neu-Cre-ErbB4(flox/null) mice in which ErbB4 was inactivated by Cre-lox-mediated recombination in the mammary gland developed MMTV-Neu-driven mammary tumors with a similar latency period to mice with one or two wild-type ErbB4 alleles. Moreover, there was no difference in the histologies of tumors that developed, nor in the propensity to form lung metastases. Taken together these results suggest that ErbB4 is not a potent, highly penetrant tumor suppressor, nor is it a factor in Neu-mediated tumorigenesis in this model.

F-MuLV acceleration of myelomonocytic tumorigenesis in SV40 large T antigen transgenic mice is accompanied by retroviral insertion at Fli1 and a novel locus, Fim4.

We describe here the development of a murine system for the identification of genes involved in myelomonocytic neoplasms. Transgenic C57BL/6J mice expressing SV40 early region under a myelomonocytic promoter develop histiocytic sarcomas with a latency of 167 days. We used retroviral proviral tagging to accelerate tumorigenesis and to uncover genetic changes that contribute to tumor development. Infection of transgenic mice with Friend murine leukemia virus (F-MuLV) shortened the latency of morbidity to 103 days (P< 0.001); this was associated with clonal proviral integrations in tumor DNA. As expected for F-MuLV, proviral insertions occurred at Fli1 in both transgenic and nontransgenic tumors. Four insertions were found at a novel locus, termed Fim4, on chromosome 6. This region is syntenic to human 7q32, a region that is commonly deleted in human myelodysplastic syndrome and acute myeloid leukemia. A murine BAC containing Fim4 was sequenced and analyzed, and while there was significant human-mouse homology in the area of the insertions, no candidate gene has been identified. Thus we have established a system to identify genes involved in myelomonocytic tumors, and have used it to identify Fim4, a new common site of proviral insertion. Study of this locus may provide insight into genes involved in AML-associated 7q32 deletions in humans.

Cooperating oncogenic events in murine mammary tumorigenesis: assessment of ErbB2, mutant p53, and mouse mammary tumor virus.

We are investigating cooperating genetic events in the genesis of breast cancer, using the mouse as a model system. We have shown cooperativity between a mutant allele of p53 (p53-172H) and overexpressed ErbB2 in mammary tumorigenesis in transgenic mice. We are now performing additional crosses to further examine oncogene cooperativity with ErbB2 and p53-172H. We attempted to test the dominant oncogenic potential of p53-172H in an in vivo setting by crossing the p53-172H transgene together with ErbB2 onto either a p53(-/-) or a p53(+/-) background. We show that the p53-172H allele and the heterozygous p53 genotype have an identical impact on the latency of ErbB2-induced mammary tumors; there was no evidence of additivity or synergy between p53-172H and the p53(+/-) genotype. On the p53(-/-) background, we obtained no mammary tumors due to the early onset of lymphomas and sarcomas, thus precluding assessment of the effect of the p53-172H transgene on mammary tumorigenesis in a p53-null background. Thus, in this in vivo model for breast cancer, we failed to find evidence that p53-172H can function as a dominant oncogenic allele, but rather found support for its being essentially equivalent to a null allele in its impact on ErbB2-induced mammary tumorigenesis. By comparative genome analysis, we showed that a common feature of tumors arising in ErbB2/mutant p53 mice (p53-null allele with or without p53-172H) is a loss of chromosome 4, a feature of many epithelial tumors in mice and one that is consistent with a role for loss of INK4a/ARF in such tumors. We also attempted to accelerate ErbB2-induced mammary tumorigenesis with mouse mammary tumor virus (MMTV) proviral tagging mutagenesis, but we were surprised to find that mice with MMTV alone had the same latency as mice with both MMTV and ErbB2, indicating no cooperativity between ErbB2 and MMTV. This may have been due to the mixed C3H/HeN x FVB strain background used in this cross.

Pathogenesis of placental site trophoblastic tumor may require the presence of a paternally derived X chromosome.

Placental site trophoblastic tumor (PSTT) is a neoplastic proliferation of intermediate trophoblasts that invades the myometrium at the placental site after a pregnancy. Less than 100 cases have been reported. Information of the sex assignment of the antecedent gestation is available in 21 cases: 18 of these were female. To explore this interesting phenomenon, we have determined the sex chromosome composition of the tumor tissue preserved in paraffin blocks for five new cases of this condition. The last documented gestational event included a normal vaginal delivery of female infants in three cases, normal vaginal delivery of an infant of unknown sex in one case and a molar gestation in one case. Using the X-linked human androgen receptor (AR) gene as a polymorphic marker, we showed that in all five cases the tumor had a likely XX chromosomal composition; and in four cases it was possible to determine that one of the X chromosomes was of paternal origin. In one case, the paternal X chromosome showed no polymorphism to either maternal X chromosomes. In addition, sensitive semi-nested PCR failed to show a human Y chromosome element in any of the five cases of PSTT. Overall, of 21 cases from the literature and 5 cases of ours, 89% (23 of 26) showed an XX genomic composition in PSTT, either by history or genetic analysis. These results suggest that most PSTT were derived from the antecedent female conceptus and were likely to have possessed a functional paternal X chromosome. Methylation status analysis at the AR locus was performed in the three PSTT in which the paternal X chromosome was identifiable. In two cases, the paternal AR locus was hypomethylated while the corresponding maternal locus was hypermethylated. The methylation status of other loci was not investigated. Collectively, sex chromosome analysis of five cases of PSTT with literature support suggests a unique genetic basis for the development of PSTT that involves the paternal X chromosome. Although largely speculative, an active paternal X chromosome may be of importance in the pathogenesis of PSTT.

Identification of candidate target genes for EVI-1, a zinc finger oncoprotein, using a novel selection strategy.

We have sought to identify and isolate target genes for the zinc finger protein, EVI-1, which has been implicated in the genesis of myelogenous leukemia both in mouse and human. We have approached this with a two-step selection: we first selected for genomic fragments of mouse DNA that bind to the protein with high affinity; second, we employed cDNA hybrid selection to identify gene sequences contained within these fragments. We show that we have constructed a sublibrary of genomic fragments that contains a significant fraction of the EVI-1-binding sites in the mouse genome. Our data has allowed us to estimate that there are approximately 4300 binding sites per haploid genome in the mouse. We further demonstrate that by using cDNA hybrid selection, it is relatively straightforward to isolate cDNAs that correspond to genes embedded in the EVI-1-binding sublibrary. Several of these are novel, but are represented in databases of anonymous human or mouse cDNAs (expressed sequence tags). One selected gene is Itpr2, encoding the inositol trisphosphate type two receptor, which is transcriptionally regulated during myelopoiesis. Finally, using a chimeric EVI-1-VP16-fusion protein under the control of a tetracycline-regulated system, we have shown that this chimeric activator can directly regulate Itpr2.

Vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge.

Since the development of a system for generating vesicular stomatitis virus (VSV) from plasmid DNAs, our laboratory has reported the expression of several different glycoproteins from recombinant VSVs. In one of these studies, high-level expression of an influenza virus hemagglutinin (HA) from a recombinant VSV-HA and efficient incorporation of the HA protein into the virions was reported (E. Kretzschmar, L. Buonocore, M. J. Schnell, and J. K. Rose, J. Virol. 71:5982-5989, 1997). We report here that VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus. Additionally, these recombinant VSVs are less pathogenic than wild-type VSV (serotype Indiana). This vector-associated pathogenicity was subsequently eliminated through introduction of specific attenuating deletions. These live attenuated recombinant VSVs have great potential as vaccine vectors.

neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice.

Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of the nontransgenic mice or the p53-172H transgenic mice developed tumors within this time period. Tumors arising in the p53-172H/neu bitransgenic mice were anaplastic and aneuploid and exhibited increased apoptosis, in distinction to tumors arising in p53-null mice, in which apoptosis is diminished. Further experiments address potential mechanisms of cooperativity between the two transgenes. In these bitransgenic mice, we have recapitulated two common genetic lesions that occur in human breast cancer and have shown that p53 mutation is an important cooperating event in neu-mediated oncogenesis.

Retroviral insertional activation of the EVI1 oncogene does not prevent G-CSF-induced maturation of the murine pluripotent myeloid cell line 32Dcl3.

Evi1 is a myeloid-specific protooncogene that encodes 145 kDa and 88 kDa proteins via alternative splicing. Overexpression of the gene via retroviral insertion in murine tumors or chromosomal rearrangement in human tumors is associated with myeloid leukemias and myelodysplasias; however, the mechanism by which such overexpression leads to transformation is not clear. It has been postulated that overexpression of evi1 acts to block normal myelopoiesis. In attempts to assess the effect of overexpression of evi1 on myelopoiesis, we chose to utilize the IL-3-dependent murine 32Dcl3 cell line, which has been shown to differentiate in culture in response to G-CSF. Previous experiments with this cell line, which we have confirmed, showed that overexpression of evi1, mediated by retroviral vector transfer, caused a block to G-CSF-induced cell survival and differentiation. We report here that the naive 32Dcl3 cell line contains a rearrangement of the evi1 locus and constitutively overexpresses evi1 mRNA and protein; this expression is downregulated only slightly during G-CSF-induced myeloid maturation. The steady state levels, molecular weight and DNA binding characteristics of the EVI1 protein in these cells is comparable to that seen in NFS 58, a myeloid leukemia cell line with retroviral insertion at evi1. The observed ability of the murine 32Dcl3 cells to fully differentiate in the presence of G-CSF while evi1 continues to be expressed indicates that, at the levels expressed in naive 32Dcl3, evi1 does not block G-CSF-induced survival and differentiation. Thus, retroviral insertions at evi1 may have been selected for in 32Dcl3 cells due to effects other than that on G-CSF-induced cell survival.

Zinc fingers 1-7 of EVI1 fail to bind to the GATA motif by itself but require the core site GACAAGATA for binding.

EVI1 is a zinc finger oncoprotein that binds via fingers 1-7 to the sequence GACAAGATAA. The target genes on which EVI1 acts are unknown. This binding motif overlaps with that for the GATA transcription factors, (T/A)GATA(A/G), and GATA-1 can bind to and activate transcription via a GACAAGATAA motif. The possibility has been raised that, when overexpressed in leukemogenesis, EVI1 may function by interfering with the differentiation-promoting action of GATA factors. To explore this, we have assessed the affinity of EVI1 for the GATA binding sites derived from erythroid-specific GATA-1 target genes, and found only low affinity interactions. We examined the contacts between EVI1 and DNA by methylation interference studies, which revealed extensive contacts between EVI1 and its binding site. The importance of the contacts for high affinity binding was shown by in vitro quantitative gel shift studies and in vivo cotransfection studies. To examine what types of sequences from mouse genomic DNA bind to EVI1, we isolated and sequenced five EVI1-binding fragments, and each showed the GACAAGATA site. The data presented contribute to our knowledge of the binding specificity of EVI1, and yield a clearer picture of what sequences can, and cannot, act as targets for EVI1 action.

Molecular analysis of Evi1, a zinc finger oncogene involved in myeloid leukemia.

Through chromosomal rearrangements and/or proviral insertions, a number of genes encoding nuclear transcription factors have been identified that play key roles in leukemogenesis. One of these is Evi1, which plays a role in both murine and human myeloid leukemia. The exact mechanism by which Evi1 exerts its leukemogenic effect is not clear, but it may involve the inhibition of terminal differentiation, through the abnormal repression of genes necessary for cellular maturation. Our analysis of the DNA binding characteristics of EVI1 indicate a high degree of specificity, which likely indicates that the protein acts on a tightly defined number of targets in the cell. We are beginning to characterize candidate target genes located in the mouse genome near EVI1 binding sites with the expectation that these will yield insight into EVI1 function both in normal cells and in leukemogenesis.

Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues.

The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice.

Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r).

Newborn mice homozygous for a targeted disruption of insulin-like growth factor gene (Igf-1) exhibit a growth deficiency similar in severity to that previously observed in viable Igf-2 null mutants (60% of normal birthweight). Depending on genetic background, some of the Igf-1(-/-) dwarfs die shortly after birth, while others survive and reach adulthood. In contrast, null mutants for the Igf1r gene die invariably at birth of respiratory failure and exhibit a more severe growth deficiency (45% normal size). In addition to generalized organ hypoplasia in Igf1r(-/-) embryos, including the muscles, and developmental delays in ossification, deviations from normalcy were observed in the central nervous system and epidermis. Igf-1(-/-)/Igf1r(-/-) double mutants did not differ in phenotype from Igf1r(-/-) single mutants, while in Igf-2(-)/Igf1r(-/-) and Igf-1(-/-)/Igf-2(-) double mutants, which are phenotypically identical, the dwarfism was further exacerbated (30% normal size). The roles of the IGFs in mouse embryonic development, as revealed from the phenotypic differences between these mutants, are discussed.

Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop.

myc genes are thought to function in the processes of cellular proliferation and differentiation. To gain insight into the role of the N-myc gene during embryogenesis, we examined its expression in embryos during postimplantation development using RNA in situ hybridization. Tissue- and cell-specific patterns of expression unique to N-myc as compared with the related c-myc gene were observed. N-myc transcripts become progressively restricted to specific cell types, primarily to epithelial tissues including those of the developing nervous system and those in developing organs characterized by epithelio-mesenchymal interaction. In contrast, c-myc transcripts were confined to the mesenchymal compartments. These data suggest that c-myc and N-myc proteins may interact with different substrates in performing their function during embryogenesis and suggest further that there are linked regulatory mechanisms for normal expression in the embryo. We have mutated the N-myc locus via homologous recombination in embryonic stem (ES) cells and introduced the mutated allele into the mouse germ line. Live-born heterozygotes are under-represented but appear normal. Homozygous mutant embryos die prenatally at approximately 11.5 days of gestation. Histologic examination of homozygous mutant embryos indicates that several developing organs are affected. These include the central and peripheral nervous systems, mesonephros, lung, and gut. Thus, N-myc function is required during embryogenesis, and the pathology observed is consistent with the normal pattern of N-myc expression. Examination of c-myc expression in mutant embryos indicates the existence of coordinate regulation of myc genes during mouse embryogenesis.

Targeting of transgene expression to monocyte/macrophages by the gp91-phox promoter and consequent histiocytic malignancies.

A component of a heterodimeric cytochrome b, designated gp91-phox, is required for the microbicidal activity of phagocytic cells and is expressed exclusively in differentiated myelomonocytic cells (granulocytes; monocyte/macrophages). In an attempt to identify cis-elements responsible for this restricted pattern of expression, we produced transgenic mice carrying reporter genes linked to the human gp91-phox promoter. Immunohistochemical and RNA analyses indicate that 450 base pairs of the proximal gp91-phox promoter is sufficient to target reporter expression to a subset of monocyte/macrophages. Mice expressing simian virus 40 large tumor antigen under control of the gp91-phox promoter develop monocyte/macrophage-derived malignancies with complete penetrance at 6-12 mo of age and provide an animal model of true histiocytic lymphoma. As these transgenes are inactive in most phagocytic cells that express the endogenous gp91-phox-encoding gene, we infer that additional genomic regulatory elements are necessary for appropriate targeting to the full complement of phagocytes in vivo.

Evi-1, a murine zinc finger proto-oncogene, encodes a sequence-specific DNA-binding protein.

Evi-1 was originally identified as a common site of viral integration in murine myeloid tumors. Evi-1 encodes a 120-kDa polypeptide containing 10 zinc finger motifs located in two domains 380 amino acids apart and an acidic domain located carboxy terminal to the second set of zinc fingers. These features suggest that Evi-1 is a site-specific DNA-binding protein involved in the regulation of RNA transcription. We have purified Evi-1 protein from E. coli and have employed a gel shift-polymerase chain reaction method using random oligonucleotides to identify a high-affinity binding site for Evi-1. The consensus sequence for this binding site is TGACAAGATAA. Evi-1 protein specifically protects this motif from DNase I digestion. By searching the nucleotide sequence data bases, we have found this binding site both in sequences 5' to genes in putative or known regulatory regions and within intron sequences.