REGULATION OF FOXC2 EXPRESSION AND FUNCTION DURING EPITHELIAL-MESENCHYMAL TRANSITION BY GSK-3β

Metastasis is the ultimate cause for the majority of cancer-related deaths. The forkhead box transcription factor FOXC2 is known to be involved in regulating metastasis as well as a variety of developmental processes, including the formation of lymphatic and cardiovascular systems. Previous studies have shown that FOXC2 protein is localized either in the nucleus and/or in the cytoplasm of human breast tumor cells. This pattern of localization is similar to that of another forkhead family member, FOXO3a. Additionally, localization of FOXO3a is known to be differentially regulated by upstream kinase AKT. Therefore, I investigated whether FOXC2 localization could also be regulated by upstream kinases. Analysis of FOXC2 protein sequence revealed two potential phosphorylation sites for GSK-3β. Furthermore, inhibition of GSK-3βsignificantly reduces FOXC2 protein. In addition, exposure of HMLE Twist cells expressing endogenous FOXC2 to the GSK-3β inhibitor, TWS119, results in accumulation of FOXC2 protein in the cytoplasm with concomitant decrease in the nucleus in a time-dependent manner. Furthermore, continued treatment with TWS119 eventually induces epithelial morphology and decreased stem cell properties including sphere formation in these cells. Further characterization of FOXC2- GSK-3β interaction and the associated signaling cascade are necessary to determine the effect of FOXC2 phosphorylation by GSK-3β on EMT and metastasis.

INVESTIGATING THE ROLES OF THE p63 ISOFORMS IN THE MICRORNA BIOGENESIS PATHWAY

MicroRNAs play roles in various biological processes like development, tumorigenesis, metastasis and pluripotency. My thesis work has demonstrated roles for p63, a p53 family member, in the upstream regulation of microRNA biogenesis. The p63 gene has a complex gene structure and has multiple isoforms. The TAp63 isoforms contain an acidic transcription activation domain. The ΔNp63 isoforms, lack the TA domain, but have a proline rich region critical for gene transactivation. To understand the functions of these isoforms, the Flores lab generated TAp63 and ΔNp63 conditional knock out mice. Using these mice and tissues and cells from these mice we have found that TAp63 transcriptionally regulates Dicer while ΔNp63 transcriptionally regulates DGCR8. TAp63 -/- mice are highly tumor prone. These mice develop metastatic mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas to distant sites including the liver, lungs, and brain. I found that TAp63 suppresses metastasis by transcriptionally activating Dicer. TAp63 and Dicer levels were very low or lost in high grade human tumors like mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas. Expression of Dicer in these tumor cell lines reduced their invasiveness. Using ΔNp63 -/- mice, I found that ΔNp63 transcriptionally activates DGCR8, resulting in a miRNA profile that is critical to reprogram cells to pluripotency. Analysis of epidermal cells derived from ΔNp63 -/- mice revealed that these cells expressed markers of pluripotency, including Sox2, Oct 4 and Nanog; however, genome-wide analysis revealed a novel profile of genes that are common between ΔNp63 -/- epidermal cells and embryonic stem cells. I also found that mouse cells depleted of ΔNp63 form chimeric mice and teratomas in SCID mice, demonstrating that ΔNp63 deficient cells are pluripotent. Further, I found that restoration of DGCR8 in ΔNp63 -/- epidermal cells reduces their pluripotency and induces terminal differentiation. I also demonstrated that iMS (induced multipotent stem) cells could be generated using human keratinocytes by knockdown of ∆Np63 or DGCR8. Taken together, my work has placed p63 and its isoforms at a critical node in controlling miRNA biogenesis.

Effects of exogeneous p53 on growth suppression, apoptosis, and differentiation in oral cancer cell lines

The p53 gene is known to be one of the most commonly mutated genes in human cancers. Many squamous cell carcinomas of the head and neck (SCCHNs) have been shown to contain nonfunctional p53 as well. The use of p53-mediated gene therapy to treat such cancers has become an intensive area of research. Although there have been varied treatment responses to p53 gene therapy, the role that endogenous p53 status plays in this response has not been thoroughly examined. Because of this, the hypothesis of this study examined the role that the endogenous p53 status of cells plays in their response to p53 gene therapy. To test this, an adenoviral vector containing p53 (p53FAd) was administered to three squamous cell carcinoma lines with varied endogenous p53. The SCC9 cell line demonstrates no p53 protein expression, the SCC4 cell line displays overexpression of a mutant p53 protein, and the 1986LN cell line displays low to no expression of wild-type p53 protein as a consequence of human papillomavirus infection. After treatment with p53FAd, the cells were examined for evidence of exogenous p53 expression, growth suppression, alterations in cellular proteins, G1 growth arrest, apoptosis, and differentiation state. Each cell line exhibited exogenous p53 protein. Growth suppression was seen most prominently in the SCC9 cells, to some extent in the 1986LN cells, and little was seen with the SCC4 cells. WAF1/p21 protein was induced in all three cell lines, while PCNA, bcl-2, and bax expression was not significantly affected in any of the lines. Apoptosis developed first in SCC9 cells, next in 1986LN cells, with little seen in the SCC4 cells. The SCC9 line was the only line to show significant GI growth arrest. No significant differences were observed in the overall expression of differentiation markers, aside from increased keratin 13 mRNA levels in all three lines indicating a possible tendency toward differentiation. This study indicates that the endogenous p53 status of squamous cell carcinomas appears to play a critical role in determining the response to p53 adenoviral gene therapy. ^

PML tumor suppressor potentiates cell death through inhibition of the NF -kappa B survival pathway

The promyelocytic leukemia protein PML is a growth suppressor essential for induction of apoptosis by diverse apoptotic stimuli. The mechanism by which PML regulates cell death remains unclear. In this study we found that ectopic expression of PML potentiates cell death in the TNFα-resistant tumor line U2OS and significantly sensitized these cells to apoptosis induced by TNFα in a p53-independent manner. Our study demonstrated that both PML and PML/TNFα-induced cell death are associated with DNA fragmentation, activation of caspase-3, -7, -8, and degradation of DFF/ICAD. Furthermore, we found that PML-induced and PML/TNFα-induced cell death could be blocked by the caspase-8 inhibitors crmA and c-FLIP, but not by Bcl-2, the inhibitor of mitochondria-mediated apoptotic pathway. These findings indicate that this cell death event is initiated through the death receptor-dependent apoptosis pathway. Our study further showed that PML recruits NF-kappa B (NF-κB) to the PML nuclear body, blocks NF-κB binding to its cognate enhancer, and represses its transactivation function with the C-terminal region. Therefore PML inhibits the NF-κB survival pathway. Overexpression of NF-κB rescued cell death induced by PML and PML/TNFκ. These results imply that PML is a functional repressor of NF-κB. This notion was further supported by the finding that the PML−/− mouse embryo fibroblasts (MEFs) are more resistant than the wild-type MEFs to TNFκ-induced apoptosis. In conclusion, our studies convincingly demonstrated that PML potentiates cell death through inhibition of the NF-κB survival pathway. Activation of NF-κB frequently occurs during oncogenesis. Our study here suggests that a loss of PML function enhances the NF-κB survival pathway and this event may contribute to tumorigenesis. ^

Survey of living conditions of Arctic indigenous peoples

Major findings of the Survey of Living Conditions in the Arctic (SLiCA) are: (1) A combination of traditional activities and cash employment is the prevailing lifestyle of Arctic indigenous peoples; (2) family ties, social support of each other, and traditional activities have a lot to do with why indigenous people choose to remain in Arctic communities; (3) well-being is closely related to job opportunities, locally available fish and game, and a sense of local control. Well-being and depression (and related problems like suicide) are flip sides of the same coin. Improving well-being may reduce social problems; and, (4) health conditions vary widely in the Arctic: three-in-four Greenlandic Inuit self-rate their health as at least very good compared with one-in-two Canadian and Alaska Inuit and one-in-five Chukotka indigenous people. Findings are based on 7,200 interviews in a probability sample of Inupiat settlement regions of Alaska, the four Inuit settlement regions of Canada, all of Greenland, and the Anadyrskij, Anadyr, Shmidtovs, Beringovskij, Chukotskij, Iujl'tinskij, Bilibinskij, Chaunskij, Providenskij, Uel'Kal' districts of Chukotka. Indigenous people and researchers from Greenland, Russia, Canada, the United States, Denmark, Norway, Sweden, and Finland collaborated on all phases of the study.

Microarray analysis of autoimmune diseases by machine learning procedures

—Microarray-based global gene expression profiling, with the use of sophisticated statistical algorithms is providing new insights into the pathogenesis of autoimmune diseases. We have applied a novel statistical technique for gene selection based on machine learning approaches to analyze microarray expression data gathered from patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS), two autoimmune diseases of unknown genetic origin that share many common features. The methodology included a combination of three data discretization policies, a consensus gene selection method, and a multivariate correlation measurement. A set of 150 genes was found to discriminate SLE and PAPS patients from healthy individuals. Statistical validations demonstrate the relevance of this gene set from an univariate and multivariate perspective. Moreover, functional characterization of these genes identified an interferon-regulated gene signature, consistent with previous reports. It also revealed the existence of other regulatory pathways, including those regulated by PTEN, TNF, and BCL-2, which are altered in SLE and PAPS. Remarkably, a significant number of these genes carry E2F binding motifs in their promoters, projecting a role for E2F in the regulation of autoimmunity.

Medicago truncatula Natural Resistance Associated Macrophage Protein1 is required for iron uptake by rhizobia infected nodule cells

Iron is critical for symbiotic nitrogen fixation (SNF) as a key component ofmultiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21WAF1/Cip-1/Sdi and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21WAF1/Cip-1/Sdi protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

FosB mutant mice: Loss of chronic cocaine induction of Fos-related proteins and heightened sensitivity to cocaine’s psychomotor and rewarding effects

Chronic exposure to cocaine leads to prominent, long-lasting changes in behavior that characterize a state of addiction. The striatum, including the nucleus accumbens and caudoputamen, is an important substrate for these actions. We previously have shown that long-lasting Fos-related proteins of 35–37 kDa are induced in the striatum by chronic cocaine administration. In the present study, the identity and functional role of these Fos-related proteins were examined using fosB mutant mice. The striatum of these mice completely lacked basal levels of the 35- to 37-kDa Fos-related proteins as well as their induction by chronic cocaine administration. This deficiency was associated with enhanced behavioral responses to cocaine: fosB mutant mice showed exaggerated locomotor activation in response to initial cocaine exposures as well as robust conditioned place preference to a lower dose of cocaine, compared with wild-type littermates. These results establish the long-lasting Fos-related proteins as products of the fosB gene (specifically ΔFosB isoforms) and suggest that transcriptional regulation by fosB gene products plays a critical role in cocaine-induced behavioral responses. This finding demonstrates that a Fos family member protein plays a functional role in behavioral responses to drugs of abuse and implicates fosB gene products as important determinants of cocaine abuse.

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-loxP-mediated gene inversion is used to change the specificity of the BCR on mature IgM+ IgD+ B cells in vivo to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive bcl-2 transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.

A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082–1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.