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Biomedical subjects

George C Prendergast

Publications and source records attributed to George C Prendergast.

At least 19 recordsLinked to original sources
PubMed2007

Bin1 attenuation in breast cancer is correlated to nodal metastasis and reduced survival.

Clinical outcomes in breast cancer are likely influenced by modifier genes that affect tumor dormancy versus progression. The Bin1 gene encodes a nucleocytosolic adapter protein that suppresses neoplastic cell transformation and that is often attenuated in human breast carcinoma. Recent mouse genetic studies indicate that Bin1 loss cooperates with ras activation to drive progression of mammary carcinoma, establishing Bin1 as a negative modifier of tumor progression in breast cancer. In this study, we investigated whether immunohistochemical losses of nuclear Bin1 proteins in cases of human breast cancer were correlated to progression status. In American and Japanese groups of low or middle grade breast cancers, losses were associated with reduced survival and increased nodal metastasis, respectively. Taken together with recent findings from mouse genetic studies, these findings encourage further evaluation of the potential utility of Bin1 as a clinical prognostic marker in breast cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2007

Bin1 ablation in mammary gland delays tissue remodeling and drives cancer progression.

Genes that modify oncogenesis may influence dormancy versus progression in cancer, thereby affecting clinical outcomes. The Bin1 gene encodes a nucleocytosolic adapter protein that interacts with and suppresses the cell transforming activity of Myc. Bin1 is often attenuated in breast cancer but its ability to negatively modify oncogenesis or progression in this context has not been gauged directly. In this study, we investigated the effects of mammary gland-specific deletion of Bin1 on initiation and progression of breast cancer in mice. Bin1 loss delayed the outgrowth and involution of the glandular ductal network during pregnancy but had no effect on tumor susceptibility. In contrast, in mice where tumors were initiated by the ras-activating carcinogen 7,12-dimethylbenz(a)anthracene, Bin1 loss strongly accentuated the formation of poorly differentiated tumors characterized by increased proliferation, survival, and motility. This effect was specific as Bin1 loss did not accentuate progression of tumors initiated by an overexpressed mouse mammary tumor virus-c-myc transgene, which on its own produced poorly differentiated and aggressive tumors. These findings suggest that Bin1 loss cooperates with ras activation to drive progression, establishing a role for Bin1 as a negative modifier of oncogenicity and progression in breast cancer.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2006

The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition.

BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Cyclin D1 functions in cell migration.

Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. A recent study reveals that cyclin D1 acts to promote cell migration by inhibiting Rho/ROCK signaling and expression of thrombospondin-1 (TSP-1), an extracellular matrix protein that regulates cell migration in many settings including cancer. Given the frequent overexpression of cyclin D1 in cancer cells, due to its upregulation by Ras, Rho, Src, and other genes that drive malignant development, the new findings suggest that cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP-1.

Animals↗
PubMed2006

Structure-activity study of brassinin derivatives as indoleamine 2,3-dioxygenase inhibitors.

A screen of indole-based structures revealed the natural product brassinin to be a moderate inhibitor of indoleamine 2,3-dioxygenase (IDO), a new cancer immunosuppression target. A structure-activity study was undertaken to determine which elements of the brassinin structure could be modified to enhance potency. Three important discoveries have been made, which will impact future IDO inhibitor development: (i) The dithiocarbamate portion of the brassinin lead is a crucial moiety, which may be binding to the heme iron of IDO; (ii) an indole ring is not necessary for IDO inhibition; and (iii) substitution of the S-methyl group of brassinin with large aromatic groups provides inhibitors that are three times more potent in vitro than the most commonly used IDO inhibitor, 1-methyl-tryptophan.

Indoleamine-Pyrrole 2,3,-Dioxygenase↗
PubMed2005

Reduction of hepatitis C virus NS5A phosphorylation through its interaction with amphiphysin II.

Hepatitis C virus non-structural protein 5A (NS5A) is a pleiotropic protein with key roles in viral RNA replication, modulation of cellular-signaling pathways and interferon (IFN) responses. To search for possible host factors involved in mediating these functions of NS5A, we adopted an affinity purification approach coupled with mass spectrometry to examine protein-protein interactions, and found that human amphiphysin II (also referred to as Bin1) specifically interacts with NS5A in mammalian cells. Pull-down assays showed that the Src homology 3 (SH3) domain of amphiphysin II is required for NS5A interaction and that c-Src also interacts with NS5A in cells. IFN-alpha treatment reduced the interaction of NS5A with c-Src, but not amphiphysin II, suggesting that the latter is independent of the IFN-signaling pathway. NS5A is a phosphoprotein and its phosphorylation status is considered to have an effect on viral RNA replication. In vitro kinase assays demonstrated that its interaction with amphiphysin II inhibits phosphorylation of NS5A. These results suggest that amphiphysin II participates in the HCV life cycle by modulating the phosphorylation of NS5A.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Marrying immunotherapy with chemotherapy: why say IDO?

Activation of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in cancer cells facilitates immune escape. A recent study now shows how small-molecule inhibitors of IDO can be used to leverage the efficacy of traditional chemotherapeutic drugs that are used to treat cancer in the clinic. By promoting antitumor immune responses in combination with cytotoxic chemotherapy, IDO inhibitors may offer a drug-based strategy to more effectively attack systemic cancer.

Animals↗
PubMed2005

Genetic response to DNA damage: proapoptotic targets of RhoB include modules for p53 response and susceptibility to Alzheimer's disease.

Knockout mouse studies indicate that the small GTPase RhoB is critical for apoptosis triggered by genotoxic stress in transformed mouse cells. However, the mechanisms used by RhoB to sensitize cells to cell death are obscure. To gain insight into this question, we compared the genetic response of cells with different rhoB genotypes to the DNA damaging anticancer drug doxorubicin (DOX). The microarray hybridization strategy focused on events occurring by 6 hr of DOX treatment, preceding the execution phase of RhoB-dependent apoptosis by 12-16 hr. Genes controlling cytoskeletal organization, adhesion, transcription, trafficking, apoptosis, and protein turnover were represented prominently. Gene clustering revealed a module of p53 target genes, suggesting that RhoB may modify the p53 response, and a module for susceptibility to Alzheimer's disease, suggesting a link between RhoB and age-associated dementia. The findings of this study suggest mechanisms by which RhoB may act to elevate the sensitivity of cells to apoptosis following genotoxic stress.

Alzheimer Disease↗
PubMed2005

Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy.

Immune escape is a crucial feature of cancer progression about which little is known. Elevation of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) in tumor cells can facilitate immune escape. Not known is how IDO becomes elevated or whether IDO inhibitors will be useful for cancer treatment. Here we show that IDO is under genetic control of Bin1, which is attenuated in many human malignancies. Mouse knockout studies indicate that Bin1 loss elevates the STAT1- and NF-kappaB-dependent expression of IDO, driving escape of oncogenically transformed cells from T cell-dependent antitumor immunity. In MMTV-Neu mice, an established breast cancer model, we show that small-molecule inhibitors of IDO cooperate with cytotoxic agents to elicit regression of established tumors refractory to single-agent therapy. Our findings suggest that Bin1 loss promotes immune escape in cancer by deregulating IDO and that IDO inhibitors may improve responses to cancer chemotherapy.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Indoleamine 2,3-dioxygenase in cancer: targeting pathological immune tolerance with small-molecule inhibitors.

Indoleamine 2,3-dioxygenase (IDO) is an interferon (IFN)-gamma-inducible, extrahepatic enzyme that catalyses the initial and rate-limiting step in the degradation of the essential amino acid tryptophan. Elevated tryptophan catabolism mediated by IDO is associated with a wide variety of human cancers and has historically been thought to be a tumoricidal consequence of IFN-gamma exposure. Evidence of a physiological requirement for IDO activity in protecting the allogeneic fetus from rejection by the maternal immune system has stimulated a radical shift in thinking about the role of IDO in cancer. Evidence now suggests that tumours can exploit IDO-mediated peripheral tolerance to promote immune escape. This review summarises key studies that implicate IDO as an important mediator of peripheral immune tolerance as well as the development of a promising new anticancer modality that incorporates the use of IDO inhibitors. The second part focuses on the current state of development of IDO inhibitory compounds as potential pharmaceutical agents.

Animals↗
PubMed2004

Targeted deletion of the suppressor gene bin1/amphiphysin2 accentuates the neoplastic character of transformed mouse fibroblasts.

The Bin1/Amphiphysin2 gene encodes several alternately spliced BAR adapter proteins that have been implicated in membrane-associated and nuclear processes. Bin1 expression is often attenuated during tumor progression and Bin1 splice isoforms that localize to the nucleus display tumor suppressor properties. While these properties may reflect the ability of these isoforms to interact with and suppress the cell transforming activity of c-Myc, the effects of Bin1 deletion on the oncogenicity of c-myc or other transforming genes has not been gauged directly. Here we report that targeted deletion of Bin1 enhances the neoplastic character of primary murine embryo fibroblasts (MEFs) cotransformed by c-myc and mutant grasg. Specifically, Bin1 loss accentuated the spindle morphology of transformed cells, increased anchorage-independent proliferation, and promoted tumor formation in syngeneic hosts. These effects were specific as they were not recapitulated in cells transformed by viral oncoproteins and mutant ras. Although some Bin1 splice isoforms associate with endocytotic complexes the effects of Bin1 loss were not correlated with a generalized defect in receptor-mediated endocytosis. However, Bin1 loss increased sensitivity to paclitaxel, a drug that can affect endocytotic trafficking by disrupting microtubule dynamics. In E1A?transformed MEFs, Bin1 loss reduced the susceptibility to apoptosis triggered by tumor necrosis factor-alpha, an effect that was associated with precocious nuclear trafficking of NF-kappaB. These findings offer a novel line of support for the hypothesized role of Bin1 in limiting malignant growth, possibly as a negative modifier or anti-progression gene.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Cyclin B1 is a critical target of RhoB in the cell suicide program triggered by farnesyl transferase inhibition.

Farnesyl transferase inhibitors (FTIs) have displayed limited efficacy in clinical trials, possibly because of their relatively limited cytotoxic effects against most human cancer cells. Therefore, efforts to leverage the clinical utility of FTIs may benefit from learning how these agents elicit p53-independent apoptosis in mouse models of cancer. Knockout mouse studies have established that gain of the geranylgeranylated isoform of the small GTPase RhoB is essential for FTI to trigger apoptosis. Here we demonstrate that Cyclin B1 is a crucial target for suppression by RhoB in this death program. Steady-state levels of Cyclin B1 and its associated kinase Cdk1 were suppressed in a RhoB-dependent manner in cells fated to undergo FTI-induced apoptosis. These events were not derivative of cell cycle arrest, because they did not occur in cells fated to undergo FTI-induced growth inhibition. Mechanistic investigations indicated that RhoB mediated transcriptional suppression but also accumulation of Cyclin B1 in the cytosol at early times after FTI treatment, at a time before the subsequent reduction in steady-state protein levels. Enforcing Cyclin B1 expression attenuated apoptosis but not growth inhibition triggered by FTI. Moreover, enforcing Cyclin B1 abolished FTI antitumor activity in graft assays. These findings suggest that Cyclin B1 suppression is a critical step in the mechanism by which FTI triggers apoptosis and robust antitumor efficacy. Our findings suggest that Cyclin B1 suppression may predict favorable clinical responses to FTI, based on cytotoxic susceptibility, and they suggest a rational strategy to address FTI nonresponders by coinhibition of Cdk1 activity.

Alkyl and Aryl Transferases↗
PubMed2004

RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane.

We have used a c-Src-GFP fusion protein to address the spatial control of Src activation and the nature of Src-associated intracellular structures during stimulus-induced transit to the membrane. Src is activated during transit, particularly in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment. Knocking out RhoB or expressing a dominant-interfering Rab11 mutant suppresses both catalytic activation of Src and translocation of active kinase to peripheral membrane structures. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner. This implies that Src may regulate an endosome-associated actin nucleation activity. In keeping with this, Src controls the actin dependence of RhoB endosome movement toward the plasma membrane. This work identifies RhoB as a component of "outside-in" signaling pathways that coordinate Src activation with translocation to transmembrane receptors.

3T3 Cells↗
PubMed2003

RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development.

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. In this study, we demonstrate a novel and critical role for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models led to apoptosis and failures in tube formation. We identified a novel link between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy revealed that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. We show that this colocalization is functionally relevant, because when RhoB was depleted, Akt was excluded from the nucleus and total cellular Akt protein was decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to only be rate limiting for endothelial cell sprouting, we propose that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable.

Active Transport, Cell Nucleus↗
PubMed2003

Expression of a MYCN-interacting isoform of the tumor suppressor BIN1 is reduced in neuroblastomas with unfavorable biological features.

PURPOSE: Amplification of the MYCN proto-oncogene is strongly correlated with poor outcome in neuroblastoma (NB), although deregulated MYCN is a potent inducer of apoptosis. BIN1 (2q14) encodes multiple isoforms of a Myc-interacting adaptor protein that has features of a tumor suppressor, including the ability to inhibit Myc-mediated cell transformation and to promote apoptosis. We hypothesized that BIN1 may function as a suppressor gene in NB, because Bin1 is highly expressed in neural tissues and binds the Myc Box motifs that are conserved in MycN. EXPERMENTAL DESIGN: Expression of MYCN, total BIN1, and BIN1 isoforms were determined in 56 primary NBs using the real-time PCR. Expression was correlated with biological and genetic features. To determine the functional significance of BIN1 expression we ectopically expressed BIN1 isoforms in NB cell lines with and without MYCN amplification, and assessed clonogenic growth. RESULTS: Four predominant BIN1 isoforms resulting from alternative splicing of exon 12A (a neural tissue-specific exon) and exon 13 (a Myc-binding domain encoding exon) were variably expressed in the 56 primary NBs. Expression of BIN1 was lower in: NBs with MYCN amplification (n = 10) compared with those without, P < 0.03; in International Neuroblastoma Risk Group high-risk NB (n = 19) compared with low- or intermediate-risk NB, P < 0.01; and in metastatic NB (n = 21) compared with localized NB, P < 0.06. BIN1 inactivation by deletion or genomic rearrangement was identified infrequently. Forced expression of BIN1 isoforms containing the Myc-binding domain (with or without exon 12A) inhibited colony formation in NB cell lines with MYCN amplification (P < 0.01) but not in those without. Forced expression of BIN1 isoforms with a MBD deletion did not inhibit colony formation in any cell line assessed. CONCLUSIONS: These data support that reduced BIN1 expression contributes to the malignant phenotype of childhood NB. As we reported previously, BIN1 may function to circumvent MycN-mediated apoptosis in NBs with MYCN amplification.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Transformation-selective apoptotic program triggered by farnesyltransferase inhibitors requires Bin1.

Neoplastic transformation sensitizes many cells to apoptosis. This phenomenon may underlie the therapeutic benefit of many anticancer drugs, but its molecular basis is poorly understood. We have used a selective and potent farnesyltransferase inhibitor (FTI) to probe a mechanism of apoptosis that is peculiarly linked to neoplastic transformation. While nontoxic to untransformed mouse cells, FTI triggers a massive RhoB-dependent, p53-independent apoptosis in mouse cells that are neoplastically transformed. Here we offer evidence that the BAR adapter-encoding tumor suppressor gene Bin1 is required for this transformation-selective death program. Targeted deletion of Bin1 in primary mouse embyro fibroblasts (MEFs) transformed by E1A+Ras did not affect FTI-induced reversion, actin fiber formation, or growth inhibition, but it abolished FTI-induced apoptosis. The previously defined requirement for RhoB in these effects suggests that Bin1 adapter proteins act downstream or in parallel to RhoB in cell death signaling. The death defect in Bin1 null cells was significant insofar as it abolished FTI efficacy in tumor xenograft assays. p53 deletion did not phenocopy the effects of Bin1 deletion. However, MEFs transformed by SV40 large T antigen+Ras were also resistant to apoptosis by FTI, consistent with other evidence that large T inhibits Bin1-dependent cell death by a p53-independent mechanism. Taken together, the results define a function for Bin1 in apoptosis that is conditional on transformation stress. This study advances understanding of the functions of BAR adapter proteins, which are poorly understood, by revealing genetic interactions with an Rho small GTPase that functions in stress signaling. The frequent losses of Bin1 expression that occur in human breast and prostate cancers may promote tumor progression and limit susceptibility to FTI or other therapeutic agents that exploit the heightened sensitivity of neoplastic cells to apoptosis.

Actins↗
PubMed2003

Role for RhoB and PRK in the suppression of epithelial cell transformation by farnesyltransferase inhibitors.

Recent genetic investigations have established that RhoB gain-of-function is sufficient to mediate the antitransforming effects of farnesyltransferase inhibitors (FTIs) in H-Ras-transformed fibroblast systems. In this study, we addressed the breadth and mechanism of RhoB action in epithelial cells transformed by oncoproteins which are themselves insensitive to FTI inactivation. Rat intestinal epithelial (RIE) cells transformed by activated K-Ras or Rac1 were highly sensitive to FTI-induced actin reorganization and growth inhibition, despite the inability of FTI to block prenylation of either K-Ras or Rac1. Ectopic expression of the geranylgeranylated RhoB isoform elicited in cells by FTI treatment phenocopied these effects. Analysis of RhoB effector domain mutants pointed to a role for PRK, a Rho effector kinase implicated in the physiological function of RhoB in intracellular receptor trafficking, and these findings were supported further by experiments in a fibroblast system. We propose that FTIs recruit the antioncogenic RhoB protein in the guise of RhoB-GG to interfere with signaling by pro-oncogenic Rho proteins, possibly by sequestering common exchange factors or effectors such as PRK that are important for cell transformation.

Actin Cytoskeleton↗