Biomedical subjects

Stuart A Aaronson

Publications and source records attributed to Stuart A Aaronson.

At least 19 recordsLinked to original sources

PubMed2006

RhoE is a pro-survival p53 target gene that inhibits ROCK I-mediated apoptosis in response to genotoxic stress.

The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton . The majority of the Rho family proteins function as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations . Unlike typical Rho-family proteins, the Rnd subfamily members, including Rnd1, Rnd2, RhoE (also known as Rnd3), and RhoH, are GTPase deficient and are thus expected to be constitutively active . Here, we identify an unexpected role for RhoE/Rnd3 in the regulation of the p53-mediated stress response. We show that RhoE is a transcriptional p53 target gene and that genotoxic stress triggers actin depolymerization, resulting in actin-stress-fiber disassembly through p53-dependent RhoE induction. Silencing of RhoE induction in response to genotoxic stress maintains stress fiber formation and strikingly increases apoptosis, implying an antagonistic role for RhoE in p53-dependent apoptosis. We found that RhoE inhibits ROCK I (Rho-associated kinase I) activity during genotoxic stress and thereby suppresses apoptosis. We demonstrate that the p53-mediated induction of RhoE in response to DNA damage favors cell survival partly through inhibition of ROCK I-mediated apoptosis. Thus, RhoE is anticipated to function by regulating ROCK I signaling to control the balance between cell survival and cell death in response to genotoxic stress.

Animals↗

PubMed2006

Discoidin domain receptor 1 receptor tyrosine kinase induces cyclooxygenase-2 and promotes chemoresistance through nuclear factor-kappaB pathway activation.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by various types of collagens and is known to play a role in cell attachment, migration, survival, and proliferation. However, little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. We report here that DDR1 induces cyclooxygenase-2 (Cox-2) expression resulting in enhanced chemoresistance. Depletion of DDR1-mediated Cox-2 induction using short hairpin RNA (shRNA) results in increased chemosensitivity. We also show that DDR1 activates the nuclear factor-kappaB (NF-kappaB) pathway and blocking this activation by an I kappaB superrepressor mutant results in the ablation of DDR1-induced Cox-2, leading to enhanced chemosensitivity, indicating that DDR1-mediated Cox-2 induction is NF-kappaB dependent. We identify the upstream activating kinases of the NF-kappaB pathway, IKK beta and IKK gamma, as essential for DDR1-mediated NF-kappaB activation, whereas IKK alpha seems to be dispensable. Finally, shRNA-mediated inhibition of DDR1 expression significantly enhanced chemosensitivity to genotoxic drugs in breast cancer cells. Thus, DDR1 signaling provides a novel target for therapeutic intervention with the prosurvival/antiapoptotic machinery of tumor cells.

Apoptosis↗

PubMed2005

Identification of mammaglobin as a novel serum marker for breast cancer.

PURPOSE: Early detection of breast cancer has implications for the management and treatment of patients with this disease. Currently, there exist no highly sensitive and specific serologic biomarkers for detection of breast cancer. Mammaglobin is predicted to be a secreted protein, and expression of this gene seems to be highly specific in breast cancer. The present studies were undertaken to develop the mammaglobin protein as a serum biomarker for detection of breast cancer. EXPERIMENTAL DESIGN: We characterized the mammaglobin protein as a secreted, 14- to 21-kDa species, which is likely post-translationally processed based on its predicted 7-kDa size. Immunostaining for mammaglobin was conducted. An ELISA was developed for the detection of the mammaglobin protein in serum, and levels were compared between women with and without breast cancer. A receiver operating characteristic curve was used to show sensitivity and specificity for cut points on the continuous mammaglobin scale. RESULTS: The protein was detectable by immunostaining in 72% of breast tumors and not in other tumor types. The ELISA was highly sensitive and specific for detection of mammaglobin protein in tissue culture fluids of breast cancer cells and sera of breast cancer patients. The ELISA differentiated healthy women from those with breast cancer with accurate, repeatable results across time and under varying storage conditions. CONCLUSION: Our results indicate that mammaglobin, as measured by the ELISA, holds significant promise for breast cancer screening with the realistic potential to impact management of this disease.

Adult↗

PubMed2005

Prostate cancer cells promote osteoblastic bone metastases through Wnts.

Prostate cancer produces painful osteoblastic bone metastases. Although prostate cancer cells produce numerous osteogenic factors, to date, none have been shown to mediate osteoblastic bone metastases in an in vivo model of prostate cancer. Wnts are a large family of proteins that promote bone growth. Wnt activity is antagonized by endogenous proteins including dickkopf-1 (DKK-1). We explored if prostate cancer cells mediate osteoblastic activity through Wnts using DKK-1 as a tool to modify Wnt activity. A variety of Wnt mRNAs were found to be expressed in prostate cancer cell lines and Wnt mRNA expression was increased in primary prostate cancer compared with nonneoplastic prostate tissue. In addition to expressing Wnts, PC-3 prostate cancer cells expressed the Wnt inhibitor DKK-1. To determine if DKK-1 masked Wnt-mediated osteoblastic activity in osteolytic PC-3 cells, the cells were stably transfected with DKK-1 short hairpin RNA. Decreasing DKK-1 enabled PC-3 cells to induce osteoblastic activity, including alkaline phosphatase production and mineralization, in murine bone marrow stromal cells indicating that DKK-1 blocked Wnt-mediated osteoblastic activity in PC-3 cells. Another prostate cancer cell line, C4-2B, induces mixed osteoblastic/osteolytic lesions. To determine if Wnts contribute to C4-2B's ability to induce mixed osteoblastic/osteolytic lesions, C4-2B cells were stably transfected with either empty vector or DKK-1 expression vector to block Wnt activity. The cells were then injected in the tibiae of mice and allowed to grow for 12 weeks. Blocking Wnt activity converted the C4-2B cells to a highly osteolytic tumor. Taken together, these data show that Wnts contribute to the mechanism through which prostate cancer induces osteoblastic activity.

Animals↗

PubMed2005

Evidence against a role for SV40 in human mesothelioma.

SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.

Animals↗

PubMed2005

Analysis of mutations in fibroblast growth factor (FGF) and a pathogenic mutation in FGF receptor (FGFR) provides direct evidence for the symmetric two-end model for FGFR dimerization.

Two competing models for fibroblast growth factor (FGF) receptor (FGFR) dimerization have recently emerged based on ternary FGF-FGFR-heparin crystal structures. In the symmetric two-end model, heparin promotes dimerization of two FGF-FGFR complexes by stabilizing bivalent interactions of the ligand and receptor through primary and secondary sites and by stabilizing direct receptor-receptor contacts. In the asymmetric model, there are no protein-protein contacts between the two FGF-FGFR complexes, which are bridged solely by heparin. To identify the correct mode of FGFR dimerization, we abolished interactions at the secondary ligand-receptor interaction site, which are observed only in the symmetric two-end model, using site-directed mutagenesis. Cellular studies and real-time binding assays, as well as matrix-assisted laser desorption ionization-time of flight analysis, demonstrate that loss of secondary ligand-receptor interactions results in diminished FGFR activation due to decreased dimerization without affecting FGF-FGFR binding. Additionally, structural and biochemical analysis of an activating FGFR2 mutation resulting in Pfeiffer syndrome confirms the physiological significance of receptor-receptor contacts in the symmetric two-end model and provides a novel mechanism for FGFR gain of function in human skeletal disorders. Taken together, the data validate the symmetric two-end model of FGFR dimerization and argue against the asymmetric model of FGFR dimerization.

Animals↗

PubMed2005

The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts.

Wnt glycoproteins are developmentally essential signaling molecules, and lesions afflicting Wnt pathways play important roles in human diseases. Some Wnts signal to the canonical pathway by stabilizing beta-catenin, while others lack this activity. Frizzled serpentine receptors mediate distinct signaling pathways by both classes of Wnts. Here, we tandemly linked noncanonical Wnt5a with the C-terminal half of Dickkopf-2 (Dkk2C), a distinct ligand of the Wnt coreceptor LRP5/6. Whereas Wnt5a, Dkk2C, or both together were incapable of stimulating endogenous canonical signaling, the Wnt5a/Dkk2C chimera efficiently activated this pathway in a manner inhibitable by specific antagonists of either frizzled or LRP receptors. Thus, activation of the canonical pathway requires ligand coupling of an endogenous frizzled/LRP coreceptor complex, rather than Wnt triggering each receptor independently. Moreover, fusion of Wnt5a with Dkk2C unmasked its ability to signal to Dishevelled through multiple frizzleds, indicating that the lack of functional interaction with LRP distinguishes noncanonical Wnt5a from canonical Wnts in mammalian cells. These findings provide a novel mechanism by which the same receptor can be switched between distinct signaling pathways depending on the differential recruitment of a coreceptor by members of the same ligand family.

Animals↗

PubMed2004

ASC is a Bax adaptor and regulates the p53-Bax mitochondrial apoptosis pathway.

The apoptosis-associated speck-like protein (ASC) is an unusual adaptor protein that contains the Pyrin/PAAD death domain in addition to the CARD protein-protein interaction domain. Here, we present evidence that ASC can function as an adaptor molecule for Bax and regulate a p53-Bax mitochondrial pathway of apoptosis. When ectopically expressed, ASC interacted directly with Bax, colocalized with Bax to the mitochondria, induced cytochrome c release with a significant reduction of mitochondrial membrane potential and resulted in the activation of caspase-9, -2 and -3. The rapid induction of apoptosis by ASC was not observed in Bax-deficient cells. We also show that induction of ASC after exposure to genotoxic stress is dependent on p53. Blocking of endogenous ASC expression by small-interfering RNA (siRNA) reduced the apoptotic response and inhibited translocation of Bax to mitochondria in response to p53 or genotoxic insult, suggesting that ASC is required to translocate Bax to the mitochondria. Our findings demonstrate that ASC has an essential role in the intrinsic mitochondrial pathway of apoptosis through a p53-Bax network.

Apoptosis↗

PubMed2004

An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells.

Autocrine Wnt signaling in the mouse mammary tumor virus model was the first identified mechanism of canonical pathway activation in cancer. In search of this transformation mechanism in human cancer cells, we identified breast and ovarian tumor lines with upregulation of the uncomplexed transcriptionally active form of beta-catenin without mutations afflicting downstream components. Extracellular Wnt antagonists FRP1 and DKK1 caused a dramatic downregulation of beta-catenin levels in these tumor cells associated with alteration of biological properties and increased expression of epithelial differentiation markers. Colorectal carcinoma cells with knockout of the mutant beta-catenin allele retained upregulated beta-catenin levels, which also could be inhibited by these Wnt antagonists. Together, these findings establish the involvement of autocrine Wnt signaling in human cancer cells.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

BRCA2 is ubiquitinated in vivo and interacts with USP11, a deubiquitinating enzyme that exhibits prosurvival function in the cellular response to DNA damage.

Individuals carrying a germ line mutation of the breast cancer susceptibility gene BRCA2 are predisposed to breast, ovarian, and other types of cancer. The BRCA2 protein has been proposed to function in the repair of DNA double-strand breaks. Using an immunopurification-mass spectrometry approach to identify novel proteins that associate with the BRCA2 gene product, we found that a deubiquitinating enzyme, USP11, formed specific complexes with BRCA2. Moreover, BRCA2 was constitutively ubiquitinated in vivo in the absence of detectable proteasomal degradation. Mitomycin C (MMC) led to decreased BRCA2 protein levels associated with increased ubiquitination, consistent with proteasome-dependent degradation. While BRCA2 could be deubiquitinated by USP11 in transient overexpression assays, a catalytically inactive USP11 mutant had no effect on BRCA2 ubiquitination or protein levels. Antagonism of USP11 function either through expression of this mutant or through RNA interference increased cellular sensitivity to MMC in a BRCA2-dependent manner. All of these results imply that BRCA2 expression levels are regulated by ubiquitination in the cellular response to MMC-induced DNA damage and that USP11 participates in DNA damage repair functions within the BRCA2 pathway independently of BRCA2 deubiquitination.

Amino Acid Sequence↗

PubMed2003

BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma.

BRAF oncogenic mutations have been identified in significant numbers of melanocytic lesions. To correlate BRAF mutation and melanoma progression, we screened BRAF mutations in 65 melanocytic lesions, including nevi, radial growth phase (RGP), vertical growth phase (VGP) melanomas, and melanoma metastases, as well as 25 melanoma cell lines. PCR and direct sequencing were used to analyze DNA samples extracted from laser capture microdissected tissues. A similar high frequency (62-72%) of BRAF oncogenic mutations was identified in melanocytic nevi, VGP, metastatic melanomas, and melanoma cell lines [H. Davies et al., Nature (Lond.), 417: 949-954, 2002; P. M. Pollock et al., Nat. Genet., 33: 19-20, 2002; and M. S. Brose et al., Cancer Res., 62: 6997-7000, 2002]. In striking contrast, we found BRAF lesions in only 10% of the earliest stage or RGP melanomas. These findings imply that BRAF mutations cannot be involved in the initiation of the great majority of melanomas but instead reflect a progression event with important prognostic implications in the transition from the great majority of RGP melanomas to VGP and/or metastatic melanoma.

Disease Progression↗

PubMed2003

p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop.

DDR1, discoidin domain receptor 1, belongs to a subfamily of tyrosine kinase receptors with an extracellular domain homologous to Dictyostellium discoideum protein discoidin 1. We showed that DDR1 is a direct p53 transcriptional target, and that DNA damage induced a p53-dependent DDR1 response associated with activation of its tyrosine kinase. We further demonstrated that DDR1 activated the MAPK cascade in a Ras-dependent manner. Whereas levels of p53, phosphoserine-15 p53, p21, ARF and Bcl-X(L) were increased in response to exogenous overexpression of activated DDR1, dominant-negative DDR1 inhibited irradiation-induced MAPK activation and p53, phosphoserine-15 p53, as well as induced p21 and DDR1 levels, suggesting that DDR1 functions in a feedforward loop to increase p53 levels and at least some of its effectors. Nonetheless, inhibition of DDR1 function resulted in strikingly increased apoptosis of wild-type p53-containing cells in response to genotoxic stress through a caspase-dependent pathway. These results strongly imply that this p53 response gene must predominately act to alleviate the adverse effects of stress induced by p53 on its target cell.

Adenoviridae↗

PubMed2003

Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors.

Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb ("b") and IIIc ("c"), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes.

Alternative Splicing↗

PubMed2003

A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor.

LDL receptor-related protein 6 (LRP6) is a Wnt coreceptor in the canonical signaling pathway, which plays essential roles in embryonic development. We demonstrate here that wild-type LRP6 forms an inactive dimer through interactions mediated by epidermal growth factor repeat regions within the extracellular domain. A truncated LRP6 comprising its transmembrane and cytoplasmic domains is expressed as a constitutively active monomer whose signaling ability is inhibited by forced dimerization. Conversely, Wnts are shown to activate canonical signaling through LRP6 by inducing an intracellular conformational switch which relieves allosteric inhibition imposed on the intracellular domains. Thus, Wnt canonical signaling through LRP6 establishes a novel mechanism for receptor activation which is opposite to the general paradigm of ligand-induced receptor oligomerization.

Allosteric Site↗

PubMed2003

Influence of induced reactive oxygen species in p53-mediated cell fate decisions.

The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.

Apoptosis↗

PubMed2002

P53-mediated induction of Cox-2 counteracts p53- or genotoxic stress-induced apoptosis.

The identification of transcriptional targets of the tumor suppressor p53 is crucial in understanding mechanisms by which it affects cellular outcomes. Through expression array analysis, we identified cyclooxygenase 2 (Cox-2), whose expression was inducible by wild-type p53 and DNA damage. We also found that p53-induced Cox-2 expression results from p53-mediated activation of the Ras/Raf/MAPK cascade, as demonstrated by suppression of Cox-2 induction in response to p53 by dominant-negative Ras or Raf1 mutants. Furthermore, heparin-binding epidermal growth factor-like growth factor (HB- EGF), a p53 downstream target gene, induced Cox-2 expression, implying that Cox-2 is an ultimate effector in the p53-->HB-EGF-->Ras/Raf/MAPK-->Cox-2 pathway. p53-induced apoptosis was enhanced greatly in Cox-2 knock-out cells as compared with wild-type cells, suggesting that Cox-2 has an abrogating effect on p53-induced apoptosis. Also, a selective Cox-2 inhibitor, NS-398, significantly enhanced genotoxic stress-induced apoptosis in several types of p53+/+ normal human cells, through a caspase-dependent pathway. Together, these results demonstrate that Cox-2 is induced by p53-mediated activation of the Ras/Raf/ERK cascade, counteracting p53-mediated apoptosis. This anti-apoptosis effect may be a mechanism to abate cellular stresses associated with p53 induction.

Apoptosis↗

PubMed2002

Induction of homologue of Slimb ubiquitin ligase receptor by mitogen signaling.

Homologue of Slimb (HOS) is the substrate-recognizing component of the SCF(HOS)-Roc1 E3 ubiquitin protein ligase. This ligase mediates ubiquitination of the inhibitor of NF-kappaB transcription factor (IkappaB). We have found that HOS is highly expressed in a number of human cancer cell lines. The rates of the HOS gene transcription as well as HOS mRNA and protein levels were up-regulated in cells treated with mitogens or transfected with the inducers of mitogen-activated protein kinase pathway. Conversely, mitogen withdrawal strikingly reduced HOS levels during differentiation of mouse myoblasts. Activators of mitogen-activated protein kinase accelerated IkappaBalpha degradation and increased NF-kappaB transcriptional activity. Inhibition of HOS function via expression of dominant negative HOS (HOS(DeltaF)) initiated mouse myoblast differentiation and prevented Ras-mediated acceleration of IkappaBalpha degradation as well as NF-kappaB trans-activation and transformation of NIH3T3 cells. These data link the induction of HOS in proliferating cells with mitogen-signaling-dependent inhibition of cell differentiation and promotion of cell transformation.

3T3 Cells↗

PubMed2002

Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence.

The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells. N-acetyl-L-cysteine, an ROS inhibitor, rescued p21-induced senescence, showing that ROS elevation is necessary for induction of the permanent growth arrest phenotype. p16(Ink4a), a CDK4- and CDK6-specific inhibitor, failed to increase ROS levels, and cell cycle arrest induced by p16 was reversible following its down-regulation, demonstrating the specificity of this p21 effect. A p21 mutant that lacked the ability to bind proliferating cell nuclear antigen (PCNA) retained the ability to induce both ROS and permanent growth arrest. All of these findings establish that p21 mediates senescence by a mechanism involving ROS accumulation which does not require either its PCNA binding or the CDK inhibitory functions shared with p16.

Acetylcysteine↗