Anne Frei

Vascular dysfunction in a murine model of severe hemolysis.

Spectrin is the backbone of the erythroid cytoskeleton; sph/sph mice have severe hereditary spherocytosis (HS) because of a mutation in the murine erythroid alpha-spectrin gene. sph/sph mice have a high incidence of thrombosis and infarction in multiple tissues, suggesting significant vascular dysfunction. In the current study, we provide evidence for both pulmonary and systemic vascular dysfunction in sph/sph mice. We found increased levels of soluble cell adhesion molecules in sph/sph mice, suggesting activation of the vascular endothelium. We hypothesized that plasma hemoglobin released by intravascular hemolysis initiates endothelial injury through nitric oxide (NO) scavenging and oxidative damage. Likewise, electron paramagnetic resonance spectroscopy showed that plasma hemoglobin is much greater in sph/sph mice. Moreover, plasma from sph/sph mice had significantly higher oxidative potential. Finally, xanthine oxidase, a potent superoxide generator, is decreased in subpopulations of liver hepatocytes and increased on liver endothelium in sph/sph mice. These results indicate that vasoregulation is abnormal, and NO-based vasoregulatory mechanisms particularly impaired, in sph/sph mice. Together, these data indicate that sph/sph mice with severe HS have increased plasma hemoglobin and NO scavenging capacity, likely contributing to aberrant vasoregulation and initiating oxidative damage.

The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases.

The small GTPase DiRas1 has tumor-suppressive activities, unlike the oncogenic properties more common to small GTPases such as K-Ras and RhoA. Although DiRas1 has been found to be a tumor suppressor in gliomas and esophageal squamous cell carcinomas, the mechanisms by which it inhibits malignant phenotypes have not been fully determined. In this study, we demonstrate that DiRas1 binds to SmgGDS, a protein that promotes the activation of several oncogenic GTPases. In silico docking studies predict that DiRas1 binds to SmgGDS in a manner similar to other small GTPases. SmgGDS is a guanine nucleotide exchange factor for RhoA, but we report here that SmgGDS does not mediate GDP/GTP exchange on DiRas1. Intriguingly, DiRas1 acts similarly to a dominant-negative small GTPase, binding to SmgGDS and inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A. DiRas1 is expressed in normal breast tissue, but its expression is decreased in most breast cancers, similar to its family member DiRas3 (ARHI). DiRas1 inhibits RhoA- and SmgGDS-mediated NF-κB transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-κB activation in breast cancer and glioblastoma cell lines. Taken together, our data support a model in which DiRas1 expression inhibits malignant features of cancers in part by nonproductively binding to SmgGDS and inhibiting the binding of other small GTPases to SmgGDS.

A novel hemoglobin-binding peptide reduces cell-free hemoglobin in murine hemolytic anemia

Hemolysis can saturate the hemoglobin (Hb)/heme scavenging system, resulting in increased circulating cell-free Hb (CF-Hb) in hereditary and acquired hemolytic disease. While recent studies have suggested a central role for intravascular hemolysis and CF-Hb in the development of vascular dysfunction, this concept has stimulated considerable debate. This highlights the importance of determining the contribution of CF-Hb to vascular complications associated with hemolysis. Therefore, a novel Hb-binding peptide was synthesized and linked to a small fragment of apolipoprotein E (amino acids 141-150) to facilitate endocytic clearance. Plasma clearance of hE-Hb-b10 displayed a rapid phase t(1/2) of 16 min and slow phase t(1/2) of 10 h, trafficking primarily through the liver. Peptide hE-Hb-B10 decreased CF-Hb in mice treated with phenylhydrazine, a model of acute hemolysis. Administration of hE-Hb-B10 also attenuated CF-Hb in two models of chronic hemolysis: Berkeley sickle cell disease (SS) mice and mice with severe hereditary spherocytosis (HS). The hemolytic rate was unaltered in either chronic hemolysis model, supporting the conclusion that hE-Hb-B10 promotes CF-Hb clearance without affecting erythrocyte lysis. Interestingly, hE-Hb-B10 also decreased plasma ALT activity in SS and HS mice. Although acetylcholine-mediated facialis artery vasodilation was not improved by hE-Hb-B10 treatment, the peptide shifted vascular response in favor of NO-dependent vasodilation in SS mice. Taken together, these data demonstrate that hE-Hb-B10 decreases CF-Hb with a concomitant reduction in liver injury and changes in vascular response. Therefore, hE-Hb-B10 can be used to investigate the different roles of CF-Hb in hemolytic pathology and may have therapeutic benefit in the treatment of CF-Hb-mediated tissue damage.

Erratum: The tumor-suppressive small GTPase DiRas1 binds the noncanonical guanine nucleotide exchange factor SmgGDS and antagonizes SmgGDS interactions with oncogenic small GTPases (Journal of Biological Chemistry (2016) 291 (6534-6545))

Carmen Bergom, Andrew D. Hauser, Amy Rymaszewski, Patrick Gonyo, Jeremy W. Prokop, Benjamin C. Jennings, Alexis J. Lawton, Anne Frei, Ellen L. Lorimer, Irene Aguilera-Barrantes, Alexander C. Mackinnon, Jr., Kathleen Noon, Carol A. Fierke, and Carol L. Williams Ref. 40 was missing from the reference list. The information for Ref. 40 should be as follows: Ogita, Y., Egami, S., Ebihara, A., Ueda, N., Katada, T., and Kontani, K. (2015) Di-Ras2 protein forms a complex with SmgGDS protein in brain cytosol in order to be in a low affinity state for guanine nucleotides. J. Biol. Chem. 290, 20245–20256. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 20, p. 10948, May 13, 2016

Abstract B07: Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses

Progress in elucidating the molecular basis of breast cancer has allowed for treatment breakthroughs such as anti-estrogen and Her2-targeted therapy. It has also shaped the approaches to both surgical and systemic therapy. However, no similar use of molecular information has been utilized to better direct the use of radiation therapy. The development of predictive tools for the radiosensitivity of tumors could allow for personally tailored radiation doses, with treatment de-escalation for radiosensitive tumors, or dose escalation or the use of adjunct treatments in the case of radioresistant tumors. Communication between malignant tumor cells and the tumor microenvironment (TME) underlies most aspects of tumor biology, including chemotherapy and radiation resistance. We have developed a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel and without cell-sorting. Here we utilize these unique techniques to identify genetic variants in the TME that can affect radiation sensitivity. In CXM, human triple negative breast cancer MDA-MD-231 cells are orthotopically implanted into immunodeficient (IL2Rγ-/-) consomic rat strains, which are rat strains in which an entire chromosome is introgressed into the isogenic background of another inbred strain by selective breeding. Because the strain backgrounds are different but the tumor cells are not varied, the observed changes in tumor progression are due to genetic differences in the non-malignant TME. We hypothesized that the tumors in SS.BN3 rats (identical to SS rats but with BN strain chromosome 3) would be more sensitive to radiation due to increased tumor vascularity via CD31 staining, and increased tumor blood volume capacity, as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Our studies demonstrate differential responses to radiation in the CXM model comparing parental SS (IL2Rγ) rats to SS.BN3 (IL2Rγ) rats treated with fractionated radiation therapy (4 Gray x 3), with altered tumor growth kinetics and tumor recurrence rates. A difference was seen in time to 5-fold increase in tumor growth, with 44 vs. >130 days for SS versus SS.BN3 rats (supra-additive, p 130 days) in the SS versus SS.BN3 rats (p=0.02). These results suggest that genetic determinants in the TME affect the radiation sensitivity of genetically identical tumor cells. Using SSRS, we identified a number of candidates on rat chromosome 3 that may potentially influence radiation sensitivity by altering the tumor vasculature. Future studies will further dissect the pathways responsible for the changes in radiation sensitivity. Determining TME factors that affect the radiation sensitivity of tumors has the potential to allow for more tailored and effective radiation treatments in breast cancer. Citation Format: Carmen Bergom, Michael Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Shirng-Wern Tsaih, Howard Jacob, Michael J. Flister. Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B07.

Abstract 3678: The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma

Glioblastoma (GB) is the most aggressive malignancy affecting the central nervous system (CNS) with a median survival of 12 to 15 months even with surgery, radiation and chemotherapy. Previous research demonstrates that increased activation of NF-κB is critical for GB growth, proliferation and the up regulation of genes involved in cytokine production, cell cycle regulation, apoptosis and cell adhesion. Understanding the molecular targets that regulate NF-κB may provide more effective therapeutic targets for GB. DiRas family small GTPases, which are homologous to pro-oncogenic Ras GTPases, are tumor suppressive rather than tumor promoting and include DiRas1, DiRas2 and DiRas3 (ARHI). DiRas1 and DiRas2 have been suggested to be tumor suppressive in CNS malignancies, but the role of DiRas3 in CNS malignancies remains unknown. Here we demonstrate that expression of DiRas3 protein in GB cell lines is absent, although DiRas3 is expressed in non-malignant glial cells. Re-expression of DiRas3 in U-87 cells reduces cell proliferation by 20%. Using a NF-κB transcriptional activity luciferase reporter assay demonstrates that DiRas3 expression reduces NF-κB transcriptional activity by 70% compared to vector control. Further experiments demonstrate that decreased NF- κB activity occurs via reduced phosphorylation of the NF-κB inhibitor IκBα. The reduced phosphorylation of IκBα could be a result of decreased AKT and ERK activity, as increased ERK and AKT activity can stimulate NF-κB pathways. Our lab has previously demonstrated that the most common binding partner for DiRas1 and DiRas2 was the small GTPase binding protein SmgGDS, and DiRas1 and DiRas2 also reduce NF- κB activation. However, DiRas3 does not interact with SmgGDS, suggesting that DiRas3 can reduce NF-κB in a SmgGDS-independent manner. Understanding the role of DiRas3 and its binding partners in mediating NF- κB activation may lead to novel therapeutics for glioblastoma. Citation Format: Amy Rymaszewski, Michael Straza, Anne Frei, Carmen Bergom. The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3678.

Abstract 4443: The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A number of malignancies are promoted by the activation of Rho GTPases. Regulators of RhoA activity, including the RhoGEF, SmgGDS, can also promote cancer development. Both RhoA and SmgGDS have been shown to enhance NF-ĸB activation in a number of tumors. Utilizing mass spectrometry, we identified the tumor suppressive small GTPase DiRas1 as a novel binding partner for SmgGDS. DiRas1 is a tumor suppressor in malignant gliomas and esophageal squamous cell carcinomas. Examining DiRas gene expression using the ONCOMINE mRNA microarray database (www.oncomine.org) revealed that DIRAS1 and DIRAS2 are markedly decreased in central nervous system tumors, including anaplastic astrocytomas and GBMs, when compared to normal tissue. The role of DiRas1 in other cancers is largely undetermined, and the ways in which DiRas1 may antagonize pro-oncogenic small GTPase signaling is largely unknown. To define how DiRas1 expression may affect SmgGDS functions, we tested the association of DiRas family proteins with SmgGDS. SmgGDS robustly bound wildtype DiRas1, and its closely-related family member DiRas2, but only weakly bound DiRas3 in vitro and in cell lines. In addition, DiRas1, DiRas2, or DiRas3 protein expression decreased proliferation of HEK293T cells. DiRas1 expression could promote less activated RhoA, as a low level of DiRas1 expression caused markedly decreased RhoA-SmgGDS binding in a number of cell lines. DiRas1 expression also abrogated SmgGDS- and RhoA-mediated NF-ĸB activation in a concentration-dependent manner. SmgGDS may exhibit GEF activity toward DiRas1, as a dominant negative DiRas1 (S21N) binds to SmgGDS more than the wildtype protein. However, DiRas1 is predominantly GTP-bound in cells, likely due to a high rate of guanine nucleotide dissociation and low intrinsic GTPase activity. Thus, DiRas1 expression may in part promote tumor suppression by non-productively binding SmgGDS, resulting in less RhoA activity. Taken together, these results suggest that the tumor suppressive small GTPase DiRas1 can antagonize RhoA signaling by competing for binding of the RhoGEF SmgGDS. Further characterizing these actions may lead to novel therapeutic targeting of RhoA activation in cancer malignancies. Citation Format: Andrew D. Hauser, Kristen M. Barr, Anne C. Frei, Patrick Gonyo, Ellen L. Lorimer, Carol L. Williams, Carmen Bergom. The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4443. doi:10.1158/1538-7445.AM2014-4443