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The TWEAK-Fn14 Ligand Receptor Axis PromotesGlioblastoma Cell Invasion and Survival Via Activation
GRADUATE INTERDISCIPLINARY PROGRAM IN CANCER BIOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2013
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THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Shannon Fortin Ensign, titled The TWEAK-Fn14 Ligand Receptor Axis Promotes Glioblastoma Cell Invasion and Survival Via Activation of Multiple GEF-Rho GTPase Signaling Systems and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy. _______________________________________________ Date: April 26, 2013 Nhan Tran, PhD _______________________________________________ Date: April 26, 2013 Suwon Kim, PhD _______________________________________________ Date: April 26, 2013 Jesse Martinez, PhD _______________________________________________ Date: April 26, 2013 Anne Cress, PhD _______________________________________________ Date: April 26, 2013 Maria Bishop, MD Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: April 26, 2013 Dissertation Director: Nhan Tran, PhD ________________________________________________ Date: April 26, 2013 Dissertation Director: Suwon Kim, PhD
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STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. SIGNED: Shannon Fortin Ensign
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ACKNOWLEDGEMENTS
I would like to express my sincerest gratitude towards everyone who has helped me reach this stage in my career and path in life. I particularly would like to thank my mentor, Dr. Nhan Tran, for his support not only in my graduate studies but as a mentor to me in my undergraduate years as well. Your passion for science and teaching has inspired me to work hard to achieve my goals, and I truly appreciate your guidance, critiques, support and friendship. In addition, I would like to thank Dr. Michael Berens for his time and devotion also as a mentor during both my undergraduate and graduate studies. Your drive for success, caring personality, and remarkable ability for collaboration are inspirational. I hope to take what knowledge I have learned from these mentors and apply it to my own future career. I feel that both of these remarkable individuals keep paramount the understanding that so many patients are suffering from the disease of cancer and that there is an urgency and precision to our work as scientists. I will strive to follow in their lead and never lose sight of this most important aspect of research. I would also like to express my appreciation for the time and devotion of my committee members, Drs. Jesse Martinez, Anne Cress, Suwon Kim, and Maria Bishop. Thank you for sharing your resources, expertise, and critiques; I value your guidance both in my research and towards my personal future career. Thank you to the amazing and devoted undergraduate interns, Ian Mathews, Molly Kupfer, and Danielle Ennesser, who have worked with me during my dissertation. For me it has been so rewarding to mentor these future scientists, and so inspirational to witness their upmost enthusiasm for science and their desire to contribute towards a project in cancer research. I have learned so much in teaching, and these interactions have brought friendship and appreciation in cultivating their passions. I wish them all the best success for their future careers as researchers and clinicians to come. Thank you to the wonderful post-doctoral fellows with whom I have had the chance to work: Dr. Timothy Whitsett and Dr. Harshil Dhruv. Your friendship and guidance have been invaluable to me during my graduate studies and I have much gratitude for your mentorship, and for sharing your critiques and expertise with me. Thank you to everyone in the Berens and Tran labs for their friendship and support during my graduate research. Thank you to the Cancer Biology Interdisciplinary Graduate Program and to Anne Cione for always being so helpful in her assistance and for her genuine caring personality. Finally, I would like to express my deepest appreciation to my friends and family, whose love, support, and encouragement have been unwavering in all my years of education and who have made it possible for me to achieve my goals.
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DEDICATION
I would like to dedicate this dissertation to those all those individuals who have suffered
from the illness of cancer, and to their family and friends who have endured this
diagnosis with them.
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TABLE OF CONTENTS
LIST OF FIGURES ........................................................................................................ 11
LIST OF TABLES .......................................................................................................... 14
catalyze intrinsic Rho GTPase GTP to GDP hydrolysis, including the removal of
phosphate and the subsequent inactivation of the Rho GTPase. Guanine nucleotide
dissociation inhibitors (GDIs) sequester Rho GTPases in their inactive GDP-bound
conformation by shielding their hydrophobic tail. Membrane interaction and attachment
is important for Rho GTPase activation and the promotion of subsequent downstream
effector binding.
42
RhoA, Rac, and Cdc42 have been the first and most well described members of
the Rho family of GTPases. These three members have been described as key regulators
of cell migration, important in promoting the formation of stress fibers, induction of
lamellipodia, and filopodia protrusion (121). RhoA was first described to promote the
assembly of contractile actomyosin filaments, and Rac promoted the assembly of a
peripheral actin meshwork (128,129). In addition to inducing peripheral actin rich
microspikes, or filopodia, Cdc42 has been described to activate Rac, demonstrating the
existence of cross-talk within the Rho family of GTPases (121,130). Furthermore, RhoA
and Rac1 have been shown to mutually inhibit each other in some studies, while other
reports postulate a positive feedback between these two GTPases (131). Another Rho
GTPase family member, RhoG, has been shown to confer downstream activity to Rac1 to
enact cell movement, but also signals to promote the robust formation of Rac1
independent lamellipodia (132-135). Moreover, there has been documented cross-talk
between the Rho and Ras families of GTPases; activated Ras can strongly activate Rac
(128).
During the process of cell migration, initial reports led to the hypothesis that
actin-driven protrusions at the leading edge of cell motility were driven by Rac
activation, while actomyosin contractility at the cell body and rear were coordinated by
active Rho (136), with Cdc42 regulating cell polarity through interpreting extracellular
directional cues (137,138). Rac and Cdc42 share an overlapping set of downstream
effectors to enact cytoskeletal changes. Signaling via Pak and MAPK activation, or via
PI5K, Formin, or IQGAP leads to actin reorganization downstream of either Rac or
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Cdc42. In addition, Cdc42 further signals via WASP and Rac further signals via WAVE
to enact cytoskeletal rearrangement (139). RhoG, which has been described to signal
both in concert with or in parallel to Rac1 and Cdc42 also shares downstream effectors
IQGAP2, MLK3, and PLD1 with Rac1 and Cdc42, but confers independent signaling as
well (135). RhoA signaling leads to phosphorylation of myosin light chain (MLC) and is
conferred by several downstream effectors including the serine/threonine kinase p160
ROCK (140,141). ROCK activity leads to increased MLC phosphorylation via the
inhibition of MLC phosphatase as well as direct MLC phosphorylation (140,142).
ROCK, together with the RhoA effector mDia, coordinates stress fiber formation (143).
While these signaling pathways have become well characterized, the
understanding of Rho GTPase regulation of cell movement has, however, become more
complex. Tumor cell motility has been characterized to occur not only in a mesenchymal
type pattern with a spindle like shape and an obvious leading cell edge, but also in an
amoeboid type fashion with cycles of expansion and contraction of the cell body,
potentially dependent upon the environment through which the cells move (144).
Moreover, biosensors capable of visualizing active Rho family members have implicated
that both Rho and Rac can be active at leading edge protrusions (145). Thus the
environmental regulation and spatiotemporal control of Rho GTPases are additional key
factor in the regulation of cytoskeletal dynamics.
The activity of Rho GTPases is tightly regulated. Activating GEFs contain a Dbl
homology (DH) domain which is the region responsible for catalyzing the exchange of
GDP for GTP (120). During nucleotide exchange, DH domain binding to the Rho
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GTPase switch region induces conformational remodeling of the nucleotide-binding
pocket (146). Pleckstrin homology (PH) domains are C-terminal adjacent associated
regions to the DH domain that regulate GEF activity. PH domains bind
phosphoinositides which facilitates plasma membrane localization, although it has also
been suggested that other domains are necessary for directing subcellular localization as
well (120). Furthermore, there is some evidence that GEFs themselves may be
negatively regulated by sites in their N-terminus. The constitutive activation of many
Rho GEFs occurs under N-terminal truncations upstream of the DH domain (147); it has
been suggested that the N-terminal region may act to auto-inhibit the DH domain, the
control of which can be relieved by phosphorylation (120). The specific mechanisms of
signal activation of GEFs appear to be complex and still remain not fully characterized.
Deregulation of RhoGTPase signaling in brain tumors
Although altered expression levels and activity of Rho GTPases have been
described in various tumor types and despite widespread reports of activating Ras
mutations across a broad spectrum of cancers, up until recently no reports had ever
classified members of the Rho family as having mutations. Rac1 activating mutations are
newly described, however, and were discovered via exome sequencing in melanoma
tissue (148,149); although to date there have yet to be reports of these mutations in other
tumor types. In brain tumors, the levels of Rac1 protein directly correlate with tumor
grade in astocytomas. In GB in particular, but not in lower grade astrocytomas, Rac1
prominent plasma membrane staining is observed (150), and Rac1 promotes an invasive
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tumor cell behavior (151). These observations support a role for Rac1 as constitutively
active in GBs, highlighting the importance of Rac1 in GB (150). Moreover, in gliomas,
Rac1 facilitation of glioma cell invasion occurs via signaling through synaptojanin 2
(152), fibroblast growth factor-inducible 14 and NF-κB (153) and Ephrin-B3 (154).
Rac1 has also been shown to regulate the formation of invadopodia, which are defined
formations of the plasma membrane that work in the capacity to promote degradation of
the extracellular matrix, an action which is critical in glioma cell invasion (155,156).
Interestingly, the Rac3 GTPase which has high homology to Rac1 has also been
described to play a role in GB cell invasion; the siRNA-mediated depletion of Rac3 led to
strong inhibition of GB cell invasion through Matrigel in vitro (155).
There have been multiple described mechanisms of Rac1 activation in
gliobalstoma. One such mechanism of Rac1 activation has been shown to be mediated
by the small Rho GTPase RhoG (133,134,157,158). RhoG protein levels are elevated in
GB (158), and RhoG has been reported to stimulate lamellipodia formation and confer
downstream activation of Rac1 with a subsequent increase in cell migration (132-
134,158). Also, the inhibition of Rho kinase led to activation of Rac1 in glioma cells and
the subsequent promotion of invasion (159). In addition, there are five Rac1 activating
GEFs, including Ect2, Vav3, Trio, SWAP-70 and Dock180-ELMO1, that have been
described to promote GB cell migration and invasion (150,157,160).
The role of GEFs in glioma may be in large part due to their expression patterns;
four of the GEFs described to promote GB cell motility, including Ect2, Vav3, Trio, and
SWAP-70, are overexpressed in GB versus non-neoplastic brain, and Dock180
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expression is higher in the tumor rim than in the tumor core (150,160). These GEFs
have been shown via RNAi knockdown to be important in glioma cell migration and
invasion assessed via radial cell and organotypic brain slice models (150,160,161). The
Dock180 GEF has been shown to signal as a bipartite GEF in connection with ELMO1,
and the expression of ELMO1 has also been characterized to be elevated in a subset of
glioblastomas (38). Furthermore, the exogenous expression of ELMO1 and Dock180 in
glioma cells enhances their migratory and invasive capacities in vitro and in brain tissue
(157). Interestingly, Ect2 has been well described for its role in the nucleus to regulate
cytokinesis (162), however in gliomas, while low-grade astrocytomas show
predominantly nuclear Ect2 staining, GBs display prominent Ect2 staining in both the
cytoplasm and nucleus (150). Thus the role of some GEFs in brain tumors may be
unique as compared to their roles in normal tissue and dependent upon subcellular
localization. Roles for additional GTPases and GEFs have yet to be defined in glioma.
TWEAK-Fn14 signaling in glioblastoma1
Cytokines are membrane bound or soluble proteins released from cells that act
mechanistically via autocrine, paracrine, or endocrine signaling. These signals regulate
important biological activities including reproduction, growth, development,
homeostasis, injury response and repair, and inflammation (163). The tumor necrosis
1 Published in (2012). Rho GTPases and Their Activators, Guanine Nucleotide Exchange Factors (GEFs): Their Roles in Glioma Cell Invasion. In A. Fatatis (Ed.) Signaling Pathways and Molecular Mediators in Metastasis. (pp 143-169). Springer Science+Business Media B.V. Publishing. <http://www.springer.com/biomed/cancer/book/978-94-007-2557-7>. The original publication is available at www.springerlink.com.
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factor (TNF) superfamily of ligands is a subgroup of cytokines heavily researched for
their role in many disease states, including human cancers and glioblastoma in particular.
The TNF cytokines are type II proteins that can exist in either a membrane
embedded or cleaved soluble form. The ligands can be active in either form, via
assembly as a noncovalent homotrimer (164). TNF receptor superfamily (TNFRSF)
members are known to have a scaffold of disulfide bridges that gives rise to the cysteine
rich domains required to be a family member. TNF receptors signal via protein-protein
interactions to promote either cell death or cell survival (165). Cytoplasmic domains may
contain TNF receptor associated factors (TRAFs) and death domains (DDs); the subset of
TNFRSF members which contain a DD are referred to as death receptors (164), and the
DD signals for cell death via caspase dependent signaling and activation of JUN kinase
pathways (165,166). TRAF mediated survival pathways have been associated with the
activation of NF-κB (166).
Of the TNF receptors characterized for their role in cancer progression,
TNFRSF12a, also known as Fibroblast Growth Factor-Inducible 14 or Fn14, is one
member of the TNF receptor superfamily that has been implicated in tumor progression
(167,168). Fn14 was first identified in murine cells as an immediate-early response gene
which encodes a cell surface localized type Ia transmembrane protein (169). Fn14 is the
smallest known TNF receptor family member and contains only one cysteine rich domain
in the extracellular region. The Fn14 cytoplasmic domain lacks a death domain but
contains TRAF binding sites specific for TRAFs 1, 2, 3, and 5 (167). Fn14 serves as a
cell surface receptor for the multifunctional cytokine tumor necrosis factor-like weak
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inducer of apoptosis (TWEAK) (170). In humans, Fn14 mRNA are expressed at the
highest relative level in heart, placenta, and kidney, at an intermediate level in lung,
skeletal muscle and pancreas, and are relatively low in brain and liver tissue. Expression
of Fn14 was found to associate with liver regeneration and hepatocarcinogenesis (171),
and endothelial growth and migration (172). Fn14 expression is induced during nerve
regeneration and tissue injury, and in several types of human cancers including brain,
breast, cervical, esophageal, hepatocellular, and testicular carcinoma in situ (168). In
brain tumors, Fn14 expression level is elevated in advanced glial tumors and is also up-
regulated in migrating glioma cells in vitro (173) and in invading glioblastoma cells in
vivo (153).
Activation of Fn14 by TWEAK promotes glioma cell migration, invasion and
survival (153,173,174). Dysregulation of the actin cytoskeleton contributes to the
migratory phenotype of cancer cells, and promotes the invasion so descriptive of GB.
Rho GTPases are a family of molecular switches that regulate the actin cytoskeleton, cell
polarity, and microtubule dynamics (175). The TWEAK-Fn14 signaling axis mediates
glioma migration and invasion via the Rac1 GTPase, and fosters a self-promoting
The central hypothesis of this dissertation is that deregulated Rho GTPase
signaling enhances TWEAK-Fn14 mediated GB progression by promoting cell
invasion and resistance to therapy. This hypothesis was investigated through the three
following specific aims:
Aim 1: Determine the role of TWEAK-Fn14 signaling to activate Rac1 in glioma
migration and invasion. Hypothesis: TWEAK-Fn14 signaling regulates multiple GEF-
Rho GTPase complexes including Ect2, Cdc42, Trio and Rac1 to enable glioma cell
motility. Activation kinetics of the Cdc42 and Rac1 Rho GTPases were determined
following TWEAK-Fn14 stimulation. Analysis of GEF mediated GTP exchange was
characterized for each Rho GTPase to determine the roles of the Ect2 and Trio GEFs in
GB.
Aim 2: Determine the signaling mechanism(s) by which SGEF promotes TWEAK-
Fn14 induced GB invasion. Hypothesis: SGEF mediates TWEAK-Fn14 signaling via
RhoG activation to promote GB cell migration and invasion via TRAF2 recruitment and
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Rac1 activation. SGEF protein regulation of RhoG activation downstream of TWEAK-
Fn14 signaling to promote GB invasion was characterized. Specifically, SGEF activity in
vitro and SGEF controlled kinetics of RhoG activation were determined, as well as Fn14
recruitment of TRAF2 for signaling through SGEF. Lastly, SGEF regulation of Rac1
activity was determined, as well as the subsequent effect of this signaling cascade on cell
motility.
Aim 3: Characterize the up-regulation of SGEF expression by TWEAK-Fn14
signaling and the role of SGEF to promote TMZ insensitivity in GB. Hypothesis:
TWEAK-Fn14 up-regulation of SGEF expression leads to SGEF promoted decreased
sensitivity to TMZ cytotoxic therapy. An analysis of the kinetics of SGEF mRNA and
protein expression following TWEAK stimulation was performed. The increased
susceptibility to TMZ treatment under depletion of SGEF was noted as well as
mechanistically characterized by assessing the role of DNA damage repair proteins in the
TMZ insensitivity phenotype.
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CHAPTER 2: PRESENT STUDY
The details of this research are presented in the appended papers, which have been
published, submitted, or are in preparation for journal submission. This chapter details
the research summary.
Glioblastoma (GB) is the highest grade and most common form of primary adult
brain tumors, characterized by a poorly defined and highly invasive cell population.
Treatment with surgical resection followed by concomitant radiation and chemotherapy
with the alkylating agent temozolomide (TMZ) is not curative and patients are faced with
a poor prognosis defined by a median survival of a mere fifteen months. GB tumors
become therapy resistant and ultimately recur, and importantly, the invading cell
population is attributed to having an intrinsic decreased sensitivity to radiation and
chemotherapy. Thus, it remains a necessity to identify the genetic and signaling
mechanisms that promote tumor invasion and therapy resistance, in order to develop new
targeted treatment strategies to combat this aggressive disease with little to no effective
treatment options once it recurs.
TWEAK-Fn14 ligand-receptor signaling is one of the mechanisms in GB that
promotes cell invasion and survival, and has been shown to be dependent upon the
activity of multiple Rho GTPases including Rac1. The molecular components of the
TWEAK-Fn14 signaling pathway have not been well understood. Here, we show that the
Rho GTPase Cdc42 is an essential signaling component in the TWEAK-Fn14 signaling
pathway that results in Rac1 activation. We first demonstrated that the TWEAK
treatment of glioma cells induced Cdc42 activity. Secondly, depletion of Cdc42
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abolished TWEAK-induced Rac1 activation. Lastly, depletion of Cdc42 abrogated
TWEAK-induced glioma cell migration and invasion.
Furthermore, we identified two guanine nucleotide exchange factors (GEFs), Ect2
and Trio, that mediated the TWEAK-induced activation of Cdc42 and Rac1, respectively,
as well as promoted TWEAK-Fn14 directed glioma cell migration and invasion. The
depletion of Ect2 abrogated both TWEAK-induced Cdc42 and Rac1 activation, whereas
the depletion of Trio inhibited TWEAK-induced Rac1 activation but not TWEAK-
induced Cdc42 activation. We additionally demonstrated that ectopic expression of Fn14
or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene
transfer in G-tva transgenic mice induced astrocyte migration within the brain,
corroborating the in vitro data of both Fn14 and Ect2 in GB invasion.
In addition, we characterized the role of another GEF, SGEF, in promoting Fn14-
induced Rac1 activation. We found that SGEF, a RhoG-specific GEF, was
overexpressed in GB tumors and promoted TWEAK-Fn14-mediated glioma invasion.
SGEF levels correlate with the invasive rim population of GB tumors analyzed via laser
capture micro-dissection, and upon TWEAK stimulation, both SGEF activity and the
SGEF-dependent activity of RhoG are increased. Moreover, we showed that TWEAK
binding to Fn14 led to the recruitment of SGEF to the Fn14 cytoplasmic tail via an
adaptor molecule TRAF2; either mutation in the Fn14-TRAF binding domain or
depletion of TRAF2 expression blocked SGEF recruitment to Fn14 and inhibited SGEF
activity. The knockdown of either SGEF or RhoG diminished TWEAK-induced
activation of Rac1 and lamellipodia formation, a ruffling extension of cell membranes
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indicative of cell motility. Moreover, we found that SGEF mRNA and protein expression
are up-regulated by the TWEAK-Fn14 signaling axis in an NF-κB dependent manner.
Thus, we demonstrated the role of SGEF/RhoG in the TWEAK/Fn14 signal pathway that
leads to GB cell motility.
Consistent with SGEF playing a role in GB cell invasion, we found that the levels
of SGEF expression in GB tumors inversely correlated with patient survival.
Additionally, we correlated high SGEF expression with TMZ resistance in some primary
glioma cell lines and defined a role for SGEF in promoting the survival of glioma cells
treated with TMZ. Inhibition of SGEF expression via shRNA shutdown sensitized
glioma cells to apoptosis and impaired colony formation under TMZ treatment. Thus, it
appears that SGEF has a dual function in aggressive GB, one that promotes cell motility
and the other one that confers TMZ resistance. It is presently unclear whether the two
functions are mechanistically related. Lastly, gene expression analysis of SGEF depleted
GB cells revealed altered expression of a network of DNA repair and survival genes,
including the down-regulation of DCLRE1C, BRCA2, XIAP, ATM, ATR, and GEN1,
among others. These results additionally supported that SGEF promotes cell survival.
In summary, we present the novel findings that the TWEAK-Fn14 ligand-receptor
pair signals through the GEF-Rho GTPase system which includes the Ect2, Trio, and
SGEF GEFs that leads to the activation of Cdc42 and Rac1, thereby increasing cell
motility and invasion. Our study results present new components in the TWEAK-Fn14
pathway that play a role in aggressive GB, and thus may represent attractive drug targets
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in glioma therapy. Additionally, SGEF signaling within the TWEAK-Fn14 pathway may
represent a novel target in TMZ refractory, invasive GB cells.
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APPENDIX A - CDC42 AND THE GUANINE NUCLEOTIDE EXCHANGE FACTORS ECT2 AND TRIO MEDIATE FN14-INDUCED MIGRATION
AND INVASION OF GLIOBLASTOMA CELLS2
Paper is published in Molecular Cancer Research
Abstract
Malignant glioblastomas (GB) are characterized by their ability to infiltrate into
normal brain. We previously reported that binding of the multifunctional cytokine tumor
necrosis factor-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth
factor-inducible 14 (Fn14) induces GB cell invasion via Rac1 activation. Here, we show
that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated
glioma cells display an increased activation of Cdc42 and depletion of Cdc42 using
siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration
and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14,
showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we
identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in
TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2
abrogates both TWEAK-induced Cdc42 and Rac1 activation as well as subsequent
TWEAK-Fn14 directed glioma cell migration and invasion. In contrast, Trio depletion
inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation.
Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an
RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces
astrocyte migration within the brain, corroborating the in vitro importance of the
2 Published in Molecular Cancer Research. 2012 Jul; 10(7):958-68.
79
TWEAK-Fn14 signaling cascade in GB invasion. Our results suggest that the TWEAK-
Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-
GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-
Rho GTPase signaling pathway present innovative drug targets for glioma therapy.
Introduction
Glioblastomas are the most malignant and common primary brain tumor in adults.
Glioblastomas are highly infiltrative, leading to a poorly defined tumor mass. As a result,
complete resection of the tumor is not feasible without compromising neurologic
function, and despite adjuvant chemo- and radiation therapy, the five year survival rate is
just under ten percent (1). The invasive nature of glioblastoma correlates to an increased
resistance to current therapeutic strategies, the mechanisms of which are complex and
remain to be fully characterized (2).
Glioma cell invasion requires adhesion to extracellular matrix, subsequent
degradation and remodeling of this matrix, as well as signaling initiated by pro-migratory
growth factors, and Rho GTPase-mediated organization and remodeling of the actin
cytoskeleton (3). Specifically, the Rho GTPase family members RhoA, Rac1, and Cdc42
are key regulators of cell migration, and have been implicated in the formation of stress
fibers, induction of lamellipodia, and filopodia protrusion (4). The regulation of Rho
GTPase activation is mediated by three classes of proteins: guanine nucleotide exchange
factors (GEFs), which are responsible for activating Rho GTPases to their GTP bound
state, GTPase activating proteins (GAPs), which enact phosphate bond hydrolysis thus
80
inactivating Rho GTPases to a GDP bound state, and GDP dissociation inhibitors (GDIs)
which bind to and stabilize Rho GTPases in their inactive GDP bound form (5). To date,
22 Rho GTPases and 80 Rho GEFs belonging to either the Dbl or DOCK families have
been identified (6).
Previous studies have shown that the fibroblast growth factor inducible -14
(Fn14) receptor can signal to induce Rac1 activation (7). Fn14 is a transmembrane
receptor belonging to the tumor necrosis factor receptor superfamily (TNFRSF), and
serves as the receptor for the multifunctional cytokine TWEAK (8). The Fn14
cytoplasmic tail lacks a death domain but contains TNFR associated factor (TRAF)
binding sites specific for TRAFs 1, 2, 3, and 5 (9). Fn14 expression is minimal to absent
in normal brain tissue but correlates with tumor grade in glioblastoma (10). Activation of
Fn14 by TWEAK promotes glioma cell migration, invasion and survival (7,10,11). The
TWEAK-Fn14 signaling axis mediates glioma migration and invasion via the Rac1
GTPase, and fosters a self-promoting feedback loop whereby Rac1 mediated TWEAK-
Fn14 signaling induces Fn14 gene expression via the NF-κB pathway (7). While Rac1 is
ubiquitously expressed among tissue types (12,13), the levels of Rac1 protein expression
in astrocytomas directly correlate with tumor grade in TMA analysis. Furthermore, in
GB, but not in lower grade astrocytomas, prominent plasma membrane staining of Rac1
is observed. These observations indicate that Rac1 is constitutively active in GBs,
underlining the importance of Rac1 in these tumors (14). To date, five GEFs that can
activate Rac1 (Ect2, Vav3, Trio, SWAP-70 and Dock180-ELMO1), have been shown to
contribute to the invasive behavior of glioblastoma (14-16). Four of these GEFs have
81
been shown to be overexpressed in GB versus non-neoplastic brain (Ect2, Vav3, Trio,
and SWAP-70) (14,16) and expression of Dock180 is higher in the tumor rim than in the
tumor core.
In this study, we describe a role for TWEAK-Fn14 signaling through a multi-
GEF, multi-Rho GTPase signaling pathway that includes Ect2, Trio, Cdc42, and Rac1.
We show that Rac1 activation via TWEAK-Fn14 signaling is dependent upon Cdc42
function. We also report that Ect2 mediates Cdc42 activation, whereas Trio mediates
Rac1 activation following TWEAK stimulation. Depletion of Ect2, Trio, or Cdc42 by
siRNA oligonucleotides suppresses TWEAK-Fn14-induced Rac1 activation and
subsequently glioma cell migration and invasion. Finally, we show that the inappropriate
expression of either Fn14 or Ect2 in the astrocyte population of glial fibrillary acidic
protein (GFAP)-tv-a transgenic mice induces astrocyte cell motility and proliferation,
suggesting that the aberrant expression of Fn14 observed in GB may play an important
role in GB progression, especially cell invasion.
Materials and Methods
Cell culture conditions. Human astrocytoma cell lines T98G and U118 (American Type
Culture Collection) were maintained in DMEM (Gibco, USA) supplemented with 10%
heat-inactivated fetal bovine serum (FBS; Gibco, USA) at 37˚C with 5% CO2. For all
assays with TWEAK treatment, cells were cultured in reduced serum (0.5% fetal bovine
serum) for 16 h before stimulation with recombinant TWEAK at 100 ng/mL in DMEM +
0.1% bovine serum albumin for the indicated time.
82
Antibodies, reagents, and Western blot analysis. A monoclonal Cdc42 antibody was
purchased from Santa Cruz (Santa Cruz, CA). Anti-myc was purchased from Cell
Signaling Technologies (Beverly, MA). A polyclonal Ect2 antibody and a monoclonal
antibody to tubulin were purchased from Millipore (Billerica, MA). Human recombinant
TWEAK was purchased from PeproTech (Rock Hill, NJ), and laminin from human
placenta was obtained from Sigma (St. Louis, MO). Lipofectamine 2000 was purchased
from Invitrogen (Carlsbad, CA).
For immunoblotting, cells were lysed in 2x SDS sample buffer (0.25 M Tris-HCl,
Grant (N.L. Tran and B.O Williams), the ARCS Foundation Eller Scholarship and
Science Foundation Arizona Fellowship (S.P. Fortin).
111
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APPENDIX B - SGEF IS OVEREXPRESSED IN HIGH GRADE GLIOMAS AND PROMOTES TWEAK-FN14-INDUCED CELL MIGRATION AND
INVASION VIA TRAF2
Paper is pending publication in the Journal of Biological Chemistry
Abstract
Glioblastoma (GB) is the highest grade of primary adult brain tumors,
characterized by a poorly defined and highly invasive cell population. Importantly, these
invading cells are attributed with having a decreased sensitivity to radiation and
chemotherapy. TWEAK-Fn14 ligand-receptor signaling is one mechanism in GB that
promotes cell invasiveness and survival, and is dependent upon the activity of multiple
Rho GTPases including Rac1. Here we report that SGEF, a RhoG-specific guanine
nucleotide exchange factor (GEF), is overexpressed in GB tumors and promotes
TWEAK-Fn14-mediated glioma invasion. Importantly, levels of SGEF expression in GB
tumors inversely correlate with patient survival. SGEF mRNA expression is increased in
GB cells at the invasive rim relative to those in the tumor core, and knockdown of SGEF
expression by shRNA decreases glioma cell migration in vitro and invasion ex vivo.
Furthermore, we showed that upon TWEAK stimulation, SGEF is recruited to the Fn14
cytoplasmic tail via TRAF2. Mutation of the Fn14-TRAF domain site or depletion of
TRAF2 expression by siRNA oligonucleotides blocked SGEF recruitment to Fn14, and
inhibited SGEF activity and subsequent GB cell migration. We also showed that
knockdown of either SGEF or RhoG diminished TWEAK activation of Rac1 and
subsequent lamellipodia formation. Together, these results indicate that SGEF-RhoG is
an important downstream regulator of Fn14-driven GB cell migration and invasion.
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Introduction
One of the key hallmarks of glioblastoma is the heightened proclivity to invade
normal brain tissue. Despite attempts at treatment with surgical resection, radiation, and
chemotherapy with the alkylating agent temozolomide, patient survival rates are less than
ten percent at five years (1). Resistance to these current treatments is inevitable with
subsequent tumor recurrence and death. Recurrent tumors develop most commonly
within two centimeters from the primary excised tumor, although single or grouped
tumor cells have frequently been observed outside this distance, as well as in the
contralateral hemisphere (2), and the possibility exists that cell invasion is an early
acquired phenotype in GB tumor formation (3). Importantly, therapy directed at
mediators of invasion has been shown to increase chemotherapeutic sensitivity (4,5) and
thus efforts are being made to identify and characterize potential drivers of cell migration.
The Rho GTPase family is comprised of critical mediators of cell migration and
invasion via cytoskeletal reorganization, most notably including Rac1, Cdc42, and RhoA
(6-8). Rho family GTPases enable downstream signaling when in their activated GTP-
bound state catalyzed by guanine nucleotide exchange factor (GEF)-mediated GTP
loading. Conversely, stimulation of Rho GTPase intrinsic GTP to GDP hydrolysis by
295), SDEE (aa 392-395), and SQEE (aa 434-437). (B) Whole cell lysates of HEK293
cells transiently transfected with plasmids encoding Fn14wt-HA, Fn14TRAFaa-HA, or
SGEF-myc were immunoblotted as indicated. (C) Whole cell lysates of HEK293 cells
transiently transfected with plasmids encoding Fn14wt-HA or Fn14TRAFaa were
collected and pre-cleared, followed by immunoprecipitation using antibodies as indicated
for anti-TRAF2 or control IP (Ctrl), and resolution via SDS-PAGE analysis using an anti-
HA antibody. (D) Whole cell lysates of HEK293 cells transiently transfected with
plasmids encoding Fn14wt-HA or SGEF-myc were collected and pre-cleared, followed
by immunoprecipitation using antibodies as indicated for anti-TRAF2 or control IP (Ctrl),
and resolution via SDS-PAGE analysis using anti-myc and anti-HA antibodies. (E & F)
HEK293 cells transiently transfected with plasmids encoding Fn14wt-HA, Fn14TRAFaa,
or SGEF-myc were collected and pre-cleared, followed by immunoprecipitation using
antibodies as indicated for anti-myc(E), anti-HA (F), or control IP (Ctrl), and resolution
via SDS-PAGE analysis using an anti-HA (E) or anti-myc (F) antibody.
144
145
Figure B.7. TWEAK-induced activation of SGEF and stimulation of migration
require TRAF2 function.
(A) U87 and U118 glioma cells were transfected with one of two independent siRNA
oligonucleotides targeting TRAF2 (TRAF2-1 & TRAF2-2), or with siRNA targeting non-
mammalian luciferase (Ctrl). After 72 hours, cells were lysed and lysates were
immunoblotted as indicated. (B) U87 glioma cells were transiently transfected with
siRNA targeting non-mammalian luciferase (Ctrl) or TRAF2 (TRAF2-1) for 48 hours.
Cells were then serum reduced (0.5% FBS) for an additional 16 hours and treated with
TWEAK for 2 minutes. SGEF activation in cell lysates was assessed using RhoG G15A-
GST constructs. (C) U87 and U118 cells were transiently transfected with siRNA
targeting non-mammalian luciferase (Ctrl) or two independent siRNA oligonucleotides
targeting TRAF2 (TRAF2-1 & TRAF2-2). After 24 hours cells were plated on glass
slides pre-coated with 10 µg/mL laminin, and cultured an additional 16 hours in reduced
serum (0.5% FBS) medium. Cells were either left untreated or treated with TWEAK and
glioma cell migration was assessed over 24 hours. Data represents the average of 10
replicates (* = p < 0.01).
146
147
Discussion
In this study, we report the importance of SGEF and RhoG signaling downstream
of TWEAK-Fn14 in promoting glioblastoma cell invasion. Levels of SGEF mRNA and
protein expression are significantly elevated in high-grade brain tumors relative to non-
neoplastic brain tissue, and elevated expression in GB samples correlates with a poorer
patient prognosis. Our data are consistent with data in the Human Protein Atlas
(www.proteinatlas.org), a publicly available portal with protein expression profiles across
human tumors, where SGEF protein staining is stronger in both glioma and liver cancer
tissues relative to normal organ tissue staining. Furthermore, SGEF expression is
elevated in cells found at the invasive edge, or rim of GB tumor specimens relative to
tumor core cells. SGEF has been shown to have the ability to confer cell invasiveness
during HPV-mediated transformation and to direct actin cytoskeleton remodeling
following infection with salmonella and in leukocyte trans-endothelial migration (30,32-
34). We have previously reported that glioma cells with the increased capacity for
migration have a decreased expression of pro-apoptotic genes and are coincidently less
sensitive to cytotoxic therapy induced apoptosis (13,14,26,35,36), arguing for a more
detailed characterization and new therapeutic strategies for the invasive cell population.
Moreover, we demonstrated that knockdown of SGEF protein inhibits TWEAK-Fn14
stimulated cell migration and invasion. Therefore, elevated SGEF signaling may
contribute to the invasive behavior of GB cells and this data highlights the potential for
targeted therapy development.
148
In glioblastoma, the Fn14 receptor is significantly overexpressed, and glioma cells
with an increased migratory capacity display elevated levels of Fn14 (12). Our data
demonstrate that TWEAK activates SGEF, followed by RhoG and subsequent Rac1
activation in glioblastoma cells. Moreover, loss of SGEF or RhoG protein inhibits
TWEAK-Fn14-induced lamellipodia formation, which is consistent with the ability of
SGEF to promote TWEAK stimulation of glioma cell migration and downstream Rac1
activation. This data corroborates our previous finding that TWEAK-Fn14 increased cell
migration is dependent on Rac1 activation (14). RhoG signaling has been described to
occur in both Rac1-dependent and -independent manners with these two GTPases sharing
overlapping signal transduction pathways (23,31,37,38). Our data support a role for
RhoG upstream of Rac1 that is dependent upon TWEAK-Fn14 stimulation of SGEF
activity.
Several studies have linked the ability of RhoG to confer downstream activation
of Rac1 with a subsequent increase in cell migration via nucleotide exchange by the
bipartite GEF Engulfment and Motility-Dedicator of Cytokinesis 180 (ELMO-Dock180)
(19,20,23). The Dock180 superfamily of proteins is an unconventional family of Rho
GTPase-specific GEFs, containing a conserved “Dock Homology Region-2” (DHR-2) or
“Docker” domain as opposed to the characteristic GEF Dbl homology (DH) domain for
catalytic nucleotide exchange (39,40), and requires the interaction with the pleckstrin
homology (PH) domain of ELMO. ELMO is a direct effector of RhoG (23). It is
possible that the dependence of TWEAK-Fn14 induced Rac1 activity on the presence of
149
SGEF and RhoG may utilize the ELMO-Dock180 GEF to facilitate guanine nucleotide
exchange for Rac1. This possibility is the focus of future studies.
We have previously shown that immunoprecipitates of Fn14 contain Rac1, and
that this interaction is dependent upon the presence of a functional Fn14 cytoplasmic tail.
The deletion of the TRAF binding site from the cytoplasmic domain results in the loss of
Fn14-Rac1 co-immunoprecipitation (14). Here we have assessed the mechanism by
which SGEF is recruited to the Fn14 cytoplasmic domain. Using predicted site analysis,
we observed that SGEF contains five TRAF2 consensus binding sites and demonstrated
that SGEF activity downstream of TWEAK-Fn14 requires the presence of a functional
TRAF binding site in the Fn14 cytoplasmic domain. Specifically, loss of TRAF2
decreases the induced levels of SGEF activity following TWEAK stimulation indicating
a requirement for TRAF2 in this signaling axis. Signaling through TRAF2, but not
TRAF1 or TRAF3, is known to promote NF-κB activity, and TRAF2 is also responsible
for promoting JNK/SAPK activity, inflammation, cell migration and chemo- and radio-
resistance of cancer cells (41-47). The specific depletion of TRAF2 in GB has been
shown to inhibit growth and confer radio-sensitization to tumor cells (48). Similarly,
signaling through Fn14 by TWEAK in glioma results in increased resistance to cytotoxic
therapy-induced apoptosis and enhanced survival via TRAF recruitment and Rac1
dependent NF-κB activation (13,14,24,36), and furthermore RhoG has been shown to
promote the activation of NF-κB (49). Thus, TRAF2 and SGEF interaction may be
necessary in the TWEAK-Fn14-TRAF signaling axis resulting in increased levels of NF-
κB activity.
150
Activated Rho GTPases are localized at the plasma membrane in close proximity
with signaling complexes (50,51). We found that the SGEF and Fn14 proteins co-
localize to areas of induced membrane ruffling (Figure B.9B). Notably, transfection of
the SGEF-myc plasmid alone into HEK293 cells resulted in robust membrane ruffling
(Figure B.9A). Furthermore, immunoprecipitation analysis demonstrated that SGEF co-
immunoprecipitated with Fn14 from cells co-expressing SGEF and Fn14 (Figure B.9D).
Thus, SGEF binds to the Fn14 receptor complex and co-localizes with Fn14 at the
leading edge of cells. Interestingly, even though levels of active SGEF are dependent
upon the presence of TRAF2, we found that the forced expression of SGEF alongside
expression of Fn14TRAFaa is still predominantly found at the cell edge (data not shown).
Other studies have also provided evidence that the overexpression of SGEF alone can
induce membrane ruffling in fibroblasts and co-localization with filamentous actin (30),
thus further supporting the role of SGEF in modulating cytoskeletal dynamics.
New treatment strategies are needed to cure glioblastoma. Patient prognosis
remains poor and actively invading cells survive current therapeutic regimens.
Importantly, therapy directed at mediators of invasion has been shown to increase
chemotherapeutic sensitivity (4,5). Our data support a role for SGEF and RhoG
activation and signaling downstream of the TWEAK-Fn14 axis to confer increased Rac1
activity and promote GB cell migration and invasion dependent upon TRAF2 recruitment
(Figure B.8). Together, these data provide a rationale for the targeting of this signaling
axis as an adjuvant therapy in glioblastoma to limit dispersion of malignant cells and
increase susceptibility to traditional radiation and chemotherapies. The role of SGEF in
151
Figure B.8. Schematic model of TWEAK-Fn14 signaling via SGEF and RhoG to
drive glioma migration/invasion.
TWEAK ligand binding to the Fn14 receptor results in the activation of RhoG by the
SGEF guanine nucleotide exchange factor. SGEF-RhoG signaling results in the
activation of Rac1 via additional guanine nucleotide exchange promoting cytoskeletal
reorganization and glioma cell migration and invasion.
152
Figure B.9. SGEF induces membrane ruffles and both co-localizes and co-
immunoprecipitates with Fn14.
(A) Non-transfected HEK293 (top panel) or HEK293 cells transiently transfected with
SGEF-myc plasmid DNA for 48 hours (bottom panel) were plated onto glass coverslips
coated with 10 µg/mL laminin and stained for actin (phalloidin) and myc. Arrows
represent membrane ruffling and areas of actin-SGEF co-localization. (B) HEK293 cells
were co-transfected with plasmids encoding Fn14wt-HA and SGEF-Myc. After 24 hours
cells, were plated on glass slides pre-coated with 10 µg/mL laminin and allowed to grow
for an additional 24 hours. Cells were stained with antibodies against HA and Myc. (C)
Immunoblot of whole cell lysates of HEK293 cells co-transfected with plasmids
encoding Fn14wt-HA and SGEF-myc. (D) HEK293 cells co-transfected with Fn14-HA
and SGEF-myc were lysed, lysates pre-cleared, and immunoprecipitated with antibodies
as indicated for anti-myc or control IP (Ctrl). Precipitates were immunoblotted as
indicated.
153
154
other disease processes is also emerging. SGEF has recently been shown to contribute to
the formation of atherosclerosis by promoting endothelial docking structures for
leukocytes at areas of inflammation (52). Thus SGEF presents a therapeutic target for
atherosclerosis and in the present study we validated SGEF as a target for glioblastoma.
Acknowledgements
This work is supported by NIH grants R01 CA130940 (N.L. Tran), the ARCS Foundation
Eller Scholarship and Science Foundation Arizona Fellowship (S.P. Fortin Ensign). The
authors would like to thank Dr. Hironori Katoh (Kyoto University) for the gift of the
pCMV-GST-ELMO-NT plasmid, and Drs. Keith Burridge and Thomas Samson
(University of North Carolina, Chapel Hill) for the gift of the pGEX4T-1-RhoG(15A)
and pCMV-SGEF-myc plasmids, respectively. The authors would also like to thank
Molly Kupfer for technical support.
155
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APPENDIX C - SGEF EXPRESSION IS REGULATED VIA TWEAK-FN14 SIGNALING THROUGH NF-ΚB AND PROMOTES CELL SURVIVAL IN
GLIOBLASTOMA
Paper was prepared to submit to a biological sciences journal not yet determined
Abstract
Glioblastoma (GB) is the highest grade and most common form of primary adult
brain tumors. Despite surgical removal followed by concomitant radiation and
chemotherapy with the alkylating agent temozolomide (TMZ), GB tumors develop
treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly
conferring a median survival of just fifteen months. Thus, it is necessary to identify the
genetic and signaling mechanisms that promote tumor resistance in order to develop
targeted therapies to combat this refractory setting. We have previously reported the
SGEF guanine nucleotide exchange factor (GEF) to be overexpressed in GB tumors and
play an important role in promoting TWEAK-Fn14 mediated glioma invasion. Here we
report a role for SGEF, a RhoG specific GEF, in glioma survival. A genome-wide
determination of NF-κB controlled genes in TMZ-resistant primary GB tumor grafts
(GB14-TMZ-R) revealed an increased occupancy of NF-κB on the SGEF gene promoter
region via ChIP-on-chip analysis. Importantly, SGEF mRNA expression is elevated in
TMZ-resistant primary GB tumor grafts compared to those with known TMZ sensitivity.
SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis
in an NF-κB dependent manner and inhibition of SGEF expression via shRNA shutdown
sensitizes glioma cells to TMZ-induced apoptosis and suppresses colony formation
following TMZ insult. Furthermore, gene expression analysis of SGEF depleted GB cells
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revealed altered expression of a network of DNA repair and survival genes, including
DCLRE1C, BRCA2, XIAP, ATM, ATR, and GEN1, among others. Understanding the
role of SGEF in promoting chemotherapeutic resistance may direct the development of
novel targeted therapeutics for TMZ refractory, invasive GB cells.
Introduction
Glioblastoma (GB) is the most common form of primary adult brain tumors
characterized by a poorly defined tumor mass resulting from highly invasive cells. The
problem of resistance to the standard anti-proliferative treatment of concomitant
radiotherapy with chemotherapy using the alkylating agent temozolomide (TMZ) is
common, and actively invading cells survive the current therapeutic regimens. Glioma
cells with the increased capacity for migration have a decreased expression of pro-
apoptotic genes and are less sensitive to cytotoxic therapy induced apoptosis (1-5); the
knockdown of several pro-invasive gene candidates in GB decreases glioma cell
migration rate and subsequently sensitizes the cells to cytotoxic therapy and importantly,
therapy directed at mediators of invasion has been shown to increase chemotherapeutic
sensitivity (6,7).
An increased capacity for cell survival results from the multi-faceted regulation of
pathways involved in promoting cell growth, replication and spread, and in importantly
preventing apoptosis in response to cytotoxic insult (8). Treatment strategies of tumor
irradiation and temozolomide administration in glioblastoma both lead to the formation
of DNA double strand breaks (DSBs), either directly, or via mismatch repair conversion
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of O(6)-methylguanine adducts into DSBs, respectively (9). DSBs are primarily repaired
through two mechanisms, homologous recombination (HR) and non-homologous end-
joining (NHEJ). HR repair makes use of a non-damaged homologous DNA template,
and thus is characterized as an error free mechanism, while NHEJ has no homologous
strand for template use leading to error development resulting in sequence modification
near the break point (10).
DNA repair is initiated via sensing of DSBs by the kinases ataxia telangiectasia
mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), and Chk2. Subsequently,
the early phosphorylation of histone H2AX (γH2AX) by ATM occurs at DNA damage
foci and leads to the phosphorylation of mediator of DNA damage checkpoint protein 1
(MDC1), with subsequent chromatin remodeling and recruitment of DNA repair proteins
(11). BRCA1 is one such key mediator of HR and NHEJ repair; after exposure to DNA
damaging agents BRCA1 is rapidly phosphorylated by ATM, ATR, and Chk2, and
relocated to sites of replication forks with γH2AX foci , where it recruits further proteins
including BRCA2 and Rad51to mediate strand exchange toward DNA repair and cell
survival (10).
One key driver in GB that has been characterized to promote both cell invasion
and cell survival is the transmembrane receptor fibroblast growth factor inducible-14
(Fn14). Fn14 is a member of the tumor necrosis factor receptor superfamily with one
known ligand, the tumor necrosis factor-like weak inducer of apoptosis (TWEAK).
Signaling through Fn14 by its cytokine ligand TWEAK activates the Rac1, Akt and NF-
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κB-pathways, and has been shown to promote increased cell invasion and resistance to
cytotoxic therapy-induced apoptosis (3,4,12).
Here we show that the src-homology 3 domain containing GEF (SGEF) promotes
cell survival in response to TMZ insult. In GB tumors, SGEF has been shown to be
significantly overexpressed, to be correlated with poor patient outcome, and to promote
glioma cell motility (Appendix B). We report that SGEF expression is increased in TMZ
resistant derived primary GB tumorgrafts and is up-regulated by TWEAK-Fn14 signaling
via NF-κB activity. SGEF and Fn14 mRNA are positively correlated in GB tumor
specimens. Depletion of SGEF impairs colony formation following TMZ insult and
increases cell susceptibility to TMZ-induced apoptosis. Moreover, the depletion of
SGEF is associated with genome wide changes in expression of DNA repair and pro-
survival pathways, and TMZ treatment of glioma cells both leads to increased nuclear
activation of SGEF and the SGEF dependent phosphorylation of the DNA damage repair
protein BRCA1. SGEF may thus be an important mediator of pro-survival signaling in
response to TMZ therapy.
Materials and Methods
Cell culture conditions. Human astrocytoma cell lines U87, U118, and T98G (American
Type Culture Collection), as well as primary tumorgraft cells (GB14 & GB14 TMZ-R)
were maintained in DMEM (Gibco, USA) supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Gibco, USA) at 37˚C with 5% CO2. For all assays with TWEAK
treatment, cells were cultured in reduced serum (0.5% fetal bovine serum) for 16 h before
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stimulation with recombinant TWEAK at 100 ng/mL in DMEM + 0.1% bovine serum
albumin for the indicated time.
ChIP on Chip Array. Analysis of the NF-κB promoter element of a primary GB
xenograft line (GB14) and its derivative line selected in vivo for temozolomide resistance
(GB14-TMZ-R) was performed using Agilent G4489a human promoter 244K array
Chips; TMZ resistance was derived as described (13). The human promoter array chip
contains ~17,000 well-defined human transcripts which encompassed -5.5 KB upstream
to +2.5 downstream from the transcriptional start site. Fresh frozen GB tumor containing
mouse brains were sliced to 20 micron thickness, formaldehyde fixed for 10min, and
collected and sheared by sonication. Samples were immunoprecipitated with the p65
antibody (IP), eluted and labeled with cy5 dye and hybridized with whole cell extracts
(WCE) labeled with cy3 dye on the chip array. GB14-TMZ-R and GB14 were processed
and hybridized independently. A “call” for a transcription factor binding event was
determined to be a region with a p-value < .05 (normalized intensity ratio) and p-Xbar <
.05 (an average of neighboring events).
RNA isolation and quantitative reverse transcriptase-polymerase chain reaction
(qRT-PCR). Total RNA was isolated as previously described (1). cDNA was
synthesized from 500 ng of total RNA in a 20 µL reaction volume using the SuperScript
III First-Strand Synthesis SuperMix Kit (Invitrogen) for 50 minutes at 50°C, followed by
85°C for 5 minutes. qPCR analysis of SGEF (sense: 5’-TGC TGA AAG GAC AAG
GAA CA-3’; anti-sense: 5’-GTA GTT TTG ATA CAG GAC AGC ATT-3’) and histone
H3.3 (sense: 5’- CCA CTG AAC TTC TGA TTC GC-3’; anti-sense: 5’-GCG TGC TAG
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CTG GAT GTC TT-3’) mRNA levels was conducted using SYBR green (Roche)
fluorescence for detection of amplification after each cycle with LightCycler analysis
software and quantified as previously described (14).
Antibodies, plasmids, reagents, and Western blot analysis. A polyclonal SGEF
antibody was purchased from Sigma (St. Louis, MO). A monoclonal tubulin antibody
was purchased from Millipore (Billerica, MA). Polyclonal antibodies for phospho-p65
(Ser536), phospho-Akt (Ser473), MCL-1, phospho-BRCA1 (Ser1524), and BRCA1, and
monoclonal antibodies for cleaved PARP, phospho-Histone H2AX (Ser139), and Histone
H3 were purchased from Cell Signaling Technologies (Beverly, MA). Anti-NF-κB p65
polyclonal antibody was purchased from Santa Cruz Biotechnology (Dallas, TX).
Human recombinant TWEAK was purchased from PeproTech (Rock Hill, NJ). Human
placental laminin and temozolomide were obtained from Sigma. In certain experiments
cells were pre-incubated for 1 hour with either 50µM SN50 or SN50M (Calbiochem)
prior to TWEAK addition. Or, in certain experiments glioma cells were infected with
either control or IκBαM expressing adenovirus (Imgenex, San Diego, CA) for 24 hours
prior to culture in reduced serum medium (0.5% FBS DMEM) for 16 hours with
subsequent addition of TWEAK for 4 hours. Plasmids: pGEX4T-1-RhoG(15A) was
obtained from Dr. Keith Burridge (U. North Carolina-Chapel Hill).
For immunoblotting, cells were lysed in 2x SDS sample buffer (0.25 M Tris-HCl,
(A) U87 glioma cells were treated with TMZ (500µM) for 24 hours followed by isolation
of nuclear proteins. SGEF activation in control and treated lysates was assessed using
RhoG G15A-GST constructs with immunoblotting for antibodies as indicated. (B) U87
glioma cells stably expressing either control empty vector (Ctrl) or shRNA targeting
SGEF (SGEF-12) were treated with TMZ (500µM) for 24 hours. Lysates were resolved
via SDS-PAGE followed by immunoblotting with the indicated anitbodies.
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preserve chromosome stability (10). This coupled with the primary role for BRCA2 to
promote HR suggests a conserved redundancy and thus heightened importance of this
repair mechanism. Thus, the combined effect of decreased BRCA2 expression and the
decreased capacity of TMZ-induced BRCA1 phosphorylation in SGEF depleted glioma
cells may help explain the observed significantly impaired capacity of glioma cells to
recover for colony formation and the increased occurrence of apoptosis in TMZ-treated
SGEF depleted glioma cells.
Despite advances in medical technology and treatment, GB prognosis has
remained largely unchanged over the last several decades (39,40). The ability of glioma
cells to survive undeterred from current treatment strategies implies that new therapeutic
avenues are necessary for treatment of this disease. There is accumulating evidence that
combinatorial therapy that includes use of treatment modalities designed to hamper the
DNA repair mechanisms of the cell may provide a significant added survival benefit over
the standard of care alone or when used in combination with inhibitors of other GB
deregulated pathways (41-45). Moreover therapy aimed at mediators of invasion can also
lead to increased chemotherapeutic sensitivity (6,7). Thus, pathways deregulated in GB
that promote both TMZ resistance and cell motility represent novel therapeutic targets in
future drug design. Our data support a role for SGEF in both the promotion of cell
invasion and cell survival signaling within GB tumors and provide a rationale for
targeting this signaling axis. Interestingly, there has been a recent report of the RhoJ
GTPase in promoting melanoma chemoresistance by suppressing DNA damage sensing
pathways including the uncoupling of ATR from its downstream effectors with resulting
200
decreased DNA damage-induced apoptosis (46). Thus the role of GEFs and GTPases in
chemoresistance via modulation of DNA repair mechanisms is an emerging field in
which we have validated a role for SGEF in GB.
Acknowledgements
The authors would like to thank Dr. Keith Burridge (University of North Carolina,
Chapel Hill) for the gift of the pGEX4T-1-RhoG(15A) plasmid.
Grant Support
This work is supported by NIH grants R01 CA130940 (N.L. Tran), the ARCS Foundation
Eller Scholarship and Science Foundation Arizona Fellowship (S.P. Fortin Ensign).
201
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APPENDIX D - CONCLUDING STATEMENTS
Deregulated pathway signaling in GB tumors occurs within multiple processes
including proliferation, metabolism, stem cells and gliomagenesis, angiogenesis, survival
and invasion. Patients ultimately die due to tumor spread and growth burden, thus the
identification of key drivers of cell invasion can inform future targeted therapy
development for use in clinical trials, with the intent to sensitize cells to combinatorial
therapy with chemotherapeutic and radiologic intervention. The overall objective of this
dissertation has been to investigate the role of the Rho family of GTPases in glioblastoma
invasion, and define which family members and regulators are key mediators of this
deadly cell process.
The acquisition of cell motility is complex and influenced by various intracellular
and extracellular signaling events. The Rho family of GTPases are well defined
regulators of actin cytoskeletal dynamics (1-3), and have been characterized to contribute
to most steps of cancer initiation and progression (4). Many Rho GTPases have been
shown to be up-regulated in several human cancers, including RhoA, RhoC, Rac1, Rac2,
Rac3, Cdc42, RhoG, Wrch2/RhoV and RhoF (4,5). Rac1 activating mutations found via
exome sequencing are newly described in melanoma tissue as a UV-signature, whereby
9.2% of sun-exposed melanomas were found to contain a proline to serine mutation at
position 29 in the highly conserved switch I domain of Rac1, which is a necessary
domain for nucleotide binding and interactions with effector molecules (6,7). However,
outside of this recent finding most reports of deregulated Rho GTPase signaling in cancer
208
involve overexpression of the GTPase itself or of upstream activators or downstream
effectors, and to date no Rac1 mutations have been described in GB. Rac1 can be
activated by multiple growth factors and cytokines. One of the mechanisms of Rac1
activation in GB is via Fn14, a transmembrane receptor of the tumor necrosis factor
receptor superfamily which has been characterized in GB for its role in promoting cell
migration, invasion, and survival (8-10). The central hypothesis of this dissertation is
that deregulated Rho GTPase signaling enhances TWEAK-Fn14 mediated GB
progression by promoting cell invasion and resistance to therapy.
In appendix A, we investigated the role of TWEAK-Fn14 signaling to activate
Rac1 in glioma migration and invasion. In our studies, we determined the kinetics of
Rac1 activation, whereby Rac1 activity increases rapidly following TWEAK treatment in
glioma cells. This activation and subsequent glioma cell migration is dependent upon the
function of Ect2. Activated GEF and Rho GTPase signaling complexes are found in
proximity to the plasma membrane and this localization is important for their function
(11,12); here we described that Fn14 wild-type, but not Fn14 cytoplasmic deleted
constructs immunoprecipitated with the Ect2 protein. In addition, we have previously
shown that Fn14 complexes with Rac1(10), and thus this data supports that within the
TWEAK-Fn14 signaling axis both the spatial and temporal intracellular regulation of the
Rac1 GTPase and its activating GEF Ect2 are important aspects of pathway control.
In GB tumors, Ect2 has been shown to localize to the cytoplasm in comparison to
a largely nuclear localization pattern in non-transformed cells (13). Moreover Rac1 has
been shown to be prominently at the plasma membrane in a significant subset of GB
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tumors (14), thus highlighting the correlation of subcellular localization with activity and
malignancy. We showed that Ect2 is necessary for TWEAK-induced Cdc42 activation,
and that Cdc42 is necessary for Rac1 activity, but Cdc42 activity does not depend on
Rac1. We discovered Trio as an additional GEF acting downstream of TWEAK-induced
Cdc42 activation that exchanges for Rac1, and thus proposed a scheme whereby
TWEAK-Fn14 stimulation confers Ect2-mediated nucleotide exchange for Cdc42 with
subsequent Rac1 activation conferred by Trio. In addition, the ectopic expression of
Fn14 or Ect2 induced astrocyte motility, and further supports a role for this axis in
promoting cell motility. Thus Ect2-Cdc42 and Trio-Rac1 activation contribute to the
TWEAK-Fn14 induced migratory capacity of GB tumor cells (Figure D.1).
Cdc42 and Rac1 share an overlapping set of activators and effectors (15). Despite
the redundancy of multiple GEFs capable of activating each GTPase, it is not clear how
each GEF is regulated to catalyze GTP loading. The Rac GEFs are differentially
expressed across tissues and at different stages of development, and thus they may be
involved in specific embryological events (16), and their regulation of GTPases may be
related to tissue specificity. Alternatively, differential GEF activation may be receptor
signaling specific; Vav2 activates Rac downstream of PDGF in fibroblasts, however
integrin mediated Rac activation in fibroblasts utilizes an unknown GEF that is not Vav2
(17). However, the Vav family is activated downstream of many receptors (18), implying
that receptor specificity may not entirely account for GEF activation. In addition, it is
also not presently clear how Rho GTPases achieve target specificity. There is some
evidence that suggests the specificity of GEF activation may determine the signal output.
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For example, PI3K constitutive activation induces some Rac and Rho downstream
activities, but not others; Rac-mediated lamellipodia and focal complex formation, and
Rho-mediated stress fibers and focal adhesions were formed, however, Rac and Rho
regulated gene transcription was unchanged (19). Moreover, the Rac and Cdc42 GEF β-
PIX binds PAK, a Rac and Cdc42 downstream effector, recruiting it to focal complexes
with the induction of membrane ruffles and Rac1 activation (20). Thus the GEF physical
association with one or more specific GTPase target effectors may help explain the
diversity of GEFs relative to Rho GTPases and support a role for GEFs to act as unique
scaffolds conferring specificity to GTPase downstream activity. Thus the underlying
importance of the central hypothesis in this dissertation is to determine which GEFs are
critical specifically for glioblastoma progression and malignancy.
In addition to Rac1 and Cdc42 activity in GB progression, the small GTPase
RhoG has been shown to play a role in promoting tumor cell invasion (5). In appendix B
we analyzed the RhoG specific GEF SGEF, to determine whether this GEF was
important in GB biology, and we reported the importance of SGEF and RhoG signaling
downstream of TWEAK-Fn14 in promoting glioblastoma cell invasion. We determined
that SGEF mRNA expression was significantly elevated among grade three anaplastic
astrocytoma and grade four glioblastoma patient tissue samples versus non-neoplastic
brain samples, and that elevated SGEF mRNA levels correlated to poor patient survival.
Furthermore, SGEF expression was elevated in cells at the invasive edge of GB tumor
specimens relative to tumor core cells. SGEF has been previously correlated to an
invasive phenotype; SGEF is necessary for the acquisition of cell invasiveness during
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HPV-mediated transformation and in actin cytoskeleton remodeling following infection
with salmonella and in leukocyte trans-endothelial migration (21-24). In this study, we
demonstrated that knockdown of SGEF abrogated TWEAK-induced cell migration and
decreased the depth of cell invasion, thus supporting a role for SGEF in GB cell motility.
The Fn14 receptor is significantly overexpressed in GB, and elevated Fn14 levels
correlate to cells with an increased migratory capacity (8). We determined components
of the signaling mechanism by which SGEF promotes TWEAK-Fn14 induced GB
migration. Our data demonstrated that TWEAK stimulation of Fn14 led to increased
SGEF activity and the SGEF dependent activation of RhoG with subsequent increased
Rac1 activity. Moreover, loss of SGEF or RhoG protein inhibits TWEAK-Fn14-induced
lamellipodia formation, which is consistent with the ability of SGEF to promote TWEAK
stimulation of glioma cell migration and downstream Rac1 activation. This data
corroborates our previous finding that TWEAK-Fn14 increased cell migration is
dependent on Rac1 activation (10). The interaction of Fn14 and Rac1 requires a
functional Fn14 cytoplasmic tail; the deletion of the TRAF binding site from the
cytoplasmic domain results in the loss of Fn14-Rac1 co-immunoprecipitation (10). In
appendix B we assessed the mechanism of SGEF recruitment to the Fn14 cytoplasmic
tail. A predicted site analysis indicated the SGEF protein contains five TRAF2 consensus
binding sites and we observed that SGEF activity downstream of TWEAK-Fn14 requires
the presence of a functional TRAF binding site in the Fn14 cytoplasmic domain. The
specific depletion of TRAF2 decreased both TWEAK-induced SGEF activity and the
TWEAK-induced migration of glioma cells. Thus our data supports that TRAF2, SGEF,
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RhoG, and subsequent Rac1 activation contribute to the TWEAK-Fn14 induced
migratory capacity of GB tumor cells (Figure D.1). It is presently unclear whether SGEF
impacts TWEAK-Fn14-induced activation of Cdc42 (Figure D.1, dotted line).
Glioma cells with a heightened migratory ability have a decreased expression of
pro-apoptotic genes and are less sensitive to cytotoxic therapy induced apoptosis
(9,10,25-27). We have shown that TWEAK-Fn14-induced Rac1 activation can be
mediated through several GEF and GTPase signaling complexes. Studies have shown
that the suppression of Rac activity selectively induces apoptosis in glioma cells but not
in normal human astrocytes (28), thus proffering a rationale for the therapeutic inhibition
of pro-migratory signaling pathways including those promoting Rac activation as an
effective clinical option for GB. Moreover, the Rac1 protein is important in promoting
TWEAK-Fn14 activation of the Akt and NF-κB-pathways, and Fn14 signaling has been
shown to promote increased cell invasion and resistance to cytotoxic therapy-induced
apoptosis (9,10,29). In appendix C we characterized the role of TWEAK-Fn14 signaling
in promoting cell survival. A CHip-Chip assay to identify differential NF-κB promoter
binding under temozolomide naïve versus resistant primary GB tumorgraft specimens
interestingly revealed increased occupancy of NF-κB on the promoter region of SGEF
under TMZ resistance. We thus formed the hypothesis that TWEAK-Fn14 signaling
regulates SGEF expression and that SGEF promotes TMZ insensitivity in GB.
We determined that TWEAK binding to Fn14 induces SGEF mRNA and protein
levels. NF-κB is capable of binding the SGEF promoter region in a TWEAK-dependent
fashion, and TWEAK-induced SGEF mRNA and protein levels are dependent upon NF-
213
κB activity. Furthermore, we showed that in a panel of 82 publicly available GB tumor
specimens there was a strong positive association between Fn14 and SGEF expression,
whereas there was no strong association noted in lower grade brain tumors, suggesting
that the co-expression of these genes is relevant specifically to GB malignancy.
Moreover, we determined that the depletion of SGEF sensitized cells to TMZ-induced
apoptosis and impaired colony formation following TMZ insult. Thus our data supports a
role for SGEF in promoting both the increased invasive capacity of glioma cells
downstream of TWEAK-Fn14 signaling and in promoting chemotherapeutic resistance
(Figure D.1).
Cancer invasion and resistance are increasingly being recognized as
interconnected processes sharing overlapping pathways that together promote disease
progression and therapy failure (30). In GB, the tumor response to external stresses
including from the tumor microenvironment or from chemotherapeutic or radiation
treatment involves the coordination of both pro-survival and pro-invasion signaling
working in concert to promote overall GB progression. The GB tumor microenvironment
is comprised of areas of hypoxia. Hypoxia, including notably areas induced by anti-
VEGF therapy, promotes increased tumor cell glycolysis and invasion into normal brain
(31). Moreover, areas of pseudopalisading necrosis, one of the histological hallmarks of
GB, have been defined to consist of tumor cells actively invading away from a hypoxic
core of tissue formed from a vaso-occlusive event. These pro-migratory cells actively
secrete proangiogenic factors, linking the formation of pseudopalisades with
microvascular hyperplasia in GB (32,33). Additionally, glioma cells promote the
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Figure D.1. TWEAK-Fn14 pro-survival and pro-invasive signaling in GB.
215
survival of endothelial cells in order to facilitate the angiogenesis necessary for tumor
survival. Hypoxia has been shown to activate NF- κB signaling in endothelial cells with
the resultant increased gene expression of antiapoptotic factors BCL2, BCL-xL, survivin,
and XIAP; the crosstalk from this endothelial cell signaling with glioma cell signaling to
release factors VEGF and TNF-alpha together promoted endothelial cell survival (34),
and increased microvessel density in astroglial brain tumors is a prognostic indicator of
poor patient survival (35). Thus, tumor hypoxia which is a common occurrence in GB
promotes both cell invasion into normal brain tissue and supports angiogenesis for tumor
survival and growth.
In addition to hypoxia, chemotherapeutic or radiation therapy promote a
coordinated pro-survival and pro-invasive tumor response. For example,
chemotherapeutic resistance in glioma cells has been shown to be promoted through Rac1
dependent Akt2 activity working upstream of the BCL2 family to promote cell survival
(29), and activation of Akt2 leads to increased MMP-9 expression and increased glioma
cell migration and invasion (36). The inhibition of NF-κB has been shown to promote
increased glioma cell death, which was synergistic under the combined treatment with
TMZ, and led to decreased migration and invasion with specifically a decreased
expression of invasion-related genes (37). Moreover, the specific depletion of TRAF2 in
GB has been shown to inhibit growth and confer radio-sensitization to tumor cells (38),
and signaling through TRAF2 promotes not only NF-κB activity, but also JNK/SAPK
activity, inflammation, and cell migration and chemo- and radio-resistance of cancer
cells (39-45). In addition, the pharmacologic auto-phosphorylation inhibition of focal
216
adhesion kinase in GB was shown to inhibit cell invasion and increase cell apoptosis, an
effect which was synergistic when treated with TMZ in vivo (46), and drug resistant
glioma cells displayed elevated expression of integrins (47), with the beta-1 integrin
shown to promote cell survival following radiation treatment via Akt signaling (48). The
combination of an mTOR inhibitor, which was shown to decrease cell invasion, with
radiotherapy prolonged survival in a syngeneic mouse glioma model through additive
cytostatic effects (49). Moreover, our data supported a relationship between invasion and
chemotherapeutic resistance; primary GB tumorgraft cells treated to develop resistance to
TMZ therapy acquired an increased migratory capacity relative to their TMZ naïve
counterparts.
Our data demonstrates a role for SGEF in the TMZ treatment resistance response.
The level of SGEF activity increased upon treatment with TMZ, and SGEF depleted
glioma cells displayed impaired expression of a network of pro-survival and DNA repair
genes. We showed that glioma cells depleted of SGEF display an impaired ability to
phosphorylate BRCA1 following treatment with TMZ, an effect that may contribute to
impaired DNA repair and explain the increased susceptibility to undergo apoptosis in
these glioma cells. Interestingly, the increase in SGEF activity following TMZ treatment
was observed to occur in the nuclear fraction of glioma cell lysates. The SGEF protein
contains two nuclear localization signal (NLS) sequences (50), as well as a site analysis
predicted nuclear export signal. Other studies have confirmed that cytoplasmic SGEF
acts in a pro-invasive fashion, while also documenting an undefined SGEF nuclear
population as well (21). We believe nuclear SGEF may act in a pro-survival fashion in
217
the coordinated response to DNA damage. Of note, several GEFs and Rho GTPases have
been identified to contain NLS sequences. The GEFs Ect2 and Net1 both contain NLS
sequences, however only Net1 contains a nuclear export signal (51,52). Moreover the
Rac1 and RhoA GTPases have been shown to contain a functional NLS, and several
additional Rho GTPases have predicted NLS sequences, including RhoC, RhoG, and
Cdc42 (53,54). Lastly, several Rho GTPase associated proteins have been shown to be
present in nuclear pools, including the GAPs p190 RhoGAP and DCL-1, and downstream
effector proteins ROCKII and LIMK (54), however a nuclear role for these proteins has
yet to be determined.
Interestingly, several recent publications have linked the role of GEFs and Rho
GTPases to the pro-survival and DNA damage responses following radiation or
chemotherapy treatment. Inhibition of Rho in vivo in glioblastoma xenografts induces
the radio-sensitivity of tumor cells, and corresponds to both improved tumor oxygenation
as a result of decreased microvessel density and to decreased MMP2 expression. Thus
Rho pathways are involved in radioresistance, via modulation of hypoxia and
angiogenesis in GB (55). The GTPase RhoA and the RhoA GEF Net1have been shown
to be activated in the nucleus following irradiation, and NET1 has been shown to regulate
RhoA-dependent actin stress fiber formation upon induction of DNA damage via p38
MAPK signaling, implicating this pathway in the DNA damage response to promote cell
survival (54,56). Moreover, the depletion of NET1 increased cell susceptibility to
apoptosis following radiation therapy (56). In addition, in melanoma cells the GTPase
RhoJ and its effector PAK1 have been shown to promote chemoresistance via modulation
218
of the ATM/ATR DNA damage response to decrease specifically DNA damage-induced
apoptosis with a corresponding increase in expression of several pro-survival genes (57).
Thus GEF and Rho GTPase signaling may promote pro-invasive signaling in concert
with pro-survival signaling by mediating the response to DNA damage.
GB tumors are characterized by deregulated pathway signaling including
proliferation, metabolism, stem cells and gliomagenesis, angiogenesis, survival and
invasion. Much effort is being placed on assessing inhibitors of these processes in
various current clinical trials, often through combinatorial treatment strategies (Table
D.1), however to date the life expectancy of a patient diagnosed with GB has not
increased significantly. Importantly, future effective treatment strategies for
glioblastoma need to address multiple hallmarks of tumor progression. The pro-invasive
and pro-survival TWEAK-Fn14 signaling pathway thus represents a novel target for
therapy; indeed drugs are currently in clinical trials which inhibit the TWEAK-Fn14
interaction, however these trials do not to date include GB (Table D.1). Moreover, the
downstream mediators of TWEAK-Fn14 signaling present new therapeutic targets for
additional future GB treatments.
Therapy directed at mediators of pro-invasive and pro-survival pathways, as well
as importantly therapy against molecules critical to tumor initiation will help prevent
tumor resistance, angiogenesis, spread and progression. NF- κB signaling, which has
been shown to be important in GB cell invasion and survival, has recently been described
to be activated during differentiation of glioblastoma-initiating cells derived from
surgical tumor specimens, whereby blockade of this signaling led to replication arrest and
219
Target Process Drugs in GB Clinical Trials ** References
*Current Clinical Trials are not specific to GB ** Data accessed from [www.clinicaltrials.gov]
Table D.1. Current signaling pathway points of intervention in GB clinical trials.
Drugs currently in use in clinical trials for GB treatment are listed above. The target
pathway for the drug and its role in tumor malignancy, as well as the list of citations
supporting a rationale for this target in GB therapy are detailed. TWEAK/Fn14 directed
therapeutics are currently in clinical trials, but are not GB specific to date.
220
senescence with results confirmed in a xenograft model (94). These findings suggest that
inhibition of NF- κB signaling may prevent these initiating cells from driving
tumorigenesis. Thus key players in NF- κB signaling should be included as future
therapeutic targets. Although to date the role of GEFs and Rho GTPases has not been
explored in cancer initiating cells in GB, there is evidence of a link between Rho GTPase
expression and tumorigenesis (95). Rac1 has been shown to play a key role downstream
of Kras in initiating early metaplastic changes in pancreatic ductal adenocarcinoma (96),
and Rac1 depletion in non-small cell lung adenocarcinoma inhibited cancer stem cell
activity, including effects on invasion, proliferation, anchorage-independent growth,
sphere formation, and lung colonization (97). Furthermore, it has been suggested that
Rac is required for the maintenance and expansion of leukemic stem/progenitor cells by
mediating stromal cell interaction (98). Lastly, during HPV-mediated cell
transformation, SGEF and RhoG are critical for the acquisition of an invasive
phenotype(21). Thus the role of GEFs and Rho GTPases in the promotion of
tumorigenesis further supports the rationale for targeting GEF-Rho GTPase signaling to
inhibit cancer progression.
Increased support for the utility of Rho GTPase pathway inhibitors in treating
cancer has led to the recent development of several strategies to identify specific drugs
that will act against Rho GTPases, or their regulation by GEFs. For example, recently a
new high-throughput flow cytometric-based assay for the identification of selective Rho
GTPase inhibitors was described which uses a fluorescent-GTP binding readout to
determine inhibition of activity (99). The development of peptide and peptidomimetic
221
inhibitors to Rho GTPase family members is also underway (100). Additionally, a new
compound targeting specifically the Rac1-Tiam GEF interaction has been described to
inhibit GTP-loading specifically of Rac1 without affecting Cdc42 or RhoA activity and
inhibited PC-3 prostate cancer cell proliferation in preliminary models (101). A
subsequently developed Rac1 inhibitor has been shown to block the association of Vav2
with Rac1 (102). Moreover, the targeting of Rho GTPases at potential allosteric non-
nucleotide binding sites has recently been described, and novel binding sites important
for these proteins have been identified (103). There is also study of Rho GTPase effector
inhibition; recently a new PAK small molecule inhibitor was described that inhibits
anchorage-independent growth of a panel of tumor cell lines (104).
Furthermore, various studies highlight the potential use of GEF inhibition and the
recent development of several therapeutics targeting GEFs supports both the rationale for
and the feasibility of future GEF inhibitors for clinical use. Recently a new method has
been described that is an adaptation of the yeast two-hybrid system, in which rapid
medium-throughput screening can be performed to identify chemical GEF inhibitors
(105). This model converts the activation of a mammalian Rho GTPase by its cognate
Rho GEF into a readout system that affects the growth of yeast. In addition, there are
reports of the identification of Trio GEF specific inhibitors that are specific either to Trio
exchange on RhoA (106) or Rac1 and RhoG (107). These preliminary studies indicate
targeting Rho GTPase signaling is a new but active field of study. Moreover,
combinatorial therapy that includes use of treatment modalities designed to hamper the
survival and DNA repair mechanisms of the cell may provide a significant added survival
222
benefit over the standard of care alone or when used in combination with inhibitors of
other GB deregulated pathways (93,108-111). Thus therapy targeting the deregulated
GEF-Rho GTPase pathways in TWEAK-Fn14 signaling in GB may act to inhibit both
cellular mechanisms of invasion and survival and in combination with chemotherapy and
radiation may improve the prognosis of patients suffering from this devastating disease.
223
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APPENDIX E - PERMISSION TO USE COPYRIGHTED MATERIAL
1) Section “TWEAK-Fn14 signaling in glioblastoma” reproduced with permission, from:
Bo Hu, Marc Symons, Bodour Salhia, Shannon P. Fortin, Nhan L. Tran, James Rutka,
and Shi-Yuan Cheng. (2012). Rho GTPases and Their Activators, Guanine Nucleotide
Exchange Factors (GEFs): Their Roles in Glioma Cell Invasion. In A. Fatatis (Ed.)
Signaling Pathways and Molecular Mediators in Metastasis. (pp 143-169). Springer
Science+Business Media B.V. Publishing.
<http://www.springer.com/biomed/cancer/book/978-94-007-2557-7>. The original
publication is available at www.springerlink.com.
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2) Appendix A reproduced with permission, from: Fortin SP, Ennis MJ, Schumacher CA,
Zylstra-Diegel CR, Williams BO, Ross JT, Winkles JA, Loftus JC, Symons MH, Tran
NL. 2012, Mol Cancer Res. 2012 Jul;10(7):958-68.
“Authors of articles published in AACR journals are permitted to use their article or parts of their article in the following ways without requesting permission from the AACR. All such uses must include appropriate attribution to the original AACR publication. Authors may do the following as applicable:
1. Reproduce parts of their article, including figures and tables, in books, reviews, or subsequent research articles they write;
2. Use parts of their article in presentations, including figures downloaded into PowerPoint, which can be done directly from the journal's website;
3. Post the accepted version of their article (after revisions resulting from peer review, but before editing and formatting) on their institutional website, if this is required by their institution. The version on the institutional repository must contain a link to the final, published version of the article on the AACR journal website. The posted version may be released publicly (made open to anyone) 12 months after its publication in the journal;
4. Submit a copy of the article to a doctoral candidate's university in support of a doctoral thesis or dissertation.”
APPENDIX F - ABSTRACTS AND PUBLICATIONS PUBLICATIONS
Bo Hu, Marc Symons, Bodour Salhia, Shannon P. Fortin, Nhan L. Tran, James Rutka, and Shi-Yuan Cheng. (2012). Rho GTPases and Their Activators, Guanine Nucleotide Exchange Factors (GEFs): Their Roles in Glioma Cell Invasion. In A. Fatatis (Ed.), Signaling Pathways and Molecular Mediators in Metastasis. (pp. 143-170). New York:Springer Publishing. Fortin SP, Ennis MJ, Schumacher CA, Zylstra-Diegel CR, Williams BO, Ross JT, Winkles JA, Loftus JC, Symons MH, Tran NL. Cdc42 and the guanine nucleotide exchange factors Ect2 and Trio mediate Fn14-induced migration and invasion of glioblastoma cells. Mol Cancer Res. 2012 Jul;10(7):958-68. Kwiatkowska A, Didier S, Fortin S, Chuang Y, White T, Berens ME, Rushing E, Eschbacher J, Tran NL, Chan A, Symons M. The small GTPase RhoG mediates glioblastoma cell invasion. Mol Cancer. 2012 Sep 11;11(1):65. Fortin Ensign S, Mathews I, Symons M, Sarkaria J, Tran N. SGEF is overexpressed in high grade gliomas and promotes Fn14-induced cell migration and invasion via TRAF2. (submitted March 2013, Journal of Biological Chemistry). Whitsett T, Fortin Ensign S, Kurywchak P, Inge L, Dhruv H, Weiss G, Tran N. Fn14 Expression Correlates with c-Met in NSCLC and Promotes Tumor Invasion and Metastasis. To be submitted summer 2013. Fortin Ensign S, Mathews I, Rollo J, Whitsett T, Symons M, Loftus J, Sarkaria J, Tran N. SGEF expression is regulated via TWEAK-Fn14 signaling through NF-κB and promotes cell survival in glioblastoma. (Manuscript in preparation). Fortin Ensign S, Mathews I, Tran N. GEFs in Glioma Review. (Manuscript in preparation).
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SGEF is over-expressed in advanced glial tumors and mediates glioma cell invasion and survival Shannon P Fortin, John Rollo, Kimberly Paquette, Amanda Chan, Juli Ross, Ian Mathews, Marc H. Symons, Nhan L. Tran. Cellular and Molecular Biology (Abstract # 3846) April 2011. American Association for Cancer Research, Orlando, FL. Glioblastoma multiforme (GBM) is the most malignant of all primary adult brain tumors, histopathologically characterized by the infiltrative capacity of glioma cells to diffuse into the surrounding normal brain. There are currently no anti-invasion therapies available, and thus the identification and functional understanding of genes that mediate malignant tumor cell dispersion could lead to the discovery of molecular targets which have the potential to respond to therapeutics. We have previously shown that several guanine nucleotide exchange factors for Rho family small GTPases are overexpressed in GBM tumors and play important roles in glioma invasion. Here we report a role for SGEF, a guanine nucleotide exchange factor (GEF) for RhoG, in mediating glioma invasion. SGEF mRNA expression increases in correlation with glioma grade and, within GBM tumors, levels of SGEF expression inversely correlate with patient survival. siRNA-mediated depletion of SGEF decreases in vitro glioma cell migration and ex vivo glioma cell invasion. In addition, genome-wide determination of NF-κB controlled genes in temozolomide-resistant primary GBM xenografts (GBM14-TMZ-R) revealed an increased occupancy of NF-κB on the SGEF gene promoter region via ChIP-on-chip analysis as compared to the parent primary line. In fact, increased phosphorylation of IkBa was detected in GBM-14-TMZ-R. Moreover, inhibition of NF-κB in GBM14-TMZ-R significantly decreases SGEF gene expression, and activation of the NF-κB pathway by TWEAK in GBM cells induces SGEF mRNA expression. Furthermore, siRNA-mediated depletion of SGEF expression enhances chemotherapy-induced cell death in glioma cells. Understanding the role of SGEF in promoting cell motility and chemotherapeutic resistance may direct the development of novel targeted therapeutics for GBM.
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SGEF is overexpressed in glioblastoma and mediates tumor cell invasion and survival Shannon P. Fortin, Ian T. Mathews, Jonathan Rollo, Marc H. Symons, Jann N. Sarkaria, Nhan L. Tran. Tumor Invasion and Metastasis (Abstract # 1642) December 2012. American Society for Cell Biology, San Francisco, CA. Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. GB tumors are characterized by a poorly defined mass due to a highly invasive cell population attributed with having a decreased sensitivity to radiation and chemotherapy with temozolomide (TMZ). Importantly, current therapies for GB do not target invading cells. Thus, the identification and characterization of genes important for cell motility could direct the development of therapies targeting invading cells in an effort to confer an increased sensitivity to radiation and TMZ. We have previously shown that several guanine nucleotide exchange factors (GEFs) for Rho family small GTPases are overexpressed in GB tumors and play important roles in glioma invasion. Here we report a role for SGEF, a RhoG specific GEF, in mediating glioma invasion and survival. A genome-wide determination of NF-κB controlled genes in TMZ-resistant primary GB tumor grafts (GB14-TMZ-R) revealed an increased occupancy of NF-κB on the SGEF gene promoter region via ChIP-on-chip analysis, and SGEF mRNA and protein expression were found to be inducible under the pro-survival and pro-invasive TWEAK-Fn14 cytokine-receptor signaling axis in an NF-κB dependent manner. Moreover, the resistant line GB14 TMZ-R migrated at an increased rate compared to non TMZ treated GB14. In addition, among human tumor specimens, SGEF mRNA expression is increased in high grade gliomas and levels of SGEF expression in GB tumors inversely correlate with patient survival. The laser capture microdissection of GB tumors showed that SGEF mRNA expression is increased at the invasive tumor rim relative to tumor core levels, and the targeted depletion of SGEF expression by shRNA oligonucleotides decreases in vitro glioma cell migration and ex vivo glioma cell invasion without affecting cell proliferation. In addition, inhibition of SGEF expression sensitizes glioma cells to TMZ-induced apoptosis and impairs colony formation following TMZ insult. Understanding the role of SGEF in promoting cell motility and chemotherapeutic resistance may direct the development of novel targeted therapeutics for invasive GB cells.
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SGEF is overexpressed in glioblastoma and mediates TWEAK-Fn14 induced cell survival Shannon P. Fortin Ensign, Ian T. Mathews, Jonathan Rollo, Julianna T.D. Ross, Marc H. Symons, Jann N. Sarkaria, Nhan L. Tran. Molecular and Cellular Biology (Abstract # 4108) April 2013. American Association for Cancer Research, Washington D.C. Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide (TMZ), GB tumors develop treatment resistance and recur, invading brain tissue. Impaired response to treatment occurs rapidly conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance and spread in order to develop targeted therapies to combat this refractory setting. Our lab has previously published a role for the TWEAK-Fn14 ligand-receptor system in GB cell invasiveness and survival. Here we report that SGEF, a RhoG specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and we hypothesize that SGEF plays an important role in promoting TWEAK-Fn14 mediated glioma invasion and survival. We show that upon TWEAK binding to Fn14, SGEF is recruited to the Fn14 cytoplasmic tail. The Fn14-SGEF interaction requires TRAF2 recruitment and leads to activation of Rac1. Importantly, SGEF mRNA expression is elevated in TMZ-resistant primary GB tumor grafts compared to those with known TMZ sensitivity. SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis in an NF-κB dependent manner and inhibition of SGEF expression via shRNA shutdown sensitizes glioma cells to TMZ-induced apoptosis and impairs colony formation following TMZ insult. Furthermore, gene expression analysis of SGEF depleted GB cells revealed impaired expression of a network of DNA repair and survival genes including ATM, ATR, BRCA2, and XIAP among others, as well as increased expression of the pro-apoptotic gene BAD. Understanding the role of SGEF in promoting chemotherapeutic resistance may direct the development of novel targeted therapeutics for TMZ refractory, invasive GB cells.