The PTEN/Akt Pathway Dictates the Direct AVB3-Dependent
Growth-Inhibitory Action of an Active Fragment of
Tumstatin in Glioma Cells In vitro and In vivo
Krystof S. Bankiewicz,
1Suzanne J. Baker,
1and Russell O. Pieper
1Brain Tumor Research Center, Department of Neurological Surgery and The University of California-San Francisco Comprehensive Cancer
Center, University of California San Francisco, San Francisco, California and
Children’s Research Hospital, Memphis, Tennessee
2Department of Developmental Neurobiology, St. Jude
The collagen type IV cleavage fragment tumstatin and its
active subfragments bind to integrin AVB3 and inhibit
activation of focal adhesion kinase, phophoinositol-3 kinase,
Akt, and mammalian target of rapamycin (mTOR) in what is
thought to be an endothelial cell–specific manner. The
resultant endothelial cell apoptosis accounts for the ability
of tumstatin to function as an endogenous inhibitor of
angiogenesis and an indirect suppressor of tumor growth.
We hypothesized that the inability of tumstatin to directly
suppress tumor cell growth might be the result of the
constitutive activation of the Akt/mTOR pathway commonly
seen in tumors. Consistent with this idea, several integrin
AVB3–expressing glioma cell lines with PTEN mutations and
high levels of phospho-Akt (pAkt) were unaffected by exposure
to an active fragment of tumstatin (T3), whereas AVB3-
expressing glioma cell lines with a functional PTEN/low levels
of pAkt exhibited T3-induced growth suppression that could
be bypassed by small interfering RNA–mediated suppression
of PTEN, introduction of a constitutively expressed Akt, or
introduction of the Akt and mTOR target eukaryotic transla-
tion initiation factor 4E. The direct tumor-suppressive actions
of T3 were further shown in an AVB3-deficient in vivo mouse
model in which T3, while unable to alter the tumstatin-
insensitive vasculature contributed by the AVB3-deficient host,
nonetheless suppressed the growth and proliferative index of
i.c. implanted AVB3-expressing PTEN-proficient glioma cells.
These results show that tumstatin, previously considered to be
only an endogenous inhibitor of angiogenesis, also directly
inhibits the growth of tumors in a manner dependent on Akt/
mTOR activation. (Cancer Res 2006; 66(23): 11331-40)
Tumors, such as glioblastoma multiforme, the most aggressive
form of human glioma, are characterized by marked endothelial
cell proliferation and extensive angiogenesis, which are in turn
believed to be driven by the high metabolic demands of the tumor,
hypoxia, and the release of factors that can lead to uncontrolled
neovascularization (1, 2). Because tumors, such as glioblastoma
multiforme, depend on vessel formation to supply oxygen
necessary for growth, a significant effort has been made to better
understand the angiogenic process in these tumors (3, 4). Results
from such studies suggest that the angiogenic process is controlled
by the balance between proangiogenic molecules that promote
vessel sprouting (5–8) and negative regulators of the angiogenic
Among the most interesting and least understood of the
antiangiogenic molecules are the endogenous inhibitors of
angiogenesis. These proteins, including canstatin, endostatin, and
tumstatin, are bioactive fragments of larger proteins commonly
found in the vascular basement membrane (VBM; refs. 9–14). As an
example, tumstatin is an NC1 domain fragment of the a3 chain of
type IV collagen, which is located in a variety of basement
membranes including those in the brain (13). Release of
endogenous inhibitors of angiogenesis from the VBM occurs by
the same matrix metalloprotease–mediated mechanism that
degrades the VBM and drives the early stages of angiogenesis.
Endogenous inhibitors of angiogenesis, however, rather than
stimulating angiogenesis, bind to integrin complexes and initiate
a cascade of events that suppress the angiogenic process. In the
case of tumstatin, the peptide can bind aVh3through amino acids
54 to 132 and 185 to 203 (13, 15). Although tumstatin does not alter
tumor cell growth directly, a NH2-terminal truncated protein or
subfragments containing amino acids 185 to 203 can block the
proliferation of melanoma cells by suppression of the focal
adhesion kinase (FAK)/phophoinositol-3 kinase (PI3K) pathway
(15). Intact tumstatin, however, has been shown to have only
antiangiogenic activity, and this activity has been localized to
fragments containing amino acids 54 to 132 (also known as Tum-5),
which bind aVh3on a variety of cells but only inhibit the PI3K/Akt/
mammalian target of rapamycin (mTOR) pathway and trigger
subsequent apoptosis in endothelial cells (13). This latter activity
has been localized to amino acids 69 to 98, and a peptide fragment
containing these amino acids (also known as T3) is considered a
promising aVh3-dependent inhibitor of angiogenesis (16).
Although the ability of the T3 peptide to bind aVh3, block the
Akt/mTOR pathway, and trigger apoptosis in endothelial cells has
been well documented, the basis for its endothelial-specific action
remains unclear, particularly in light of the fact that many tumor
cells, including those at the invading edges of glioblastoma
multiforme, express aVh3and rely on Akt activation for growth
(17, 18). Although a variety of aVh3-expressing tumor cells have
been shown to be insensitive to T3-mediated suppression of cell
growth and /or apoptosis, it is worth noting that most of these
tumors have also been reported to be PTEN deficient and/or to
Note: Supplementary data for this article are available at Cancer Research Online
Requests for reprints: Russell O. Pieper, Brain Tumor Research Center,
Department of Neurological Surgery and The University of California-San Francisco
Comprehensive Cancer Center, University of California San Francisco, San Francisco,
CA 94115-0875. Phone: 415-502-7132; Fax: 415-502-6779; E-mail: email@example.com.
I2006 American Association for Cancer Research.
Cancer Res 2006; 66: (23). December 1, 2006
exhibit constitutive activation of the Akt/mTOR pathway. If these
cells have alterations that activate the Akt pathway downstream of
the point at which T3 blocks Akt activation, these cells might seem
insensitive to T3. Conversely, tumors that do not have PTEN
alterations or high levels of phospho-Akt (pAkt; a group that
includes the majority of glioblastoma multiforme) might, along
with endothelial cells, be directly targeted by T3. In such tumors,
T3 might have a broader range of action than previously reported.
To address this possibility, we collected a variety of transformed
glial and glioblastoma multiforme cells, genetically modulated the
PTEN/Akt/mTOR pathway, and examined the effects of T3 in vitro
and in vivo in response to these modulations. The results of the
studies show that at concentrations that suppress the growth of
endothelial cells, T3 also suppresses the growth of glioblastoma
multiforme cells, but only if the cells express aVh3and have a
functional PTEN/low levels of pAkt. These results suggest that in a
subset of glioblastoma multiforme and potentially genetically
similar tumors, T3 might have a dual mechanism of action, both
directly and indirectly suppressing tumor growth.
Materials and Methods
Reagents. T3 peptide (amino acids 69-88 of human tumstatin) was
obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA). For distribu-
tion studies, mono-5-(and-6)-carboxyfluorescein (FAM)-labeled T3 was
used. The maximum excitation/emission of FAM is 495/519 nm,
Cell lines. Genetically modified human astrocytes containing constructs
encoding HPV16 E6, HPV16 E7, hTERT, V12 H-Ras, and either a blank vector
or constructs encoding human integrin h3[wild-type (WT) or constitutively
active (CA) D723R; Ras + blank vector, Ras + h3WT, and Ras + h3CA cells,
respectively] were created as described (19, 20). Cells were further infected
with a blank vector, with pWZL-hygro encoding CA myristilated Akt
(myrAkt D4-129), or with pWZL-hygro encoding eukaryotic translation
initiation factor 4E (eIF4E). All established human glioblastoma multiforme
cell lines were obtained from the University of California San Francisco
(UCSF) Brain Tumor Research Center Tissue Bank, and select lines were
also infected with blank vector, pMXI-egfp-human integrin h3WT, and/or
pWZL-hygro-myrAktD4-129. An immortalized human dermal microvascular
endothelial cell line (HDMEC) was created from cryopreserved cells isolated
from human dermal tissue (PromoCell, Heidelberg, Germany) and
immortalized with SV40 Tag (21) and was provided by Dr. Gabriele Bergers
(UCSF Department of Neurological Surgery). These cells were further
infected with blank vector or pMXI-egfp-human integrin h3WT. Mouse
astrocytes derived from h3WT or h3 knockout (h3KO) animals were
transformed by retroviral infection with V12 H-Ras and HPV16 E6/HPV16
E7 as described (2, 19, 20, 22). The cells were further infected with blank
vector, pWZL-hygro-myrAktD4-129, or pMXI-egfp-human integrin h3WT,
after which expression of h3WTor myrAkt was verified by PCR and Western
blot. Mouse astrocytes derived from WT or PTEN conditional KO mice [P2
animals with genotypes Pten+/+;GFAP-cre (WT) and PtenloxP/loxP;GFAP-
cre (Pten cKO; ref. 23] and transfected with SV40 large T antigen and
mutant V12 H-Ras were also provided by Dr. Gabriele Bergers.
For enhanced green fluorescent protein (egfp) selection, retrovirally
infected egfp-positive cells (>10,000 per population) were sorted 5 days after
infection on a FACSVantage (Becton Dickinson, San Jose, CA) and pooled.
For Hygromycin B selection, cells were grown for 5 days in Hygromycin B
(300 Ag/mL) beginning 72 hours after infection.
Cells were maintained as monolayers in a complete medium consisting
of DMEM supplemented with 10% fetal bovine serum (FBS). HDMEC cells
were cultured in MCDB131 (Life Technologies, Carlsbad, CA) with 10% FBS
and 1:100 L-Glutamine. All cells were cultured at 37jC in a humidified 5%
Small interfering RNA studies. Cells were seeded at 2.0 ? 105per six-
wellplate24 hoursbeforetransfection andtransfectedwith50 or 100nmol/L
small interfering RNA (siRNA) targeting PTEN (5¶-AACAGTAGAG-
GAGCCGTCAAA-3¶; B-Bridge International, Inc., Sunnyvale, CA) or control
non-targeting siRNA (Dharmacon, Lafayette, CO), using Oligofectamine
(Invitrogen, Carlsbad, CA). After 4 hours of transfection, the media were
replaced with complete growing media containing 10% FBS. Cells were
harvested 48 or 96 hours after transfection and analyzed by immunoblot-
ting or plated for further assays.
Cell viability/growth assays. For cell viability/growth assays, pre-plated
cells (1,000 cells per 96-well plate) were serum starved for 24 hours, after
which medium was changed to normal growing medium containing 10%
FBS with or without T3 peptide (0-50 Amol/L). Seventy-two hours after
(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent (Promega, Mad-
ison, WI) was added and used to analyze the ability of the cells to
chemically reduce the MTS reagent to formazan, which was subsequently
detected spectrophotometrically (490 nm) according to manufacturer’s
protocol. Absorbance of control cultures (which was not significantly
influenced by 24 hours of growth in serum-free conditions) was defined as
1.0 and compared with that of T3-treated cultures.
Western blot analysis. Cells were serum starved for 24 hours, after
which medium was changed to normal growing medium containing 10%
FBS with or without T3 peptide (10 Amol/L). After 4 hours of incubation,
cells were harvested and lysed as described previously (24). Whole-cell
lysate (10 Ag) was subjected to gel electrophoresis and electroblotted onto
an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was
blocked in 5% skim milk and incubated with antibodies against pAkt, total
Akt, PTEN, integrin h3, phospho-S6 kinase (pS6K), total S6K, eIF4E-binding
protein 1 (4E-BP1), eIF4E (1:1,000; all from Cell Signaling Technology,
Danvers, MA), or a-tubulin (1:2,000; Santa Cruz Biotechnology, Santa Cruz,
CA) overnight at 4jC. Bound antibody was detected with horseradish
peroxidase–conjugated secondary antibody using Enhanced Chemilumi-
nescence Western blotting detection reagents (Pierce, Milwaukee, WI).
Bromodeoxyuridine cell cycle analysis. Cells were serum starved for
24 hours, after which medium was changed to normal growing medium
that contained 10% FBS with or without 10 Amol/L T3 peptide. After 24
hours of incubation, bromodeoxyuridine (BrdUrd; 10 Amol/L) was added to
medium, and the cells were incubated for 1 hour at 37jC. The cells were
then trypsinized, fixed, and stained according to the manufacturer’s
instruction (BD Biosciences PharMingen, San Diego, CA).
Analysis of cell cycle distribution/Annexin V-FITC assay. Cells were
trypsinized after T3 incubation, washed in PBS, and fixed in 70% ethanol at
?20jC. Following incubation in PBS containing 40 Ag/mL propidium iodine
and 200 Ag/mL RNase A (Sigma, St. Louis, MO) for 1 hour at room
temperature in the dark, stained nuclei were analyzed on a FACScan
machine (Becton Dickinson) with 10,000 events per determination as
described previously (25). ModFit LT software (Verity Software House, Inc.,
Topsham, ME) was used to assess cell cycle distribution. The Annexin
V-FITC binding assay was done using an ApoAlert Annexin V kit (Clontech,
Palo Alto, CA) as described previously (26), with 20,000 events per
Analysis of cell surface expression of integrin AVB3. To confirm
integrin aVh3expression on the cell surface, cells were trypsinized, washed
with PBS, and incubated with anti-human integrin aVh3antibody (1:200 in
PBS with 1% bovine serum albumin; Chemicon, Temecula, CA) for 1 hour at
room temperature. After washing with PBS, cells were incubated with
phycoerythrin-conjugated anti-mouse IgG secondary antibody (1:200;
Vector Lab, Burlingame, CA) for 1 hour at room temperature in the dark.
Stained cells were then analyzed on FACScan machine (Becton Dickinson).
Mice genotyping. To confirm the integrin h3genotype, three-primer
PCR was carried out as described previously (27) using the following
conditions: 35 cycles of 94jC (1 minute)/60jC (1 minute)/72jC (1 minute)
followed by 72jC (10 minutes). The primer sets were 5¶-CTTAGACACCTGC-
TACGGGC-3¶ (primer 1; common forward primer), 5¶-CTGAGGCTGAGTGT-
GATGG-3¶ (primer 2; WT specific), and 5¶-CACGAGACTAGTGAGACGTG-3¶
(primer 3; mutant specific). PCR products were electrophoresed in 2%
agarose gel and observed under UV light. PCR products were 538 bp
(mutant) and 446 bp (WT).
Cancer Res 2006; 66: (23). December 1, 2006
effectively delivered in vivo across the entire i.c. tumor volume by
CED at higher concentrations than could likely be achieved
systemically, thereby bypassing possible concerns about blood-
brain barrier impermeability (30, 31). As the CED technique
becomes more refined and applied in clinical settings, prolonged
localized delivery of T3 might allow long-term control of glioma
growth and progression, whereas avoiding the autoimmune
responses possible following long-term systemic administration
(13, 48). The identification of a dual mechanism of action of T3 in
the present study, therefore, not only provides insight into the
function of endogenous inhibitors of angiogenesis but also may
help influence how patients are selected for such therapy and
ultimately may suggest alternative ways to use such agents
Received 4/26/2006; revised 7/21/2006; accepted 9/14/2006.
Grant support: NIH grant CA94989 (R.O. Pieper), NIH (S.J. Baker), and American
Lebanese and Syrian Associated Charities (S.J. Baker).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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