Overexpression of ST6GalNAcV, a ganglioside-specific α2,6-sialyltransferase, inhibits
glioma growth in vivo
Author(s): Roger A. Kroes, Huan He, Mark R. Emmett, Carol L. Nilsson, Franklin E. Leach
III, I. Jonathan Amster, Alan G. Marshall, Joseph R. Moskal and Senitiroh Hakomori
Source: Proceedings of the National Academy of Sciences of the United States of America,
Vol. 107, No. 28 (July 13, 2010), pp. 12646-12651
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/20724308
Accessed: 26-05-2016 15:09 UTC
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Overexpression of ST6GalNAcV, a ganglioside-specific
?2,6-sialyltransferase, inhibits glioma growth in vivo
Roger A. Kroesa, Huan Hebf Mark R. Emmettbc, Carol L Nilssond, Franklin E. Leach IIIe, I. Jonathan Amster6,
Alan G. Marshallbc, and Joseph R. Moskal3'1
aThe Falk Center for Molecular Therapeutics, Department of Biomedicai Engineering, Northwestern University, Evanston, IL 60201; bNational High Magnetic
Field Laboratory, Florida State University, Tallahassee, FL 32310; department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306;
dPfizer Global Research and Development, San Diego, CA 92121; and department of Chemistry, University of Georgia, Athens, GA 30602
Edited* by Senitiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved June 10, 2010 (received for review August 31, 2009)
Aberrant cell-surface glycosylation patterns are present on virtually
all tumors and have been linked to tumor progression, metastasis,
and invasivity. We have shown that expressing a normally quies
cent, glycoprotein-specific a2,6-sialyltransferase (ST6Gal1) gene in
gliomas inhibited invasivity in vitro and tumor formation in vivo. To
identify other glycogene targets with therapeutic potential, we cre
ated a focused 45-mer oligonucleotide microarray platform repre
senting all of the cloned human glycotranscriptome and examined
the glycogene expression profiles of 10 normal human brain speci
mens, 10 malignant gliomas, and 7 human glioma cell lines. Among
the many significant changes in glycogene expression observed,
of particular interest was the observation that an additional ot2,6
sialyltransferase, ST6 (ot-/V-acetyl-neuraminyl-2,3-?-galactosyl-1,3)
/V-acetylgalactosaminide a2,6-sialyltransferase 5 (ST6GalNAcV), was
expressed at very low levels in all glioma and glioma cell lines exam
ined compared with normal brain. ST6GalNAcV catalyzes the forma
tion of the terminal a2,6-sialic acid linkages on gangliosides. Stable
transfection of ST6GalNAcV into U373MG glioma cells produced (/') no
change in a2,6-linked sialic acid-containing glycoproteins, (//') in
creased expression of GM2a and GM3 gangliosides and decreased
(9, 10) and Nakamura et al. (11) has shown that human neuro
blastomas, medulloblastomas, and astrocytomas shed ganglio
sides that can be detected in patient serum and cerebrospinal
fluid. Li et al. (12) showed that shed ganglioside GD2 was
markedly immunosuppressive and, as such, may facilitate tumor
formation and progression.
We used focused 45-mer oligonucleotide microarrays coupled
with quantitative RT-PCR (qRT-PCR) analyses to comprehen
sively query the human glyco-transcriptome (13) and found
significantly lower levels of ganglioside-selective ot2,6-sialyl
transferase, ST6 (a-N-acetyl-neuraminyl-2,3-?-galactosyl-l,3)-./V
acetylgalactosaminide a2,6-sialyltransferase 5 (ST?GalNAcV),
in malignant glioblastomas (92 primary tumor specimens and 7
human glioma cell lines) relative to normal brain (34 specimens).
GDI a is the terminal structure of a-series gangliosides and is
synthesized from GM lb through the transfer of sialic acid to its
?-acetylgalactosamine residue through an oc2,6-linkage with
GMlb (x2,6-sialyltransferase ST?GalNAcV, which, in mice, is
only active toward GMlb (12). Significant down-regulation of
ST?GalNAcV has been demonstrated in lung squamous cell
carcinoma (14). Its involvement in the inflammatory breast
cancer phenotype (15) and in breast cancer m?tastases to the
brain (16) has been reported. Alterations in the adhesive prop
(v) increased adhesion-mediated protein tyrosine phosphorylation of
erties of HeLa cells by modulation of ST?GalNAcV expression
HSPA8, and {vi) inhibition of tumor growth in vivo. These results has also been demonstrated (17). Interestingly, there is little to
strongly suggest that modulation of the synthesis of specific glioma no a2,6-sialytransferase (ST?Gall) activity, the enzyme that
cell-surface glycosphingolipids alters invasivity in a manner that may sialylates glycoprotein-associated, N-linked Oligosaccharides, in
malignant glioblastomas. Moreover, studies have shown that
have significant therapeutic potential.
expression of this sialyltransferase in human glioma model sys
expression of G M 1b, Gb3, and Gb4, (//'/) marked inhibition of in vitro
invasivity, (/V) modified cellular adhesion to fibronectin and laminin,
glycosyltransferase | glycosphingolipid | glioblastoma | heat shock 70 kDa
protein 8 | glycosynapse
Aberrations
in cell-surface
glycosylation
patterns
a dis
tinguishing
feature of all
vertebrate tumor
cells,are
including
brain tumors (1). Cell-surface glycoproteins have been identified
that are associated with the invasive potential of malignant gliomas
(2). The overexpression of the glycosyltransferase, GnT-V in gli
omas, led to increased invasivity of these tumor cells in vitro,
suggesting that ?l,6-N-acetylglucosamine-bearing N-glycans play
a role in glioma invasivity (3), consistent with earlier reports
showing a strong correlation between metastatic potential and the
expression of tri- and tetra-antennary ? 1,6NAG-bearing N-glycans
tems inhibited both tumor growth and invasivity, by altering the
signal transduction capability of the oc3?l integrin, because of its
altered terminal sialic acid composition, and reflected in changes
in focal adhesion kinase activity (5, 6).
Thus, studies were undertaken to evaluate the role of
ST?GalNAcV in glioma invasivity by creating stable trans
fectants and evaluating them for alterations in cell-surface Oli
gosaccharide expression, invasive potential in vitro, intracellular
protein phosphorylation, and tumorigenicity in vivo.
Results
Stable Transfection. We have demonstrated the relative absence
of ST?GalNAcV mRNA in clinical gliomas and glioma cell lines,
including the U373MG cell line (13). The coding region of the
(4). And recently, direct demonstration of a role for the Oligo ST?GalNAcV gene (UnigenelD: 181819), subcloned into the
saccharide component of the glioma-associated integrin, a3?l, in pcDNA3 expression vector, was used for the generation of
regulating glioma invasivity has been reported (5, 6).
a panel of stable ST?GalNAcV-expressing cell lines. We chose
Gangliosides are sialic acid-bearing glycosphingolipids that are
an important part of the cell surface glycoconjugates expressed
on all mammalian cells. They influence tumor growth and pro Author contributions: R.A.K, and J.R.M. designed research; R.A.K., H.H., M.R.E., C.L.N., and
F.E.L. performed research; M.R.E., U.A., and A.G.M. contributed new reagents/analytic
gression through modulation of adhesion, migration, and an
giogenesis, and their expression is markedly altered in a variety tools; R.A.K., H.H., M.R.E., C.L.N., F.E.L, and J.R.M. analyzed data; and R.A.K, and J.R.M.
wrote the paper.
of brain tumors and brain tumor model systems. Hamasaki et al.
The authors declare no conflict of interest.
(7) have reported that GTlb ganglioside can be used as a brain
metastasis marker. Mennel and Lell (8) also corroborated earlier *This Direct Submission article had a prearranged editor.
reports that simpler ganglioside patterns were associated with 1To whom correspondence should be addressed. E-mail: j-moskal@northwestern.edu.
the malignancy progression, but not the proliferation patterns, of This article contains supporting information online at www.pnas.org/lookup/suppl/doi: 10.
astrocytomas. And a series of reports by Ladisch and coworkers 1073/pnas.0909862107/-/DCSupplemental.
12646-12651 | PNAS | July 13,2010 | vol.107 | no. 28
www.pnas.org/cgi/doi/10.1073/pnas.0909862107
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S25) by use of a detailed, sensitive, semiquantitative method
using lipid extraction, one-step nano-liquid chromatography
(nLC) separation, and high-resolution mass analysis (18,19). We
observed significant modulation of specific gangliosides in the
a-series pathway (Fig. 2). As ST?GalNAcV directs the synthesis
of GDla in mice, we initially expected that the increased cell
A
c
surface SNA reactivity was due to specific increases in this rare,
brain-specific ganglioside. The lipid profiling data clearly dem
onstrate that, despite no change in GDla content in the trans
fectants, there was an ?7-fold increase in another a2,6
D
sialoganglioside, GM2a, with a concomitant ?3-fold decrease in
GMlb and ?2-fold decreases in Gb3 and Gb4. A 2-fold increase
in GM3 was also observed in the transfectant clones. Sialic acid
linked to GalNAc is thought to be relatively rare in most tissues.
U373 pcDNA3 S32 S23 S8 S25
Clone
Fig. 1. ST6GalNAcV mRNA expression in U373MG/ST6GalNAcV clones. (A)
Transcript abundance, normalized to GAPDH, was calculated by qRT-PCR. Data
represent mean (? SD) of triplicate analyses. < 0.01; ***P < 0.001; two
tailed, unpaired Student's t test), n/s, not significant (P > 0.05). (B-E) Immu
nohistochemical analysis after incubation with 10 pg/mL FITC-SNA (Matreya)
and 1% BSA. Staining of U373MG parental cells (B), pcDNA3-transfected
control cells (C), and ST6GalNAcV-transfected cells, clones S8 and S25 (D and ?).
four individually isolated clones for further study and quanti
Although GM2a may constitute only a minor ganglioside com
ponent, the lack of appropriate standards does not allow mea
surement of its absolute concentration. Nonetheless, the 7-fold
increase in GM2a containing the dl8:l-C16:0 ceramide structure
common in many tumor cells had the highest ion abundance
measured across all transfectant samples (Table S2 and Fig. S2).
Electron-induced dissociation (EID) experiments, performed
on selected ions from the nLC effluent after peak-parking to
differentiate between globo-series Gb4 and iso-globoseries iGb4
isomers, confirmed the presence of Gb4 and not iGb4 (Fig. S3).
EID fragmentation of Gb3/iGb3 was also performed, but be
cause of the highly symmetrical nature of the glycan residue, we
were unable differentiate between Gb3 and iGb3. Because Gb4
is biosynthesized from Gb3 and not iGb3, Gb3 is the most likely
isomer detected.
tated ST?GalNAcV mRNA expression by qRT-PCR (Fig. 1).
The morphology of these clones was more rounded and less
In Vitro Invasivity. The four clones examined represented a spec
trum of ST?GalNAcV overexpression ranging from 2- to 25-fold.
The decrease in in vitro invasivity was directly related to the level
pcDNA3 transfectants. The proliferation rates of the trans
fectants did not significantly differ from controls, with doubling of expression of ST?GalNAc mRNA, with clones S8 and S25, the
dendritic than the parental U373MG cells or the control
highest expressers, displaying ?20% of the invasivity of the
times of ?30 h (Table SI).
control cells (Fig. 3). Thus, within the limits of quantitation used
in these analyses, it appears compelling that the greater the
immunohistochemical staining of the transfectant clones with ST?GalNAcV mRNA levels, the greater the quantity of cell
FITC-SNA was detectable only in the highest expressers (S8 and surface-expressed a2,6-linked glycolipids and the lesser the in
Expression of Cell Surface Glycoconjugates. Prominent cell surface
S25; Fig. 1). The effects of ST?GalNAcV overexpression on vitro invasivity of the transfectants.
overall glycoprotein sialylation, evaluated by using SNA lectin
Western blot analysis, demonstrated no alterations in terminal
a2,6-sialylation (Fig. SI).
In Vitro Adhesion. One of the primary mechanisms to modulate
cellular invasivity is via differential adhesion to extracellular ma
We compared polar lipid changes in control U373MG cells trix (ECM). Compared with controls, a marked reduction in ad
and the two highest expressing ST?GalNAc transfectants (S8 and hesion to fibronectin (?20-40%) and increase (?40-60%) in
GM2a CID spectrum
a-series Ganglioside Biosynthetic Pathway
O
r
MS2 spectrum
[M-H]* miz 1354.7857
|GM2a (d18:1/C16:0)
Cer#OD*
Q?3/ GMIg_
Cer Ceramide (d18:1/C16:0)
# Glc
O Gal
GalNAc
o
?3>GMI
+o3Qgr CDIo
i
X
GTltc
o3O?3
a8 )0P<#eCer
a3 CQIba
I
a8^a30 3 ?4,
+ Neu5Ac
<7 o
Symbol Key
Gal O
GalNAc
1100 1200 1300
Glc ?
Neu5Ac
Fig. 2. a-Series ganglioside profile of ST?GalNAcV transfectant (S25) via nLC-MS analysis {Left) with gangliosides demonstrating significant overexpression in
the transfectants outlined in red, and those demonstrating significantly decreased expression outlined in green. The approximate fold-difference in expression
is indicated. Linear quadrupole ion trap product ion mass spectrum for precursor ions of miz 1354.783 (Right), with fragmentation pattern consistent with the
c+rnr+iir? r\f C^?'im
Kroesetal. PNAS | July 13,2010 | vol.107 | no. 28 | 12647
This content downloaded from 129.105.72.174 on Thu, 26 May 2016 15:09:15 UTC
All use subject to http://about.jstor.org/terms
f, kD 1 2 3 4
IM
Clone
Fig. 3. In vitro invasion assay. Evaluation of the relative invasivity of the
transfected subclones compared with pcDNA3-transfected controls. The data
are the average ? SD (bars) values of assays performed in triplicate. (**P < 0.01;
***P< 0.001; two-tailed, unpaired Student's ttest). n/s, not significant (P>0.05).
adhesion to laminin was observed in the two clones (S8 and S25)
demonstrating comparatively high levels of ST?GalNAcV mRNA
expression and cell surface a2,6-linked gangliosides (Fig. 4). No
significant difference in adhesion to collagen was demonstrated.
Adhesion-Mediated Protein Tyrosine Phosphorylation. Altered ad
hesivity of U373MG cells to fibronectin by modulation of cell
surface N-linked glycoconjugates is mediated by altered tyrosine
phosphorylation of pl25F (5). Regulation of laminin-mediated
signaling in other systems has also been described (20). After
adhesion to laminin, both the qualitative and quantitative pattern
of phosphorylated proteins in each of the clones was identical to
that observed in control cells (Fig. S4). After adhesion to fibro
nectin, however, there was a striking difference in the level of
phosphorylation of a 75-kDa protein in all of the transfectants.
The abundance of this 75-kDa phosphoprotein directly correlated
with the level of ST?GalNAcV expression (Fig. 5A). The identity
of this 75-kDa phosphoprotein (pp75) and the signaling pathway
altered by aberrant glycolipid expression in these transfectants
was examined.
Identification of pp75. After Off-Gel Electrophoretic (OGE) and
antiphosphotyrosine Western blot analyses, pp75 was identified
in the fraction corresponding to an approximate pi range of
pcDNA3 S8_S26 pcDNA3 _S8_S25_
CLONE
CLONE
00003*?5 (100%). 70 Mi Da
IPI:IPW00038C5.1 Tax_ld>9606 0?n?_Symbol?HSPA8 Itoform 1 of H?at shock cognate 71 kDa protoln
11 unlquo popttdos. 12 uniqu* spoctra. 19 total spactra. 212?4? amino acids (33?. covorago)
MSKOPAVOIO
i gdaakn qva
v q v e y kge
nosorqatkd
gggtfdvsil
kdisenkrav
rfeelnadlf
qdffngk 6 l
lcietaccvm
kdnnllgkfe
i ti tnokgrl
atvedeklqg
kvcnpii tkl
L c Y S V G V
mn TVF0A
TKSFYPEEVS
a g i aglnvl
tieogifevk
rrlrtacera
rgtldpveka
KSINPDEAVA
tvlikrntti
ltgi a r g
s e d i ermvq
kinde dkqk i
S A G G ? G
f v e i i a
krligrrfdd
tlvLTKMKE i
ri i e a a a
s tagdthlgg
krtlssstqa
lroakloksq
yoaavoaa i l
ptkototftt
vpqievtfoi
eaekykaede
l 0 c e i i w
pgcfpgcga
noqgnrttps
avvqsomkhw
aeaylgktvt
IAYGLDKKVG
eofonrmvnh
sieidslyeg
ihdivlvggs
SGDKSENVOD
YSONOPOVLI
d a g i l v s a
kqrdkvsskn
loknqtaeke
PPSGGASSGP
yvaftoterl
pfBvvnoagr
navvtvpayf
aernvl i fol
f i aefkrkhk
i dfytsi tra
tripkiqkll
lllldvtpls
ovyegeramt
vokstgkenk
slesyafnmk
ef ehqqkele
TI e e v d
Fig. 5. Adhesion-mediated protein tyrosine phosphorylation. (A) Cells were
incubated in fibronectin-coated plates, unattached cells were removed, and
phosphotyrosine-modified proteins were detected as described in Methods.
Lane 1, parental U373MG cells; lane 2, pcDNA3 control cells; lane 3, clone S8;
lane 4, clone S23; lane 5, clone S25; lane 6, clone S32. (B) Identification of
HSPA8 by nanoLC-MS/MS. The precursor ion shown is m/z 740.88, and the
resultant peaks searched against the IPI human database by using both the
Mascot 2.2 search engine and the SEQUEST algorithm. Eleven unique tryptic
Peptides, as shown, matched the heat shock cognate 71-kDa protein. (C)
Immunoprecipitation analysis of phospho-HSPA8 in the ST6GalNAcV-trans
fected cells. Lanes 1 and 2: pcDNA3-transfected control cells and ST6Gal
NAcV-transfected clone (S25) were incubated in fibronectin-coated plates.
HSPA8 protein content was detected by Western blot analysis using HRP
conjugated anti-HSPA8 antibody (Millipore) and visualization by ECL. Lanes
3 and 4: After adhesion to fibronectin, pcDNA3-transfected control cells and
ST6GalNAcV-transfected clone (S25) were lysed in RIPA buffer, 200 g of
precleared lysate protein immunoprecipitated with antibody to HSPA8 (1B5;
AbCam) and analyzed by using an antiphosphotyrosine antibody, as de
scribed in Methods.
PCDNA3 SB_S25
CLONE
Fig. 4. In vitro adhesion assay. Relative adhesion of the two highest
analysis 5.3-5.6. After excision of the corresponding region of
a sister Coomassie-stained gel, trypsin digestion and nano LC
MS/MS analyses identified two major proteins: the clathrin un
from a representative experiment, n/s, not significant; *P < 0.05; **P < 0.01;
coating ATPase HSPA8/HSC70 (with 11 unique peptides rep
resenting 212 of 646 amino acids spanning >99.5% of the
protein; Fig. 5B) and KEAP1 (with 3 unique peptides repre
senting 66 of 624 amino acids spanning 48% of the protein).
***P < 0.001 from a Student's ttest (unpaired).
Immunoprecipitation of extracts with the respective primary
ST6GalNAcV-expressing transfectants (clones S8 and S25) on fibronectin- (A),
laminin- (?), or collagen type l-coated (C) plates compared with control
pcDNA3 cells. Data are average ? SEM (bars) values of three values taken
12648 I wvvw^nas.org/cgi/doi/10.1073/pnas.0909862107 Kroes et al.
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onstrated compensatory shunting of ganglioside biosynthesis
toward the normally quiescent "0" pathway in murine cells but
not in human cells, with differing alternative glycosphingolipid
pathways used to functionally compensate for aberrant ganglio
side biosynthesis (21, 22). The reduced expression of Gb3 and
Gb4 observed in the transfectants is also consistent with such
potential shunting of ganglioside biosynthetic pathways toward
the synthesis of GM2a. At any rate, the stable overexpression of
ST?GalNAcV leads to the production of nascent oc2,6-linked
sialoglycoconjugates on the cell surface. It also follows that the
relatively low, yet significant, cell surface SNA reactivity observed
in the clones exhibiting the highest levels of ST?GalNAcV
expression is likely due to the low affinity of this lectin for sialic
acid that is a2,6-linked to 7V-acetylgalactosamine, as opposed
to galactose.
The modest increase in GM3 synthesis, a ganglioside pos
sessing modulatory effects on glioma growth, adhesion, and
invasivity (23), is also intriguing. At high concentrations, GM3
inhibits glioma cell proliferation predominantly by inhibition of
tyrosine phosphorylation of EGF, PDGF, or FGF receptors. In
our studies, no changes in cellular proliferation or in tyrosine
phosphorylation of these receptors in the transfectant clones
were observed. High concentrations of GM3 also inhibit tumor
cell invasion predominantly via interference with integrin re
cantly smaller tumors, with tumors primarily surrounding the needle-tract
ceptor function, which would also be reflected in events down
(arrows). (D) Differences in tumor maximal cross-sectional area between
stream of the receptor. The predominant integrin in U373MG
groups were determined by ANOVA followed by Fisher's PLSD post hoc test.
cells is a3?l, whose primary downstream effect is tyrosine
Ten mice per cell line were used in each group. Data are expressed as av phosphorylation of pl25FAK (5). We also did not observe ad
erage ? SEM (bars). (*** < 0.0001).
hesion-mediated alterations in pl25FAK expression in the trans
fectants. We interpret these observations to suggest that the 2
fold increase in GM3 content in the transfectants was not suffi
antibodies followed by antiphosphotyrosine Western blot analy cient to explain the differences in in vitro invasivity or inhibition
sis demonstrated a significant increase in HSPA8 phosphoryla
of in vivo tumor growth that we have demonstrated.
tion after adhesion to fibronectin (Fig. 5C). No change in Keapl
In addition to their well studied roles as epitopes/cell surface
phosphorylation was observed (Fig. S5).
markers, gangliosides participate in multiple biological functions,
including inter- and intracellular signaling functions (24). Aber
Intracranial Tumorigenicity. Intracranial tumor growth by ST6Gal rantly expressed cell surface gangliosides have been demon
NAcV-transfected U373MG (clones S8 and S25), parental
strated to directly impact intracellular signaling. Alteration of
U373MG, and pcDNA3 vector-transfected control cells was glycosylation in glycolipids affects intracellular localization of
examined after stereotactic implantation into SCID mice (Fig. 6 integrin, src, and caveolin into or out of glycolipid-enriched
A-C). Both parental and vector-transfected U373MG cells microdomains (25). The association of GD2 with the integrin/
formed large tumors in vivo. Tumors formed by the ST6GalNAcV FAK macromolecular complex has also been demonstrated (26).
transfected U373MG clones were, on average, 20-25% the size of GD3-mediated inhibition of laminin 5-dependent cell motility is
those formed by vector-transfected U373MG cells (Fig. 6D).
due to disruption of specific interactions between CD9 and oc3
Fig. 6. Intracranial tumorigenicity. Glioma cells (1.25 106) were stereo
tactically injected into the basal ganglia of SCID mice. Eight weeks later,
brains were harvested, and sections stained with H&E (A-C). The pcDNA3
vector-transfected U373MG cells (A) formed large tumors (arrow). The
ST6GalNAcV transfectants [clone S8 (B) and clone S25 (C)] formed signifi
integrin in GD3-containing microdomains (27). Ganglioside
Discussion
In this study, we have demonstrated that ST6GalNAcV over
expression in tumorigenic, human U373MG glioma cells leads to
(/) increased expression of GM2oc and GM3 gangliosides, and de
creased expression of GM lb, Gb3, and Gb4, (//) no change in De
alterations in epithelial cells leading to changes in (/) adhesion to
specific extracellular matrix components, (ii) relative rates of
cellular proliferation and apoptosis, (///) protease activation and
function, and (iv) disruption of cell surface integrimgrowth factor
receptor. associations have also been described (28). Signifi
linked sialic acid-containing glycoproteins, (///) marked inhibition cantly, GTlb-induced apoptosis in SCC12 cells by direct binding
of in vitro invasivity with no effect on cellular proliferation, (iv)
to oc5?l leads to decreased activity of the integrin-linked kinase/
modified cellular adhesion to fibronectin leading to significantly protein kinase B/AKT pathway (29). Although the detailed
increased adhesion-mediated protein tyrosine phosphorylation of molecular mechanisms of specific glycosphingolipid modulation
HSPA8 that directly correlated with decreased in vitro invasivity, on glioma invasivity may differ, it is nonetheless clear that ab
and (vi) significant inhibition of in vivo tumor growth. These results errant signal transduction plays a pivotal role.
strongly suggest that modulation of the synthesis of specific glioma
The opposing effects of ST?GalNAcV overexpression on ad
cell-surface glycosphingolipids alters adhesion and invasivity in
a manner that may play a significant role in gliomagenesis.
In mice, ST6GalNAcV encodes an enzyme catalyzing the for
mation of GDI a, a relatively minor brain-specific ganglioside in
the "0" biosynthetic pathway (21). The lack of GDloc and the
elevated expression of GM2a in the U373MG stable transfectants
described here is unexpected and may result from compen
satory mechanisms after long-term ST?GalNAcV expression. In
humans, the "a" and "b" biosynthetic pathways are far more ac
tive than the "0" pathway, which would result in synthesis of GTla
hesion to laminin vs. fibronectin are seemingly paradoxical. Up
regulated expression of ST?GalNAcV may result in signal
transduction cascades that culminate in different membrane
protein expression profiles that, on one hand, are favorable to
adhesion to laminin, and on the other hand, not favorable to
adhesion to fibronectin. In support of this hypothesis, we did not
measure any significant changes in signal transduction (measured
at the level of laminin-mediated tyrosine phosphorylation) in the
transfectants. Another possibility is that the effects on the
receptors responsible for adhesion to different substrates may be
from GDla and GQlb from GTlb. Because we did not detect any differentially susceptible to glycolipid-associated alterations in
of these products in our lipidomic analysis, it may be more likely membrane microdomains in the transfectants. Altered glioma
that the synthesis of GM2a results from altered substrate avail cell adhesion to laminin has been demonstrated not to be suffi
cient to affect invasion (30).
ability. Recent studies in GM3-deficient fibroblasts have dem
Kroes et al. PNAS | July 13, 2010 | voi. 107 | no. 28 | 12649
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UU
(
s
Unlike the effects of adhesion to fibronectin on protein tyrosine
phosphorylation in glioma cells expressing the glycoprotein
ST?Gall (5), no decrease in pl25F phosphorylation was ob
served in these studies. There was, however, a direct relationship
between the level of overexpression of ST?GalNAcV and the level
of protein tyrosine phosphorylation of a unique 75-kDa (p75)
protein after adhesion to fibronectin. Detailed phosphoproteomic
analysis demonstrated that overexpression of ST?GalNAcV leads
to enhanced de novo phosphorylation of HSPA8/HSC70 in re
sponse to attachment to fibronectin. We hypothesize that this
Methods
Cell Culture and Stable Transfection. Cells were maintained exactly as de
scribed (5). The 1.3-kb protein coding region of the human ST6GalNAcV
gene (UnigenelD: 181819), subcloned into the pcDNA3 expression vector,
was transfected into human U373MG glioma cells, and G418-resistant
transfectants screened for ST6GalNAcV expression by qRT-PCR.
Immunohistochemistry. Cell surface a2,6-linked sialoglycoconjugate expres
sion was confirmed by using fluorescein isothiocyanate (FITC)-conjugated
Sambucus nigra agglutinin (SNA) (Matreya) exactly as described (5). SNA
phosphorylation is caused by the expression of GM2oc ganglioside
recognizes epitopes containing a2,6-linked sialic acid linked to either Gal or
GalNAc (45).
synthesized by this enzyme and plays a key role in regulating al
tered adhesivity. It seems reasonable to generalize that these data
imply that ganglioside signal transduction is an important part of SNA Lectin Blot Analysis. Expression of cell-surface glycoproteins was evalu
the mechanisms that underlie glioma invasivity.
ated by SNA lectin blot analysis, as described (5), by using the DIG Glycan
Further supporting this hypothesis, it has been reported that Differentiation Kit (Roche Molecular Biochemical), exactly as per the
HSPA8 is colocalized with cytoskeletal-associated proteins in the manufacturer's recommendations.
extended pseudopodial protrusions of highly invasive variants of
canine kidney tumor cells (31). There is increasing evidence that nLC-MS Detection of Polar Lipids. Polar lipids were extracted from cells and
phosphorylation of cell-surface-expressed heat shock proteins
(HSPs) are intimately involved in tumor cell adhesion, motility,
and invasion (32, 33). The effect of specific tyrosine phosphory
lation of HSPA8 in mitotic and tumor cells (34) on cellular lo
calization and function (35) has been demonstrated. In human
analyzed by nLC-MS as described (18, 19) and detailed in SI Methods. Data
dependent MS/MS was performed in the linear quadrupole ion trap (CID)
during collection of the ICR time-domain data. Mean fold change was cal
culated by direct comparison of average ion signal magnitude in trans
expression is associated with decreased glioma cell migration
fectants vs. control cells, the combined data representing different ceramide
chain compositions. To differentiate between Gb4/iGb4 isomers, EID exper
iments were also performed as detailed in SI Methods.
phosphorylation has been demonstrated in integrin-mediated
Invasion Assay. In vitro invasivity was examined by using Biocoat Matrigel
D54MG glioma cells, siRNA-mediated knockdown of HSPA8
through vitronectin-coated membranes (36). Alteration in HSPA8
platelet adhesion to collagen (37). The authors proposed that heat
Invasion Chambers, exactly as described (5).
shock proteins, including HSPA8, act as signaling scaffolds regu
lating platelet adhesion and spreading (38). HSPA8 is coexpressed Cell Adhesion Assay. Cell adhesion to human fibronectin, laminin, and col
with, and anchored by, cytoskeletal ?-actin on the cell surface of
lagen type I was measured, also as described (5).
HTLV-susceptible cells and is an integral part of a complex
supported by palmitoyl (16:0)-oleoyl (18:l)-phosphatidylglycerol Adhesion-Mediated Protein Tyrosine Phosphorylation. Cells were incubated on
in the lipid bilayer (39). The N-terminal ATPase-containing do fibronectin-coated plates, unattached cells were removed, and Western blot
main of HSPA8 involved in uncoating of clathrin-coated vesicles analysis was performed by using a HRP-conjugated antiphosphotyrosine
has been shown to directly bind to cell surface 3'-sulfogalactoli antibody (clone 4G10; Millipore) performed as described (5).
pids, sulfogalactosyl ceramide, and sulfogalactoglycerolipid (40).
Because membrane localization of HSPs appears to be restricted Phosphoproteomic Analysis. After adhesion to fibronectin as above, 300 pg of
to lipid rafts/glycosynapses on transformed cells (41) and is spe
cell lysate protein was fractionated on an Agilent 3100 OFFGel Electropho
cifically associated with Gb3 in colon and pancreatic tumor
resis Apparatus by using 24-cm IPG strips, pi 3-10 (GE Healthcare) in urea,
cells (42), perhaps the differential phospho-HSPA8-mediated
thiourea, DTT, glycerol, and the corresponding GE ampholine under con
signal transduction modulated by specific membrane ganglioside ditions recommended by Agilent. The buffer in each well, containing fo
content provides the scaffold for signal transduction cascades
cused proteins, was recovered for Western blot analyses to identify
in glial tumors.
appropriate phosphoprotein-containing fractions by using an anti
Hakomori (43, 44) has discussed models of how altered mem
phosphotyrosine antibody. Protein identification in the corresponding band,
brane glycosphingolipids (GSL) can influence the tumorigenic excised from Coomassie-stained sister gels, was performed by nanoLC-MS/
phenotype, based on their ability to form clusters at the cell surface
MS of tryptic fragments, as described (46) on a Thermo Instruments LTQ-FT
functional membrane proteins, including integrins, growth factor
Immunoprecipitation. After adhesion to fibronectin-coated plates, cells were
through this framework that modulation of cell surface carbohy
tured, and analyzed by Western blot analysis using antiphosphotyrosine
antibody, as above.
in cellular phenotype.
From the "glycosynapse" perspective, our results can be sum
marized in the following way: Manipulation of the synthesis of
Intracranial Tumorigenicity. Intracranial tumorigenicity of ST?GalNAcV-,
pcDNA3 vector-transfected cells, and parental U373 MG cells was examined in
SCID mice (C.B-17 scid/scid, 6 wk of age; Charles River Laboratories), as de
the composition of specific membrane domains. Alteration in the
animal facility of Northwestern University, Evanston, IL, and approved by
their Institutional Animal Care and Use Committee.
and interact with various functional components on the cell
equipped with a Dionex Ultimate 3000 2D microcapillary HPLC system.
membrane. These clusters (or "glycosynapses") interact with such
receptors, tetraspanins, and nonreceptor cytoplasmic protein lysed in RIPA buffer and 200 pg of precleared lysate protein was incubated
kinases (e.g., src kinases and small G proteins) that ultimately with 5 pg/mL antibody to HSPA8 (1B5; AbCam). After incubation with Protein
control GSL-modulated cell adhesion, growth, and motility. It is A/G Plus Agarose (Santa Cruz Biotechnology), beads were washed, dena
drate expression and subsequent disorganization of cell mem
brane components can mediate cell signaling, leading to changes
cell surface gangliosides by ST?GalNAcV gene transfer alters
composition of specific membrane domains, in turn, alters ad
hesivity to ECM components, increases the phosphorylation of
HSPA8 protein, and decreases the invasive potential and in vivo
tumorigenicity of glioma cells. Based on this and previous studies
(3, 5, 6), modulating the expression of cc2,6-sialic acids on either
gangliosides or the N-linked Oligosaccharides expressed on cell
surface signaling molecules (i.e., integrins) by modulating glyco
gene expression may have therapeutic potential for the treat
ment of malignant gliomas.
scribed (6) and detailed in SI Methods. All mice were maintained in the
ACKNOWLEDGMENTS. We thank Mary Schmidt and Dr. Jeffrey Burgdorf for
expert technical assistance. Proteomics and informatics services Were pro
vided by the CBC-UIC Research Resources Center Proteomics and Informatics
Services Facility established by a grant from The Searle Funds at the Chicago
Community Trust to the Chicago Biomedicai Consortium. This research was
supported by a grant from the Falk Foundation (Chicago) (to J.R.M),
National Science Foundation Grant DMR 0654118, and a grant from the
State of Florida.
12650 I www.pnas.org/cgi/doi/10.1073/pnas.0909862107 Kroes et al.
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Kroes et al. PNAS I Ju|y 13? 2010 I '? 107 I no- 28 I 12651
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