Báo cáo khoa học: Tumor suppressor p16INK4a: Downregulation of galectin-3, an endogenous competitor of the pro-anoikis effector galectin-1, in a pancreatic carcinoma model pptx

Tumor suppressor p16
INK4a
: Downregulation of galectin-3,
an endogenous competitor of the pro-anoikis effector
galectin-1, in a pancreatic carcinoma model
Hugo Sanchez-Ruderisch
1,
*, Christian Fischer
1
, Katharina M. Detjen
1
, Martina Welzel
1
,
Anja Wimmel
1
, Joachim C. Manning
2
, Sabine Andre
´
2
and Hans-Joachim Gabius
2
1 Medizinische Klinik m.S. Hepatologie und Gastroenterologie, Charite
´
-Universita
¨
tsmedizin Berlin, Germany
2 Institut fu
¨
r Physiologische Chemie, Ludwig-Maximilians-Universita

¨
tMu
¨
nchen, Germany
Introduction
The tumor suppressor p16
INK4a
, a frequent target for
deletion mutations underlying carcinogenesis, is known
as a binding partner of cyclin-dependent kinases
CDK4 and CDK6, interfering with their association
with D-type cyclins [1,2]. Emerging evidence broadens
the spectrum of p16
INK4a
functionality beyond cell-
cycle control. In fact, recent experiments have unrav-
eled an effect on distinct aspects of gene expression.
One functional consequence is to restore the suscepti-
bility of tumor cells to anoikis (the category of apopto-
sis caused by inadequate or inappropriate cell–matrix
contacts). In detail, enhanced production of the
a
5
-integrin subunit and also an increased cell-surface
presence of a
5
b
1
-integrin (the fibronectin receptor)
were detected in p16

INK4a
-restituted Capan-1 pancre-
atic carcinoma cells [3]. Routing of this glycoprotein
and the levels of its cell-surface presentation, binding
activity and capacity for downstream signaling may, in
principle, depend not only on the protein part, but
also on its glycosylation. Because the fibronectin recep-
tor is heavily glycosylated with 26 sites for N-glycosyl-
ation [4] and variations in the structures of glycan
Keywords
anoikis; galectin; glycosylation; integrin;
pancreatic carcinoma
Correspondence
K. M. Detjen, Charite
´
-Universita
¨
tsmedizin
Berlin, Campus Virchow-Klinikum, Med.
Klinik m.S. Hepatologie und
Gastroenterologie, Augustenburger Platz 1,
D-13353 Berlin, Germany
Fax: +49 30 450 559939
Tel: +49 30 450 559679
E-mail:
*Present address
Center for Cardiovascular Research, Charite
´
-
Universita

¨
tsmedizin Berlin, Germany
(Received 20 May 2010, revised 1 July
2010, accepted 5 July 2010)
doi:10.1111/j.1742-4658.2010.07764.x
The tumor suppressor p16
INK4a
has functions beyond cell-cycle control via
cyclin-dependent kinases. A coordinated remodeling of N- and O-glycosyla-
tion, and an increase in the presentation of the endogenous lectin galectin-
1 sensing these changes on the surface of p16
INK4a
-expressing pancreatic
carcinoma cells (Capan-1), lead to potent pro-anoikis signals. We show
that the p16
INK4a
-dependent impact on growth-regulatory lectins is not lim-
ited to galectin-1, but also concerns galectin-3. By monitoring its expression
in relation to p16
INK4a
status, as well as running anoikis assays with galec-
tin-3 and cell transfectants with up- or downregulated lectin expression, a
negative correlation between anoikis and the presence of this lectin was
established. Nuclear run-off and northern blotting experiments revealed an
effect of the presence of p16
INK4a
on steady-state levels of galectin-3-spe-
cific mRNA that differed from decreasing the transcriptional rate. On the
cell surface, galectin-3 interferes with galectin-1, which initiates signaling
toward its pro-anoikis activity via caspase-8 activation. The detected oppo-

site effects of p16
INK4a
at the levels of growth-regulatory galectins-1 and -3
shift the status markedly towards the galectin-1-dependent pro-anoikis
activity. A previously undescribed orchestrated fine-tuning of this effector
system by a tumor suppressor is discovered.
Abbreviations
Gal-1, galectin-1; Gal-3, galectin-3; PCNA, proliferating cell nuclear antigen; poly-HEMA, poly(2-hydroxyethyl methacrylate).
3552 FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS
chains, such as status of sialylation, have a bearing on
protein functions in general and on integrins in partic-
ular [5–8], glycan remodeling affords an attractive level
of regulation of integrin functionality. The assumption
of a modulatory impact of p16
INK4a
at the level of gly-
cosylation was a reasonable and testable hypothesis.
This hypothesis was verified by combining glycogene
microarray analysis, chromatographic glycan profiling
and lectin binding [9]. Influencing N- and O-glycan
galactosylation and sialylation, salient biochemical sig-
nals in protein–carbohydrate ⁄ protein interplay [10–12],
have thus become a new aspect of p16
INK4a
functional-
ity. Hereby, the tumor suppressor can affect integrin
processing and routing, as well as its affinity for
protein ligands.
In addition, modified glycan chains can be engaged
in recognitive interactions at the cell surface with

endogenous lectins, which translate sugar-encoded
messages into cellular responses [13–15]. Even seem-
ingly small modifications can act as potent switches of
affinity. In fact, altering branch-end positions, the sta-
tus of core substitutions and glycan density strongly
affects lectin reactivity, as measured for example for
members of the family of adhesion ⁄ growth-regulatory
galectins [16–19]. Fittingly for a functional implication,
microarray analysis on a set of 1996 cancer-associated
genes, proteomic profiling and cytofluorometry and
anoikis assays revealed the upregulation of homo-
dimeric galectin-1 (Gal-1) as a carbohydrate-binding
effector for anoikis induction under the control of
p16
INK4a
[9]. Thus, this tumor suppressor sensitizes
cells for the onset of anoikis by increasing the comple-
mentary sides of pro-anoikis protein–carbohydrate
interactions.
As the term Gal-1 implies, the functionality of this
protein might be embedded in a network with other
family members [20]. This raises the possibility of addi-
tive or antagonistic effects. Exploring this issue, model
studies on SK-N-MC neuroblastoma cells illustrated
the potential of other galectins, especially galectin-3
(Gal-3), to factor in growth inhibition by Gal-1 at the
level of cell-surface ligand binding [21,22]. Having
recently delineated a direct connection between
p16
INK4a

and Gal-1 [9], the question arises as to
whether the influence of p16
INK4a
is restricted to this
family member. The following four lines of evidence
direct the focus of attention on Gal-3. First, it is func-
tionally antagonistic to Gal-1 in the neuroblastoma
system by blocking access to ganglioside GM1, the
common galectin ligand on the cell surface in these
cells [21]. Second, activating K-ras mutations are com-
mon in pancreatic cancer, and Gal-3 (but not Gal-1)
interacts with oncogenic K-ras–GTP to promote Raf-1
and phosphoinositide 3-kinase activities, as well as a
signal attenuating extracellular signal-regulated kinase
[23]. This is in accordance with the lack of aberrantly
enhanced extracellular signal-regulated kinase signaling
in pancreatic tumors which harbor the mentioned gene
defect [24]. Third, Gal-3 protects BT549 breast cancer
cells from anoikis [25]. Fourth, Capan-1 wild-type cells
are known to express Gal-3 [26]. On this basis, we
investigated the influence of the presence of p16
INK4a
on the level of Gal-3 production and possible func-
tional competition between galectins-1 and -3. The
reported results document the relevance of Gal-3 in
interfering with anoikis induction and, more impor-
tantly, shed light on the intriguing capacity of this
tumor suppressor to coordinately exploit this aspect of
the galectin network.
Results

Expression of p16
INK4a
decreases the level of
Gal-3
In order to delineate any impact of the tumor suppres-
sor p16
INK4a
on Gal-3 expression we worked with
Capan-1 pancreatic carcinoma wild-type and vector-
transfected cells (mock) as well as three independently
generated clones stably transfected with p16
INK4a
tumor
suppressor cDNA (p16 1-3). Of note, these clones had
been studied previously, revealing increased expression
and cell-surface presentation of the a
5
-integrin subunit
and Gal-1 [3,9]. Western blot analyses first resulted in
the expected pattern for the p16
INK4a
protein, then con-
firmed the reported presence of Gal-3 in Capan-1 wild-
type cells [26] and excluded an effect of the control
transfection on Gal-3 and general protein synthesis
(Fig. 1A). In contrast to the mock process, the presence
of p16
INK4a
protein had an effect. A clear decrease in
the intensity of signals for Gal-3 was invariably

observed, although proliferating cell nuclear antigen
(PCNA) staining as a control for loading remained
rather constant (Fig. 1A,B). To examine whether such a
negative correlation between Gal-3 and p16
INK4a
can
also be seen in vivo, we processed routinely fixed sec-
tions from normal pancreas tissue and pancreatic can-
cer immunohistochemically. The resulting staining
profiles, shown in Fig. S1, confirmed an inverse expres-
sion pattern in accordance with the in vitro data. In
principle, Gal-3 can act as an anti-anoikis effector in
the cell and ⁄ or at the cell surface, for example, blocking
Gal-1 functionally as seen previously in a neuroblas-
toma model [21]. In view of the proven pro-anoikis
cell-surface activity of Gal-1, the cultured cells provided
the opportunity to test the hypothesis of reduced
H. Sanchez-Ruderisch et al. p16
INK4a
downregulates anti-anoikis effector Gal-3
FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS 3553
cell-surface expression of Gal-3. Indeed, cytofluorimet-
ric monitoring of mock-transfected and p16
INK4a
-resti-
tuted Capan-1 cells revealed that cell-surface Gal-3 was
downregulated (Fig. 1C). As reported previously [9],
Gal-1 cell-surface presentation determined in the con-
trol increased (not shown). The presence of the tumor
suppressor thus reduced the level of Gal-3 and its cell-

surface presentation. To further test whether Gal-3 pro-
tein abundance was regulated by prolonged culture in
suspension, we maintained cells for up to 24 h under
this condition, with the percentage of cells undergoing
anoikis increasing as expected in p16
INK4a
-expresssing
cells (Fig. 2A). Loss of anchorage did not notably
change the level of Gal-3 detected in western blots
(Fig. 2B). These results document a significant decrease
in the presence of Gal-3 in tumor-suppressor-positive
cells, which is not further enhanced in suspension cul-
ture. This suggests regulation at the level of transcrip-
tion, as previously detected for Gal-1 [9]. To test this,
we determined the steady-state mRNA concentration
by northern blotting and the de novo transcription rate
by run-off assays.
p16
INK4a
negatively affects Gal-3 mRNA
availability
Northern blotting using samples from p16
INK4a
-positive
cells revealed a conspicuous decrease in the availability
of Gal-3-specific mRNA, when compared with mock-
transfected controls, whereas similar mRNA levels for
the housekeeping gene GAPDH were present irrespec-
tive of cell status (Fig. 2C, left). This diminished steady-
state amount may be because of alterations in de novo

production or availability. Nuclear run-off assays,
which report on de novo synthesis, revealed rather simi-
lar signal intensities in samples from mock- and
p16
INK4a
-transfected cells for the two tested housekeep-
ing genes and for Gal-3 (Fig. 2C, right). De novo Gal-1
gene transcription, by contrast, increased significantly in
the presence of the tumor suppressor (not shown). Thus,
the transcriptional rate is a major factor in increasing
the production of Gal-1, whereas post-transcriptional
processes decrease Gal-3 production. We propose a
functional relevance for this alteration. In order to
prove an effect of Gal-3 on anoikis in this cell system,
especially regarding a functional antagonism with Gal-1
at the level of the cell surface, we followed three routes
of investigation to test the validity of this assumption.
Gal-3 is an inhibitor of anoikis
In the first set of experiments, we tested the capacity
of Gal-3 to block anoikis in p16
INK4a
-expressing cells
1000
1000
10
0
10
1
10
2

10
3
10
4
10
0
10
1
10
2
10
3
10
4
A
B
C
Fig. 1. p16
INK4a
restitution inhibits Gal-3 expression. (A) Western
blot after 1D SDS ⁄ PAGE [15% gel, proteins blotted onto poly(vinyli-
dene fluoride)] with protein extracts from Capan-1 wild-type (wt),
mock-transfected and three p16
INK4a
-expressing clones incubated
with antibodies to p16
INK4a
, Gal-3 and PCNA, respectively. The level
of Gal-3 is reduced in p16
INK4a

-expressing clones. (B) Western blot
after 2D gel electrophoresis with protein extract from mock-trans-
fected (1,2: 200, 400 lg) and p16
INK4a
-transfected cells (3,4: 200,
400 lg). Residual protein in gels was visualized by silver staining
(lower part of each panel) and each blotting procedure included a
positive control with Gal-3 (top right). (C) Quantitation of cell-sur-
face presentation of Gal-3 in mock-transfected (left) and p16
INK4a
-
expressing Capan-1 pancreatic cancer cells (right). The control for
antigen-independent staining by omitting the incubation step with
the lectin-specific antibody from the protocol is given in each panel
(gray area), as are the percentage of positive cells and the mean
fluorescence intensity of staining when incubating with 20 lgÆmL
)1
non-cross-reactive anti-galectin-3 Ig. Standard deviation did not
exceed 11% in experimental series with four different experiments
run in triplicates.
p16
INK4a
downregulates anti-anoikis effector Gal-3 H. Sanchez-Ruderisch et al.
3554 FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS
when added to the medium. As shown in Fig. 3, expo-
sure of cells to Gal-3 was inhibitory in this experimen-
tal setting. The percentage of p16
INK4a
-positive cells
undergoing anoikis after 20 h in suspension was

reduced when kept in the presence of suitable concen-
trations of Gal-3. To further strengthen the link
between Gal-3 and anoikis, the availability of this
lectin was upregulated deliberately by generating
p16
INK4
-positive cells, which additionally express Gal-3
at a high level via a second transfection. Thus, the
presence of Gal-3 was increased, counteracting the
p16
INK4a
-dependent downregulation. A series of five
clones was obtained with notably enhanced Gal-3 con-
centration, an unspecific influence of the second trans-
fection step rendered unlikely by a mock control
(Fig. 4A). The level of anoikis in these control cells
was used as a reference, and four Gal-3-overexpressing
clones revealed a trend towards reduced anoikis sus-
ceptibility. This reached statistical significance in clone
Gal-3 ⁄ 2 (Fig. 4B). Results showing that exogenous
addition and vector-directed overexpression of Gal-3
can reduce the level of anoikis shaped the notion that
Gal-3 physically hampers the induction of this cell
death program. In this case, even p16
INK4a
-negative
cells should become sensitized toward anoikis if their
Gal-3 production is downregulated. This reasoning led
to the third set of experiments.
To test the given hypothesis, we generated clones

harboring an antisense vector for Gal-3. Because of
this engineering, the clones contained a lower level of
Gal-3 than wild-type and mock-transfected cell popula-
tions (Fig. 4C). Measurements of the corresponding
cell-cycle profiles and percentages of cells undergoing
anoikis revealed a Gal-3-dependent increase in this
200100 15050080
120160 200
Counts
400
80
120 160
400
0 102030405060
020406080
0 200
0 200 400 600 0 200 400 600 0 200 400 600
400
FL2-H
5121231
5
A
B
C
56 6
FL2-H FL2-H FL2-H
FL2-H
Mock
p16-3
0Time (h) 8 16 24

FL2-H FL2-H FL2-H
600
0 200 400 600 0 200 400 600
0 200 400 600
0
200
400 600
200100 150500
30 60 90120 1500
30 60 90 1200
Fig. 2. The level of Gal-3 is diminished in p16
INK4a
-expressing clones but remains constant over the 24 h anoikis induction period. (A) Repre-
sentative FACS histograms illustrating the increased extent of anoikis induction in Capan-1 ⁄ p16
INK4a
-positive cells compared with a mock-
transfected clone. Cells were harvested following incubation on poly-HEMA to preclude attachment and trigger anoikis for the indicated
time. The given numbers indicate the percentage of cells with subdiploid DNA content (pre-G1 fraction). (B) Western blot analysis from
extracts obtained under the conditions described above and analyzed with anti-Gal-3 or anti-PCNA Ig as indicated. (C) Poly-(A
+
) RNAs were
isolated from mock-transfected and p16
INK4a
-expressing clones and northern blots were probed for the presence of mRNA specific for Gal-3
or GAPDH (left). In vitro elongation of de novo RNA transcripts was performed in isolated nuclei from mock-transfected and p16
INK4a
-
expressing clones in the presence of [
32
P]UTP[aP], and the radioactively labeled RNA was hybridized to immobilized cDNA for Gal-3, GAPDH

and b-actin (right). One representative of three experiments that yielded similar results is shown.
H. Sanchez-Ruderisch et al. p16
INK4a
downregulates anti-anoikis effector Gal-3
FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS 3555
parameter (Fig. 4D,E). Evidently, the level of Gal-3 is
important to protect wild-type cells from anoikis
induction, and the forced expression of Gal-3 in
p16
INK4a
-positive cells reduces their susceptibility to
anoikis.
Because this pattern is inverse to the Gal-1-induced
effects reported previously [9], it is reasonable to pro-
pose functional competition between galectins-1 and -3
at the cell surface. To probe for such an effect, we
stimulated anoikis by addition of Gal-1 to the culture
medium. Should Gal-3 be an inhibitor, the extent of
Gal-1-dependent anoikis induction via glycan binding
will decrease. Stepwise increases in the Gal-3 concen-
tration progressively diminished the Gal-1-dependent
effect (Fig. 5A), and galectin binding, shown to be
carbohydrate dependent [9], was reduced in cross-com-
petition assays (Fig. 5B,C). The presence of Gal-3 can
thus impair the pro-anoikis effect of Gal-1 at the level
of the cell surface, here most likely targeting the
fibronectin receptor as had been shown in this and
other carcinoma cell systems [9,27]. If Gal-3 interferes
with Gal-1 binding to and cross-linking the a
5

-subunit,
then Gal-3 should also negatively affect post-binding
signaling by the integrin. We tested and established the
involvement of caspase-8 activation for Gal-1-depen-
dent anoikis induction (Fig. 6A) and revealed a nega-
tive impact of Gal-3 at this level (Fig. 6B,C).
Discussion
The term ‘tumor suppressor’ summarizes an obvious
function of a protein on malignancy. Naturally, a sup-
pressor engages secondary effectors at different levels,
which turn its presence as a master organizer into ren-
ormalization of phenotypic characteristics. Because of
growing insights into the role of glycosylation in cellu-
lar communication, including growth control [6,28], we
hypothesized that p16
INK4a
is capable of taking advan-
tage of this network, explicitly by modulating the
lectin reactivity of glycans and ⁄ or lectin expression. As
a consequence, further study provided a novel explana-
tion for why this tumor suppressor restores susceptibil-
ity to anoikis in Capan-1 pancreatic carcinoma cells
[9]. Having detected coordinated upregulation of both
Gal-1 and suitable cell-surface ligands, the results
provoked a question regarding orchestration of effects
on expression in the lectin family beyond Gal-1. Evi-
dently, the chimera-type Gal-3, known as a functional
antagonist of Gal-1 in a tumor model, offered a prime
target for study.
Cumulatively, the experiments reported herein

revealed p16
INK4a
-dependent regulation of the presence
of Gal-3 in this cell system. They also delineated a
strong influence of Gal-3 on anoikis induction. Of spe-
cial note, we detected functional competition at the cell
surface with the recently proven pro-anoikis activity of
Gal-1, which involves caspase-8 activation. Combining
previous results on Gal-1 ⁄ a
5
b
1
-integrin co-immuno-
precipitation, and the effects of p16
INK4a
on a
5
b
1
-
integrin, cell-surface glycosylation and Gal-1 [3,9] with
the presented data enabled us to set up a scheme of
p16
INK4a
-orchestrated changes that favor Gal-1-depen-
dent anoikis (Fig. 7). Whether and how a reduction in
the intracellular activities of Gal-3 documented in
other tumor cell types (e.g. interaction with oncogenic
K-ras and transcriptional modulation of cell-cycle reg-
ulators such as cyclins A, D

1
and E as well as p21 ⁄ p27
[23,25,29]) will cooperate with the functional interfer-
ence of cell-surface binding of homodimeric Gal-1, is
not clear. At the cell surface, the particular profile of
the chimera-type galectin for ligand cross-linking
shown recently [30] will definitely not elicit pro-anoikis
0
0
0 40 80 120 160
20 40 60 80
200 400
39
12
DNA
No Gal-3
Counts
+ 125 µg·mL
–1
Gal-3
DNA
0 200 400
A
B
Fig. 3. Addition of Gal-3 to the culture medium inhibits anoikis
induction. (A) Gal-3 was added to p16
INK4a
-expressing cells (clone
p16-3) in the concentrations given. Following 20 h incubation on
poly-HEMA, anoikis rates were determined from DNA histograms

based on the fraction of cells with subdiploid DNA content. The
data are expressed as percentage of the control without the addi-
tion of Gal-3 (n =3,**P < 0.01, ***P < 0.005). (B) Representative
DNA histograms in the absence (no Gal-3) or in the presence of
125 lgÆmL
)1
Gal-3. The given numbers indicate the percentage of
cells in the pre-G1 fraction.
p16
INK4a
downregulates anti-anoikis effector Gal-3 H. Sanchez-Ruderisch et al.
3556 FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS
signaling of Gal-1 in this cell system. This functional
competition between galectins, described initially for
SK-N-MC neuroblastoma cells and ganglioside GM1
[21], may well play a more general role in tumor
growth control. Its detection gives a clear direction to
further research. In this respect, such a study is
warranted on the p27-dependent downregulation of
carcinoma cell growth triggered by Gal-1 and most
likely the a
5
b
1
-integrin [27], as well as the Gal-1 ⁄ GM1-
dependent communication between effector T and reg-
ulatory T cells, which involves a
4
⁄ a
5

b
1
-integrins and
Ca
2+
influx via TRPC5 channels [31]. Eventually,
these investigations will unravel the mostly unexplored
intricacies of the galectin network monitored in tumors
by RT-PCR and by immunohistochemistry [32–34].
Such cell biological studies will then shed light on
additive ⁄ synergistic, as opposed to antagonistic, activi-
ties in malignancy and immune regulation.
0
0
02040
60 80 100 120
0
0306090120150
20 40
60 80 100 120
30 60 90
Counts
120
200
DNA
Wt
9101722
Mock asG3-A asG3-B
DNA DNA
DNA

400 0 200 400 0 200 400
0200400
A
B
C
E
D
Fig. 4. Upregulation of Gal-3 in clones changes anoikis susceptibility. (A,B) Overexpression of Gal-3 protects p16
INK4a
-expressing clones from
anoikis. (A) Increased Gal-3 content was confirmed via detection of Gal-3 in western blots of clones that were stably transfected with a Gal-
3 expression construct. Blots were conducted on whole-cell lysates of Capan-1 wild-type cells (wt), a p16
INK4a
-expressing clone (p16) and
p16
INK4a
-positive clones following mock transfection and with additional overexpression of Gal-3 (p16 ⁄ Mock and Gal-3 ⁄ 1-5). Blots were
probed with anti-Gal-3 or anti-PCNA Ig. (B) Determination of anoikis level in five p16
INK4a
-positive clones with overexpression of Gal-3 and a
respective mock control. Extent of anoikis is expressed as a percentage relative to the mock-transfected control clone (n =3,*P < 0.05).
(C–E) Reduction in the level of endogenous Gal-3 stimulates anoikis induction. (C) Downregulation of cellular Gal-3 was ascertained by detec-
tion of Gal-3 in western blot analyses of protein extracts from wild-type (wt) and mock-transfected cells, as well as from two clones trans-
fected with an antisense (as) Gal-3 cDNA construct (asG3A ⁄ B). Additional immunoblotting for PCNA (lower) was conducted to control for
unspecific effects on protein synthesis. (D) Determination of level of anoikis in clones with reduced Gal-3. Summary of anoikis rates obtained
in clones with decreased Gal-3 and mock controls. Anoikis rates are given as a percentage of the total number of cells (n =3,*P < 0.05,
**P < 0.01). (E) Representative cell-cycle histograms from cultures kept on poly-HEMA for 20 h. The given numbers indicate the percentage
of cells in the pre-G1 fraction.
H. Sanchez-Ruderisch et al. p16
INK4a

downregulates anti-anoikis effector Gal-3
FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS 3557
Equally important, our study broadens the basis for
galectin involvement in tumor suppressor activity.
Here, the key reference point to date had been p53. In
detail, SAGE screening on DLD-1 colon carcinoma
cells expressing p53 identified galectin-7 as a
p53-induced gene 1 in a group of 14 genes markedly
upregulated from a total of 7202 tested transcripts
[35]. Along this line, genotoxic stress by UVB irradia-
tion of human keratinocytes afforded a second system
in which a connection between p53 and galectin-7 was
200
A
B
C
**
**
*
*
#
##
150
50
Gal-3 binding (log fluorescence)
Gal-1 bindin
g
(lo
g
fluorescence)

40
10
0
10
0
10
1
10
2
10
1
10
2
80 1200
40 80 1200
Control
Gal-1 (100 µg·mL
–1
)
Gal-1 + Gal-3 (25 µg·mL
–1
)
Gal-1 + Gal-3 (50 µg·mL
–1
)
Gal-1 + Gal-3 (100 µg·mL
–1
)
Gal-1 + Gal-3 (150 µg·mL
–1

)
0
100
Anoikis (% control)
CountsCounts
Fig. 5. Gal-3 inhibits Gal-1-stimulated anoikis and Gal-1 binding.
(A) Determination of Gal-1-stimulated (100 lgÆmL
)1
) anoikis in
p16
INK4a
-restituted cells in the presence of increasing concentra-
tions of Gal-3. Anoikis rates were determined following 20 h of
culture on poly-HEMA and were calculated based on the pre-G1
fraction from cell-cycle analyses. Data are expressed as the per-
centage of vehicle-treated mock control cells (n =4, *P < 0.05,
**P < 0.01, compared with control without Gal-1; #P < 0.05,
##P < 0.01 compared with data for Gal-1). (B) Binding of biotiny-
lated Gal-3 in the presence (light line) or absence (bold line) of
100 lgÆmL
)1
Gal-1. The dashed line signifies the control with
fluorescent reagent, without prior incubation with biotinylated
Gal-3. (C) Binding of biotinylated Gal-1 in the presence (light line)
or absence (bold line) of 150 lgÆmL
)1
Gal-3. The dashed line sig-
nifies the control.
A
Control

40
30
20
10
0
616
Time (h)
Apoptotic cells (%)
24
FAM-LETD-FMK
*
B
C
Fig. 6. Gal-3 affects the onset of anoikis by inhibiting Gal-1-depen-
dent caspase-8 activation. (A) Anoikis after 6, 12 or 24 h incubation
on poly-HEMA in the presence or absence of the caspase 8 inhibi-
tor FAM-LETD-FMK. (B) Caspase 8 activation expressed as the per-
centage of cells with active caspase 8 in the absence (control, filled
circles) and presence (open circles) of 100 lgÆmL
)1
Gal-3
(**P < 0.01, ***P < 0.001, compared with control). (C) Anoikis
(percentage of cells in the pre-G1 fraction) in the absence (control,
filled circles) and presence (open circles) of 100 lgÆmL
)1
Gal-3
(**P < 0.01, compared with control).
p16
INK4a
downregulates anti-anoikis effector Gal-3 H. Sanchez-Ruderisch et al.

3558 FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS
made likely [36]. When expressed in HeLa cell transfec-
tants, the intracellular function of galectin-7 appeared
to relate to affecting gene expression profiles. Conspic-
uous changes were attributed to pro-apoptosis signal-
ing upstream of c-Jun N-terminal kinase activation
and cytochrome c release [37]. When tested as an
extracellular effector, carbohydrate-dependent binding
led to growth inhibition of activated T cells or neuro-
blastoma cells via caspase-dependent or -independent
pathways [38,39]. Of further relevance, the recently
documented effect of compensation of loss of supres-
sor genes in microsatellite instability on glycosylation,
including a2,6-sialylation of N-glycans, broadens the
scope for co-regulation [40]. Further work to
strengthen this connection between a tumor suppressor
and lectin ⁄ glycan remodeling as an effector pathway,
especially by examining clinical samples, is clearly jus-
tified.
Examining galectin expression in clinical samples of
pancreatic cancer by gene-expression profiling uncov-
ered a consistent, albeit quantitatively variable, upreg-
ulation of Gal-3 expression [41–45]. The similarly
consistent upregulation of Gal-1 gene expression and
protein production may at first seem puzzling. Looking
at its immunohistochemical pattern, it could mostly be
accounted for by a desmoplastic reaction around the
tumor cells, with a Gal-1 ⁄ tissue plasminogen activator
interaction being involved [44–50]. Taking our data
literally, an absence or low level of Gal-1, combined

with an abundance of Gal-3 in the tumor cells, seems
to reflect impairment of p16
INK4a
. As shown in the
Supporting Information, preliminary immunohisto-
chemical analysis on a limited number of cases focus-
ing on the presence of p16
INK4a
and Gal-3 indicated a
tendency for a negative correlation, in accordance with
the in vitro data. These results encourage thorough
investigation of this aspect to strengthen clinical
relevance. By following this line of research, the
orchestration of galectin expression described here may
become instrumental in devising a novel therapeutic
strategy to rationally shift the balance between resis-
tance and susceptibility to favor the anoikis process.
Materials and methods
Galectins
Human galectins were produced by recombinant expression,
isolated by affinity chromatography on lactosylated Sepha-
rose 4B as crucial step followed by gel filtration, and tested
for purity using 1D and 2D gel electrophoresis and nano-
electrospray ionization mass spectrometry, as well as for
activity by hemagglutination [9,38,51,52]. Biotinylation
labeling was performed under activity-preserving conditions
using the commercial N-hydroxysuccinimide ester derivative
of biotin (Sigma, Munich, Germany). Its incorporation into
the galectins was determined using a proteomics protocol,
and the activity of the labeled proteins was checked using

carbohydrate-dependent solid-phase and cell binding assays
[52,53]. Polyclonal antibodies were raised in rabbits and rig-
orously checked for lack of cross-reactivity using enzyme-
linked immunosorbent assays and western blotting [54,55].
No experimental animals were used in this study. Monitor-
ing the cell-surface presentation of galectins was carried out
by flow cytofluorometry using fluorescent goat anti-rabbit
IgG with 20 lgÆmL
)1
galectin-type-specific IgG fractions
and FACS equipment (Becton-Dickinson, Heidelberg,
Germany) [9].
Cell culture
Human Capan-1 pancreatic carcinoma cells and clones with
stable vector-directed p16
INK4a
expression were established
and cultured as described previously [3].
Fig. 7. Glycobiology of p16
INK4a
functionality in Capan-1 pancreatic
carcinoma cells in vitro. The tumor suppressor orchestrates: an
increase in the cell-surface presentation of the fibronectin receptor
(involving transcriptional upregulation of a
5
-subunit gene expres-
sion), regulation of glycogenes enabling increased Gal-1 cell-surface
reactivity, and an increase in the cell-surface presentation of Gal-1
(involving transcriptional upregulation of Gal-1 gene expression)
[3,9]. Formation of Gal-1 ⁄ a

5
b
1
-integrin complexes with ensuing
cross-linking appears to lead to anoikis induction via caspase-8
activation. Gal-3 can interfere with Gal-1 binding and ⁄ or Gal-1-
dependent cross-linking at the level of the cell surface, decreasing
the pro-anoikis activity of Gal-1. p16
INK4a
-dependent Gal-3 downre-
gulation, with potential bearing also on intracellular anti-anoikis
activity of Gal-3, favors the pro-anoikis effect of cell surface Gal-1.
H. Sanchez-Ruderisch et al. p16
INK4a
downregulates anti-anoikis effector Gal-3
FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS 3559
Antibodies
Antibodies to PCNA and p16
INK4a
were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA, USA) and
NeoMarkers (Fremont, CA, USA), respectively.
Protein extraction and western blotting
Cells were lyzed in radioimmunoprecipitation assay buffer
(50 mm Tris ⁄ HCl at pH 7.5, 0.15 m NaCl, 0.25% SDS,
0.05% sodium deoxycholate, 1% NP-40, 1 mm dithiothrei-
tol, 1 lgÆmL
)1
aprotinin, 2 mm leupeptin, 1 mm Na
3

VO
4
,
1mm NaF, 1 mm phenylmethylsulfonyl fluoride), and soni-
cated on ice. Aliquots (5–10 lg) were subjected to
SDS ⁄ PAGE and electroblotted onto poly(vinylidene fluo-
ride) membranes (NEN, Cologne, Germany). Blots were
incubated overnight at 4 °C with the respective antibodies
(diluted 1 : 1000 in 5% non-fat dried milk in phosphate
buffered saline with 0.5% Tween-20). Immunoreactive
bands were visualized by enhanced chemoluminescence
(NEN). Cell processing, blotting and signal generation
when using 2D gel electrophoresis followed the procedure
previously used to detect Gal-1 [9].
Immunohistochemistry
Immunohistochemistry was performed on cryosections with
antibodies controlled for specificity and lack of intergalectin
cross-reactivity, as described previously [3,56,57]. Briefly,
sections were fixed in 4% paraformaldehyde and primary
antibodies were applied at a dilution of 10 lgÆmL
)1
(Gal-3)
or 1.25 lgÆmL
)1
(p16
INK4a
). Immunoreactivity was detected
with biotinylated secondary antibodies and avidin–biotin–
peroxidase complex, with 3-amino-9-ethylcarbazole as the
chromogenic substrate. Sections were counterstained with

hemalaun.
Northern blot analysis
Total RNA from 10
8
cells was isolated using RNAzolÔB
(WAK-Chemie Medicals GmbH, Bad Homburg, Germany),
and the poly-(A
+
) fraction was purified using the PolyA-
Track
Ò
System 1000 (Promega, Mannheim, Germany)
according to the manufacturer’s protocol. Aliquots were
separated on a 1% agarose ⁄ 3-(N-morpholino)propanesulf-
onic acid ⁄ formaldehyde gel, blotted to Hybond N
+
filters
(Amersham Pharmacia, Freiburg, Germany) and linked to
the membrane using UV light. Full-length cDNA prepara-
tions for human Gal-3 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were labeled with [
32
P]dCTP[aP]
by random priming (Megaprime DNA labeling kit; Amer-
sham Pharmacia). Residual nucleotides were removed and
hybridization was carried out in Quick-Hyb buffer (Strata-
gene, La Jolla, CA, USA) at 65 °C for 2 h. Following
hybridization, membranes were washed at 65 °C to a strin-
gency of 0.1· NaCl ⁄ Cit, 0.1% SDS and exposed to X-ray
films at –70 °C.

Nuclear run-off
Blots were prepared using isolated and denatured cDNA
fragments immobilized on Hybond N
+
nylon membranes
(Amersham Pharmacia). The amount of denatured cDNA for
Gal-1, GAPDH and b-actin blotted was 5, 2 and 1 lgÆslot
)1
,
respectively. Nuclear RNA preparation, labeling and hybrid-
ization were performed as described previously [3]. Blots were
washed twice for 10 min at 42 °C with 40 mm NaH
2
PO
4
⁄
Na
2
HPO
4
pH 7.2, 1% SDS. Labeled de novo mRNA tran-
scripts were detected on autoradiographs.
Induction and detection of anoikis
For determination of anoikis, 2 · 10
5
cells were cultured as
suspension cultures in plates coated with poly(2-hydroxy-
ethyl methacrylate) (poly-HEMA) (Sigma, Deisenhofen,
Germany) for the indicated times. Apoptotic cells were then
quantitated from the pre-G1 fraction in cell cycle analyses

as described [3].
Stable transfection of Gal-3 sense/antisense
cDNA
Full-length cDNA obtained from amplification of human
Gal-3-specific mRNA of human DLD-1 colon carcinoma
cells in either the sense (pcDNA–Gal-3S) or antisense
(pcDNA–Gal-3AS) orientation was subcloned into the
pcDNA3.1 vector, and the EffecteneÔ Transfection Reagent
(Quiagen, Hilden, Germany) was used to generate stably
transfected cells following the manufacturer’s protocol.
Determination of binding of labeled galectins
Cells were incubated with 100 lgÆmL
)1
of biotinylated
Gal-1 and -3, washed and surface-bound probe was
detected by flow cytometry using an indocarbocyanine–
streptavidin conjugate. Fluorescence intensity was recorded
on a FACSCaliburÔ (Becton Dickinson) and analyzed with
cellquestÔ software [9,58]. Cells incubated with the
fluorescent indicator only were used to determine the back-
ground fluorescence.
Determination of caspase-8 activity
Cells with activated caspase-8 were detected using the
carboxyfluorescein-labeled derivative of the caspase-8 in-
hibitor Z-LETD-FMK (FAM-LETD-FMK) (Biocarta,
Hamburg, Germany), which irreversibly binds to activated
caspase-8. Fluorescence intensity was evaluated by flow
cytometry.
p16
INK4a

downregulates anti-anoikis effector Gal-3 H. Sanchez-Ruderisch et al.
3560 FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS
Statistical analysis
Unless indicated, unpaired Student’s t-test analyses (two-
tailed distribution, two-sample unequal variance) were per-
formed using prism software (Prism, San Diego, CA,
USA). Data were considered significant at P-values < 0.05.
Acknowledgements
This work is dedicated to Prof. Dr Stefan Rosewicz
(1960–2004), who was crucial to start this project line.
We are grateful to Drs B. Friday, G. Ippans and S.
Namirha for helpful comments, to L. Mantel for excel-
lent technical assistance as well as to the Dr Mildred
Scheel Stiftung, Sonnenfeld Stiftung, the Wilhelm-San-
der-Stiftung, LMUexcellent program, the Verein zur
Fo
¨
rderung des biologisch-technologischen Fortschritts
in der Medizin e.V. and the EC program on Marie Curie
Research Training Networks (contract no. MRTN-
CT-2005-019561) for generous financial support.
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Supporting information
The following supplementary material is available:
Fig. S1. Evidence for negative correlation between

Gal-3 and p16
INK4a
in human pancreatic tissue and
pancreatic cancer.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
H. Sanchez-Ruderisch et al. p16
INK4a
downregulates anti-anoikis effector Gal-3
FEBS Journal 277 (2010) 3552–3563 ª 2010 The Authors Journal compilation ª 2010 FEBS 3563