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: katharina.detjen@charite.de *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. 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Tumor suppressor p16 INK 4a : Downregulation of galectin-3, an endogenous competitor of the pro-anoikis effector galectin-1, in a pancreatic carcinoma model Hugo Sanchez-Ruderisch 1, *,. glycogene microarray analysis, chromatographic glycan profiling and lectin binding [9]. In uencing N- and O-glycan galactosylation and sialylation, salient biochemical sig- nals in protein–carbohydrate ⁄. 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