Purpose:

Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide–Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells.

Experimental Design:

sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling.

Results:

We found sGal-3 preferentially binds to β1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-β1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival.

Conclusions:

We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin β1–dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Translational Relevance

Tumor-selective targeting is a critical requirement for successful cancer therapeutics. A major difference between normal and cancer cells is the presence of aberrantly enhanced glycans on tumor cells. Yet, successful exploitation of augmented glycosylation in cancer for the design of new cancer therapy has remained elusive. Here, we show that an engineered chimeric form of Galectin-3 can potently induce cancer-specific cell death by targeting aberrantly enhanced N-glycans on tumor cells. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets that can be successfully targeted for therapeutic gain.

Altered glycosylation of cell surface proteins is a characteristic of cancer and leads to abnormal glycoproteins with N- and O-glycans on human tumors (1). Tumor-associated glycosylation occurs through two mechanisms: “incomplete synthesis,” which leads to truncated forms of normal glycan structures (2), and “neo-synthesis,” where abnormal cancer-specific glycans are generated through cancer-associated changes in expression of glycosyltransferases (3). Aberrantly enhanced glycosylation contributes to tumorigenesis and tumor immune evasion (4). Carcinomas show increased expression of N-acetylglucosaminyltransferase V (MGAT5), an enzyme that catalyzes the synthesis of GlcNAcβ1,6-Mannose glycan branches, leading to the formation of tetra-antennary N-glycans (5), which carry poly-N-acetyl-lactosamine, the preferred ligand for Galectin-3 (Gal-3). Consequently, there is a strong interest in exploiting aberrantly glycosylated tumor cell surface proteins as targets for cancer therapy (3), but this objective has not been realized to date (6).

Integrins are cell surface adhesion molecules composed of dimers of α and β subunits and their signaling is important for cell proliferation, survival, cytoskeletal reorganization, and migration. Upon activation, integrins undergo rapid, reversible conformational changes, which promote recruitment of intracellular signaling molecules (7). Modification of integrin extracellular N-termini through glycosylation is required for dimer formation, ligand binding, and functional activation (8–10). Alteration in N-linked glycans on cell surface integrins increases the migration and metastasis potential of several cancer types (11).

Lectins are carbohydrate-binding proteins that recognize distinct glycan moieties on glycoproteins or glycolipids through carbohydrate recognition domains (CRD). Gal-3 belongs to the β-galactoside binding galectin family, is secreted through a nonclassical secretion pathway, and is unique among galectins in that it carries a long collagen-like N-terminal domain that permits oligomerization, and for its dual role in apoptosis (12). Intracellular Gal-3 has a well-documented role as antiapoptotic factor in cytoplasm and at the mitochondrial membrane through carbohydrate-independent mechanisms (13, 14). In contrast, carbohydrate-dependent proapoptotic responses of extracellular Gal-3 have been observed at supraphysiologic concentrations (1–10 μmol/L) in lymphocytic cells (15), but not in other adherent cell types where Gal-3 regulates migration through the modulation of cell adhesion molecules and extracellular matrix (16).

Here, we sought to determine whether the weak proapoptotic activity of extracellular Gal-3 could be enhanced through a peptide conjugate approach in its N-terminus, and harnessed for targeting aberrantly glycosylated cancer cells. Peptide conjugates have been developed to selectively enhance cell surface receptor binding (17). Our study demonstrates that aberrant β1-integrin glycosylation can be exploited for tumor-specific targeting with a signal peptide conjugated Gal-3, supporting its further development for therapeutic applications.

Chemicals, cell lines, and primary cells

Recombinant human galectin-3, sugars (lactose, sucrose, and melibiose), apoptosis inhibitors [caspase-3 (Ac-DEVD-CHO), caspase-8 (Z-IETD-FMK), and caspase-9 (Z-LEHD-FMK)], glycosylation inhibitors (kifunensine for N-linked glycosylation and Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside for O-linked glycosylation), signaling pathway inhibitors (LY294002 for PI3K/Akt, Cpd22 for ILK, MDL28170 for Calpain, and Verapamil hydrochloride for calcium channels), transfection reagents (GenePorter and GeneSilencer), and glycan purification material [unconjugated Phaseolus vulgaris leucoagglutinin (PHA-L), agarose-bound PHA-L, and agarose-bound Ricinus communis agglutinin (RCA)] were purchased from commercial sources (see Supplementary Data). Human glioblastoma cell lines LN-Z308 (p53 null) parental (18); LN229 parental; clone LN229-L16 (tet-on; grown with 1 μg/mL Puromycin) (19); U87MG and SF767 (20); embryonic kidney (293HEK), breast (MCF7 and MD468), lung (A549), colon (HCT116), and prostate (LnCaP) cancer cell lines; and human foreskin fibroblasts (HFF1) were grown in DMEM supplemented with 5%–10% tetracycline-free FCS (Gibco). Primary cultures of human dermal fibroblasts (HDF), human dermal microvascular endothelial cells (HDMEC), human normal breast epithelial cells (MCF10), human astrocytes, and glioma neurospheres (N08-74) were grown as described in Supplementary Data. Cell lines were tested for Mycoplasma and their identity verified by SNP analysis.

Generation of secreted Gal-3 (sGal-3) expression vectors

The generation of sGal-3 transient (pUMVC7-Gal3) and stable vectors (pTRE2-sGal3) is described in Supplementary Data.

Transient cDNA or siRNA transfection studies

For β1 integrin knockdown, cells were transfected with β1 integrin (sc-35674 and sc-44310) or control (RNA-A; sc-37007, Santa Cruz Biotechnology) siRNAs (100 nmol/L) for 72 hours with siRNA-specific transfectant (Gene Silencer, Genlantis) after which the cells were seeded at 5,000 cells per well in 96-well plates. For sGal-3 treatment, 24 hours after cell splitting, 200 μL of 1× control or sGal-3 conditioned media (CM) were added to each well. For MGAT5 overexpression or knockdown studies, the pCXN2-MGAT5 and pSUPER-MGAT5 expression vectors were used (11, 21).

Generation of stably transfected cells

Doxycycline-inducible sGal-3–transfected clones were generated in LN229-L16 glioma cells (Tet-on clone derived from LN229; ref. 22) by transfecting them with pCMV-Neo and pTRE-sGal3 plasmids (1:10 ratio) and selecting stable clones with 1,000 μg/mL of G418. 293 cells stably expressing MGAT5 were generated as described previously (23).

Production of sGal-3

293 cells were transiently transfected using GenePORTER reagent (Genlantis) with the pUMVC7-sGal-3 expression vector or pCMV-LacZ as control, and switched to serum-free media 16 hours later. The CM was collected after 48 hours, purified, and was either stored in frozen aliquots at −20°C or used undiluted (1×) on target cells in cell viability assays. Purification of sGal-3 from CM was performed using a lactosyl-Sepharose column as described previously (24). Preparation and purification of His-tag sGal3 was performed as described previously (25); see details in Supplementary Materials and Methods.

RT/PCR and Western blot analyses

Detailed information for immunoblot preparation is provided in Supplementary Materials and Methods.

Antibody-mediated activity blocking assays

Neutralization of β1 integrin on cells was achieved by addition of 5 μg/mL anti-human β1 integrin inhibitory antibodies (clones P5D2 and AIIB2; The Developmental Studies Hybridoma Bank) or control immunoglobulins (normal mouse IgG, Santa Cruz Biotechnology) 24 hours before sGal-3 CM treatment.

GST–Gal-3 pull-down assays

Recombinant GST–Gal-3 constructs were used for the production of recombinant Gal-3 (rGal-3) in bacteria and used for pull-down assays as described previously (26) with some modification (see Supplementary Materials and Methods).

Coimmunoprecipitation assays

To examine the interaction of sGal-3 with tumor cell surface integrins, sGal-3 CM was added to live cultured cells. Briefly, 3 mL of 2× sGal-3 CM was added to 5 × 106 cells grown in 10-cm diameter culture dishes (serum-starved for 24 hours), and rocked for 4 hours at room temperature. After sGal-3 CM removal, cells were washed three times with PBS and lysed with CHAPS buffer [30 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.25% CHAPS (A.G. Scientific)] supplemented with a protease inhibitor cocktail (Roche). Aliquots of 750 μg of whole-cell extracts at 1 μg/μL were precleared with protein G agarose beads at 4°C for 20 minutes. Subsequently, anti-human galectin-3, β1 integrin, α5 integrin antibodies, or nonspecific immunoglobulins (1.5 μg/mL, Santa Cruz Biotechnology) were used for immunoprecipitation overnight at 4°C. Protein G agarose beads [20 μL; 50% (w/v), Roche] were added and incubated for another 4 hours at 4°C. Beads were recovered by centrifugation, washed three times with lysis buffer, boiled in Laemmli sample buffer, and the immunoprecipitated proteins analyzed by Western blotting.

For the detection of MGAT5-synthesized glycans on cell surface integrin β1, PHA-L cell surface integrin β1 coimmunoprecipitation assays were performed as above, except the cultured cells were pretreated with a pure PHA-L solution (2.5 μg/mL; Vector Laboratories) for 2 hours at room temperature. After washing 3× with PBS, a cell extract was prepared by lysis in CHAPS buffer and anti–PHA-L antibodies (1.5 μg/mL Vector Laboratories) used to immunoprecipitate the PHA-L–bound glycan residues, followed by Western blot for β1 integrin.

Plant lectin pull-down assays

For the detection of glycans synthesized by MGAT5 on β1 integrin, PHA-L agarose-integrin β1 pull-down assays were performed. For the detection of β4GalT-transferred glycan branches, RCA-agarose-β1 integrin pull-down assays were used with RCA agarose beads (Vector Laboratories). Detailed procedures are described in Supplementary Data.

Apoptosis assays

For apoptosis analysis, cells were treated with either CM containing sGal-3 or with purified sGal-3, with or without 100 nmol/L AC-DEVD-CHO caspase-3/7–specific inhibitor (BD Bioscience), 20 μmol/L of Z-IETD-FMK caspase-8 inhibitor, or 20 μmol/L of Z-LEHD-FMK caspase-9 inhibitor (MBL International). Caspase-3/7 or -9 Glo Assays (Promega) were performed on sGal-3–treated cells as per the manufacturer's instructions. The luminescence value (RLU, blank subtracted) was converted to fold induction and the values from control vector–transfected CM-treated cells were considered as 1.

Calpain GLO protease assays

For calpain protease activity analysis, cells were treated with either CM containing sGal-3 alone or supplemented with 500 nmol/L of calpain inhibitor III (MDL28170, Cayman Chemical). As controls, cells were treated with rGal-3 or sGal-3 CM pretreated with 25 mmol/L lactose or 25 mmol/L melibiose for 30 minutes. Calpain GLO Protease Assays (Promega) were performed on sGal-3–treated cells as per the manufacturer's instructions. The luminescence value (RLU, blank subtracted) was converted to fold induction and the value from 0 hour sample was considered as 1. All assays were repeated three times independently (n = 3) in triplicate.

Calcium colorimetric assays

For calcium influx accumulation analysis, cells were treated with sGal-3 CM for indicated times. As controls, cells were pretreated with 50 μmol/L of verapamil (calcium channel blocker, Sigma Aldrich) for 24 hours or with sGal-3 CM pretreated with 25 mmol/L lactose for 30 minutes. Calcium colorimetric assay was performed as per the manufacturer's instructions (Cayman Chemical). For further details see Supplementary Data.

Crystal Violet cytotoxicity assays

Cells were plated at 5,000 cells/well in 96-well plates and treated with 1× control or doxycycline-induced sGal-3 CM (∼500 ng/mL sGal-3) for 24 to 120 hours. Thereafter, the cells were fixed in a Crystal Violet (0.2%)/ethanol (2%) solution for 10 minutes, washed in water, and solubilized in 1% SDS. Relative cell number was quantified by acquiring absorbance at 575 nm using a spectrophotometer.

Soft-agar colony formation assays

Six-well plates were layered with 2 mL of 1% agar in DMEM medium supplemented with 10% Tet-free serum. This bottom layer was overlaid with 5,000 cells mixed in 0.33% agar with DMEM and 10% Tet-free serum. One mL of 10% Tet-tested serum containing media ± 5 μg/mL of doxycycline was added on top of the agar and replaced every 72 hours. After 21 days the colonies were fixed using 100% methanol and visualized using Giemsa stain according to the manufacturer's protocol (Sigma). The plates were air-dried to flatten the agar discs, the colonies counted, and photographed at 20×. The experiment was repeated three times in triplicate (n = 3).

In vivo tumorigenicity experiments

All animal experiments were performed under Institutional Animal Care and Use Committee (IACUC) guidelines. For the subcutaneous tumor growth experiments, 6-week-old female athymic nude mice (NCI, Bethesda, MD; 8–10/group) were injected subcutaneously with 5 × 106 cells of the indicated cell lines. Mice with LN229–sGal-3 Tet-on gliomas received oral doxycycline (2 mg/mL) in drinking water containing 4% sucrose to induce expression of sGal-3 1 week postinjection of tumor cells until termination of the experiment. Lung cancer cells were preincubated with His-tag sGal3 (500 ng/mL) for 20 minutes at room temperature, then mixed with an equal volume of Matrigel (Corning Life Sciences; catalog no. 356234), and injected subcutaneously. Tumor volume was calculated in mm3 = (length × width2)/2.

For the orthotopic brain tumor experiments, 6-week-old female athymic nude (NCI, Bethesda, MD) mice were injected intracranially with 5 × 105 LN229-L16 sGal-3 Tet-on cells (clone #11) and divided into two groups (± doxycycline) of 11 mice each. Sixty-three days after the intracranial tumor injection, 10 nmol/L of IR-labeled 2-deoxyglucose (LI-COR) was tail-vein injected and the intensity of dye-stained brain tumor was analyzed 24 hours later with Olympus FV-1000 microscopy (IR wavelength = 750 nm). Mice were terminated as per IACUC criteria. The Kaplan–Meier survival curve was established using SPSS and MedCalc statistical software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism v6.01 Software (GraphPad Software Inc.). Results are presented as mean ± SEM. For comparison of sample versus control, unpaired t test was used. For Kaplan–Meir survival study, P value was calculated by log-rank test. A P value less than or equal to 0.05 was considered significant. For results P values are presented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Study approval

All animal work was performed according to the guidelines for animal experimentation and welfare and approved by the Emory University IACUC (Atlanta, GA).

N-terminus–modified Gal-3 reduces cancer cell viability in vitro

To study the importance of the N-terminus in the proapoptotic function of Gal-3 (15), we previously made a series of expression constructs that either shortened or extended the N-terminus of Gal-3 and tested them for in vitro cytotoxicity against cancer cells (27). One construct produced a form of Gal-3 with dramatically increased cytotoxicity as compared with wild-type Gal-3. This construct generates an approximately 33 kDa Gal-3 protein (sGal-3) due to the N-terminus conjugation of the signal peptide from tissue-plasminogen activator protein (tPA; Supplementary Fig. S1A). The engineered signal peptide of sGal-3 functions as a classical secretion signal, but is incompletely processed by signal peptidases, and only the uncleaved form is efficiently secreted. Upon transient expression in mammalian cells (HEK293), sGal-3 is produced abundantly in the CM and can easily be distinguished from the endogenous ∼27 kDa Gal-3 (Fig. 1A). While Gal-3 is now secreted through the Golgi apparatus, there is no evidence for glycosylation (Supplementary Fig. S1B).

Figure 1.

sGal-3 inhibits tumor cell viability in vitro. A, Western blot analysis showing levels of endogenous (Gal-3) and sGal-3 forms of Galectin-3. Whole-cell extracts (WCE) and supernatants (CM) from 293 cells 48 hours after transient transfection with an expression vector encoding LGALS3 cDNA fused to a classical secretion signal (pUMVC7) or control vector pCMV-LacZ (Ctrl) were analyzed (left). BSA, Ponceau staining of BSA. Coomassie blue staining shows lactosyl-Sepharose-purified sGal-3 from CM (right). B, Crystal Violet cell viability assay showing tumor cell–specific toxicity of sGal-3. Human glioma cells (LN229, top), and human fibroblasts (HFF-1, bottom) were treated in triplicate for 30–120 hours with CM from 293 cells either untransfected (UT), or transfected with control (LacZ) or sGal-3–expressing vectors. **, P < 0.01; ***, P < 0.001 (unpaired t test). C, Comparison between sGal-3 CM and rGal-3–mediated cytotoxicity in LN229 and HFF-1 cells after 48 hours treatment. Quantification is presented as percent of sGal-3/vector control CM crystal violet staining from triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control CM (unpaired t test). D, Dose response of purified sGal-3 on LN229 and HFF-1 cell viability by Crystal Violet assay after 48 hours. sGal-3 CM was purified through lactosyl-Sepharose column and quantified by ELISA. Quantification from triplicates as above. **, P < 0.01; ***, P < 0.001 (unpaired t test). E, Crystal Violet assay demonstrating sGal-3 CM–induced death at 48 hours in genetically and biologically heterogeneous malignant human cancer cell lines (black) derived from brain (SF767, LN-Z308, and LN229), breast (MD468 and MCF7), lung (A549 and H1289), and prostate (LnCaP and PC3) tumors. In contrast, primary cultures of HDMEC or fibroblasts (HDF and HFF-1) or normal breast epithelial cells (MCF10) or embryonic neuroepithelial 293 cells (gray) did not show significant decreases in cell viability. Cell viability is expressed as percentage sGal-3 over control CM. Three independent experiments were performed in triplicate (n = 3). ***, P < 0.001; ****, P < 0.0001 compared with control CM (unpaired t test). F, Crystal Violet assay showing neutralization of sGal-3 CM with lactose. sGal-3 CM was pretreated for 1 hour with 20 mmol/L (final concentration) of lactose, sucrose, or melibiose, then used to treat tumor (LN229 and A549) and normal (HFF-1) cells for 48 hours. Quantified as percent of sGal-3/control CM Crystal Violet staining from triplicates. ***, P < 0.001 compared with control CM (unpaired t test).

Figure 1.

sGal-3 inhibits tumor cell viability in vitro. A, Western blot analysis showing levels of endogenous (Gal-3) and sGal-3 forms of Galectin-3. Whole-cell extracts (WCE) and supernatants (CM) from 293 cells 48 hours after transient transfection with an expression vector encoding LGALS3 cDNA fused to a classical secretion signal (pUMVC7) or control vector pCMV-LacZ (Ctrl) were analyzed (left). BSA, Ponceau staining of BSA. Coomassie blue staining shows lactosyl-Sepharose-purified sGal-3 from CM (right). B, Crystal Violet cell viability assay showing tumor cell–specific toxicity of sGal-3. Human glioma cells (LN229, top), and human fibroblasts (HFF-1, bottom) were treated in triplicate for 30–120 hours with CM from 293 cells either untransfected (UT), or transfected with control (LacZ) or sGal-3–expressing vectors. **, P < 0.01; ***, P < 0.001 (unpaired t test). C, Comparison between sGal-3 CM and rGal-3–mediated cytotoxicity in LN229 and HFF-1 cells after 48 hours treatment. Quantification is presented as percent of sGal-3/vector control CM crystal violet staining from triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control CM (unpaired t test). D, Dose response of purified sGal-3 on LN229 and HFF-1 cell viability by Crystal Violet assay after 48 hours. sGal-3 CM was purified through lactosyl-Sepharose column and quantified by ELISA. Quantification from triplicates as above. **, P < 0.01; ***, P < 0.001 (unpaired t test). E, Crystal Violet assay demonstrating sGal-3 CM–induced death at 48 hours in genetically and biologically heterogeneous malignant human cancer cell lines (black) derived from brain (SF767, LN-Z308, and LN229), breast (MD468 and MCF7), lung (A549 and H1289), and prostate (LnCaP and PC3) tumors. In contrast, primary cultures of HDMEC or fibroblasts (HDF and HFF-1) or normal breast epithelial cells (MCF10) or embryonic neuroepithelial 293 cells (gray) did not show significant decreases in cell viability. Cell viability is expressed as percentage sGal-3 over control CM. Three independent experiments were performed in triplicate (n = 3). ***, P < 0.001; ****, P < 0.0001 compared with control CM (unpaired t test). F, Crystal Violet assay showing neutralization of sGal-3 CM with lactose. sGal-3 CM was pretreated for 1 hour with 20 mmol/L (final concentration) of lactose, sucrose, or melibiose, then used to treat tumor (LN229 and A549) and normal (HFF-1) cells for 48 hours. Quantified as percent of sGal-3/control CM Crystal Violet staining from triplicates. ***, P < 0.001 compared with control CM (unpaired t test).

Close modal

Treatment of malignant human glioma cells (LN229) with sGal-3–containing CM mediated tumor cell killing in a time- and dose-dependent fashion, while human foreskin fibroblasts (HFF-1) remained unaffected (Fig. 1B; Supplementary Fig. S1C). Purification of recombinant sGal-3 from the CM using a lactosyl-Sepharose column showed that sGal-3 per se induced tumor cell killing with an IC50 of approximately 250 ng/mL (Fig. 1C). In contrast, bacterially produced rGal-3 (full length, CRD only, or artificially elongated to 33 kDa by duplicating collagen repeats 4, 5, and 6) had no tumor killing effect at the same concentration (Fig. 1D; Supplementary Fig. S1D), even when mixed with control CM (Supplementary Fig. S1E). Only 100- to 200-fold higher concentrations of rGal-3 (30–60 μg/mL) were able to induce apoptosis in Jurkat cells, consistent with the literature (ref. 15; Supplementary Fig. S1F). sGal-3 showed tumor-specific killing in a panel of genetically and biologically heterogeneous cancer cell lines, while a variety of nontumoral cells were resistant to sGal-3 cytotoxicity (Fig. 1E).

The CRD of Gal-3 is known to interact with multiple cell surface receptors carrying glycosylated branches containing β-galactoside modifications and this binding can be competed with high concentrations (>1 mmol/L) of lactose [β-D-galactopyranosyl-(1→4)-D-glucose]. Addition of lactose neutralized sGal-3–mediated tumor killing, while the control sugars sucrose [α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside] and melibiose [α-D-galactopyranosyl-(1→6)-D-glucose], which do not bind Gal-3, had no effect (Fig. 1F). The neutralizing effect was not due to a direct survival effect mediated by lactose cell binding per se, as preincubation of the tumor cells with lactose did not have a protective effect (Supplementary Fig. S1G). Altogether, these data showed that sGal-3 mediates tumor cell–specific killing, and this is dependent upon its β-galactoside binding function.

sGal-3 reduces in vitro and in vivo tumorigenicity

To examine whether the presence of sGal-3 in the extracellular milieu would reduce tumor growth, we generated human glioblastoma cells with inducible sGal-3 expression (clones LN229–sGal-3 Tet-on Med and High). Both clones showed tight doxycycline-mediated induction with abundant secretion of sGal-3 in the CM under induced conditions (Fig. 2A). To examine sGal-3 effect on anchorage-independent growth, we performed soft-agar colony formation assays. sGal-3 induction resulted in an approximately 4- to 6-fold decrease in colony number, proportional to the amount of sGal-3 produced in the CM (Fig. 2B). Reduced growth upon sGal-3 treatment was not related to a reduction in cell proliferation as there were no changes in cell-cycle distribution (Supplementary Fig. S2A). Induction of sGal-3 strongly reduced subcutaneous and intracranial in vivo tumor formation and increased animal survival while being well tolerated (Fig. 2C and D; Supplementary Fig. S2B and S2C). Furthermore, sGal-3–mediated in vivo antitumor effects were not limited to brain tumors as a short exposure of aggressive lung cancer cells to recombinant His-tagged sGal3 completely prevented subcutaneous tumor development (Supplementary Fig. S2E and S2F). These data show that sGal-3 has potent antitumor activity in vitro and in vivo.

Figure 2.

Gal-3 secretion reduces the tumorigenicity of malignant glioma cells in vitro and in vivo. A, Western blot analysis showing doxycycline (dox)-dependent sGal-3 expression in CM of two sGal-3 inducible clones (Med. and High) derived from LN229-L16-tet-on cells after 48 hours of induction. Thrombospondin-1 (TSP-1) was used as loading control. B, Soft-agar colony formation assays show dose-dependent inhibition of colony formation upon doxycycline-inducible sGal-3 expression in both clones. Representative pictures of colonies (top) and quantification (bottom) are shown. * P < 0.01 (unpaired t test). C, sGal-3 inhibits subcutaneous tumor growth. Athymic nude mice were injected subcutaneously with 5 × 106 cells of each clone (Med. and High) and divided into two groups (9 mice/group; two tumors/mouse). One group/cell line was left untreated, while the second group was given 2 mg/mL doxycycline in drinking water containing 5% sucrose to induce expression of sGal-3 one week after tumor cell implantation until termination of the experiment. Average tumor volumes at termination were 4- to 6-fold smaller in dox group versus controls (P < 0.02; unpaired t test). Doxycycline had no effect on LN229-L16 control tumor growth (data not shown). D, Kaplan–Meier survival curve for nude mice intracranially injected with 5 × 105 High clone #11 cells (n = 12/group; left). Mean survival time: 63 days for control and 78 days for sGal-3 group (P = 0.0026; log-rank test). Near-infrared imaging 24 hours after injection of fluorescent 2-deoxy-glucose (10 nmol/L) showed increased tumor burden in uninduced mice at day 63, which preceded their rapid decline (right and Supplementary Fig. S2A). No systemic or brain toxicity were observed with production of sGal-3 by the tumor cells (not shown). Doxycycline treatment per se had no effect on survival of mice implanted with parental LN229-L16 cells (Supplementary Fig. S2B).

Figure 2.

Gal-3 secretion reduces the tumorigenicity of malignant glioma cells in vitro and in vivo. A, Western blot analysis showing doxycycline (dox)-dependent sGal-3 expression in CM of two sGal-3 inducible clones (Med. and High) derived from LN229-L16-tet-on cells after 48 hours of induction. Thrombospondin-1 (TSP-1) was used as loading control. B, Soft-agar colony formation assays show dose-dependent inhibition of colony formation upon doxycycline-inducible sGal-3 expression in both clones. Representative pictures of colonies (top) and quantification (bottom) are shown. * P < 0.01 (unpaired t test). C, sGal-3 inhibits subcutaneous tumor growth. Athymic nude mice were injected subcutaneously with 5 × 106 cells of each clone (Med. and High) and divided into two groups (9 mice/group; two tumors/mouse). One group/cell line was left untreated, while the second group was given 2 mg/mL doxycycline in drinking water containing 5% sucrose to induce expression of sGal-3 one week after tumor cell implantation until termination of the experiment. Average tumor volumes at termination were 4- to 6-fold smaller in dox group versus controls (P < 0.02; unpaired t test). Doxycycline had no effect on LN229-L16 control tumor growth (data not shown). D, Kaplan–Meier survival curve for nude mice intracranially injected with 5 × 105 High clone #11 cells (n = 12/group; left). Mean survival time: 63 days for control and 78 days for sGal-3 group (P = 0.0026; log-rank test). Near-infrared imaging 24 hours after injection of fluorescent 2-deoxy-glucose (10 nmol/L) showed increased tumor burden in uninduced mice at day 63, which preceded their rapid decline (right and Supplementary Fig. S2A). No systemic or brain toxicity were observed with production of sGal-3 by the tumor cells (not shown). Doxycycline treatment per se had no effect on survival of mice implanted with parental LN229-L16 cells (Supplementary Fig. S2B).

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sGal-3 selectively induces apoptosis in cancer cells

Next, we investigated the mechanism of sGal-3–induced tumor cell death. sGal-3–treated glioma cells displayed activation of caspases-3 and -9 and PARP cleavage, and these effects were inhibited by inhibitors of caspase-3 and -9, but not caspase-8 (Fig. 3AD; Supplementary Fig. S3A). In contrast, sGal-3 elicited no caspase activation in normal fibroblasts (Fig. 3BD; Supplementary Fig. S3B). sGal-3 treatment also induced caspase-3/7 activation in glioma stem–like cells (GSC) and blocked neurosphere formation, while normal neural progenitor cells (NPC) were unaffected (Fig. 3E and F). sGal-3 also efficiently induced caspase-7 activation in caspase-3–deficient MCF-7 cells (Supplementary Fig. S3C). To identify apoptosis-related proteins altered by sGal-3 in tumor cells, we performed a protein array and found upregulation of cleaved caspase-3 and proapoptotic Bax in the presence of sGal-3, while survivin, a member of the inhibitor of apoptosis family, was downregulated (Supplementary Fig. S3D). Western blot analysis confirmed these results (Supplementary Fig. S3E and S3F). These data suggest that sGal-3 induces cancer cell apoptosis through a caspase-9–dependent mitochondrial pathway by disrupting the balance between pro- and antiapoptotic proteins.

Figure 3.

sGal-3 induces apoptosis specifically in tumor cells. A, Western blot showing cleaved caspase-3 (cCasp-3; 17 and 19 kDa) and cleaved PARP (cPARP; 89 kDa) in sGal-3–treated LN229 cells at indicated times. Cleavage was inhibited by a caspase-3 inhibitor (C3i; Ac-DEVD-CHO, 100 nmol/L). B, Caspase-3/7 GLO assay shows kinetics of induction of caspase-3/7 cleavage following treatment with sGal-3 CM in LN229 but not HFF-1 cells. C, Crystal Violet cell survival assay showing that caspase-3 and -9 inhibitors prevent sGal-3 CM–mediated cancer cell (LN229 glioma and LnCaP prostate cancer) death at 72 hours. Inhibitors: caspase-3 (Ac-DEVD-CHO; 100 nmol/L), caspase-8 (C8i; Z-IETD-FMK; 20 μmol/L), and caspase-9 (C9i; Z-LEHD-FMK; 20 μmol/L). Quantification is percent of sGal-3/control CM Crystal Violet staining. n = 2 (in triplicates). *, P < 0.05; **, P < 0.01 compared with control CM (unpaired t test). D, Caspase-9 GLO assay shows induction kinetics of caspase-9 cleavage following treatment with 2× sGal-3 CM in LN229, but not HFF-1 cells. (n = 2). E, sGal-3 CM reduces neurosphere formation by CD133+ GSCs (N08-74) in a dose-dependent fashion. Number of neurospheres formed is expressed as percent in sGal-3/control CM groups. Scale bar, 100 μm. F, Caspase-3/7 GLO assay shows induction kinetics of caspase-3/7 cleavage after GSC treatment with sGal-3 (600 ng/mL). Normal human neural progenitor cells (NPCs) were not affected.

Figure 3.

sGal-3 induces apoptosis specifically in tumor cells. A, Western blot showing cleaved caspase-3 (cCasp-3; 17 and 19 kDa) and cleaved PARP (cPARP; 89 kDa) in sGal-3–treated LN229 cells at indicated times. Cleavage was inhibited by a caspase-3 inhibitor (C3i; Ac-DEVD-CHO, 100 nmol/L). B, Caspase-3/7 GLO assay shows kinetics of induction of caspase-3/7 cleavage following treatment with sGal-3 CM in LN229 but not HFF-1 cells. C, Crystal Violet cell survival assay showing that caspase-3 and -9 inhibitors prevent sGal-3 CM–mediated cancer cell (LN229 glioma and LnCaP prostate cancer) death at 72 hours. Inhibitors: caspase-3 (Ac-DEVD-CHO; 100 nmol/L), caspase-8 (C8i; Z-IETD-FMK; 20 μmol/L), and caspase-9 (C9i; Z-LEHD-FMK; 20 μmol/L). Quantification is percent of sGal-3/control CM Crystal Violet staining. n = 2 (in triplicates). *, P < 0.05; **, P < 0.01 compared with control CM (unpaired t test). D, Caspase-9 GLO assay shows induction kinetics of caspase-9 cleavage following treatment with 2× sGal-3 CM in LN229, but not HFF-1 cells. (n = 2). E, sGal-3 CM reduces neurosphere formation by CD133+ GSCs (N08-74) in a dose-dependent fashion. Number of neurospheres formed is expressed as percent in sGal-3/control CM groups. Scale bar, 100 μm. F, Caspase-3/7 GLO assay shows induction kinetics of caspase-3/7 cleavage after GSC treatment with sGal-3 (600 ng/mL). Normal human neural progenitor cells (NPCs) were not affected.

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β1 integrin is required for sGal-3–mediated cancer cell death

Extracellular Gal-3 interacts with a variety of cell surface proteins mediating its multiple functions (12). In search of candidate cell surface ligands as mediators of the proapoptotic signal of sGal-3, we tested death receptors (Fas, DR4, and DR5) and members of the integrin family. We did not find involvement of the death receptors (data not shown), consistent with the lack of caspase-8 activation upon sGal-3–induced apoptosis (Fig. 3C). In contrast, siRNA and neutralizing antibodies against integrin β1 largely prevented sGal-3–mediated activation of caspase-3/7, PARP cleavage, and tumor cell killing (Fig. 4A; Supplementary Fig. S4A and S4B). Immunofluorescence staining confirmed colocalization of sGal-3 and integrin β1 at the tumor cell surface (Supplementary Fig. S4D). Conversely, overexpression of either α5 or β1 or both integrins in 293 cells induced β1–Gal-3 interaction in GST pull-down experiments (Supplementary Fig. S4E), and sensitized the cells to sGal-3–induced apoptosis (Supplementary Fig. S4F). This data demonstrates that sGal-3–mediated tumor cell death requires β1 integrin expression; however, β1 expression alone is not sufficient to explain cancer-specific cytotoxicity. Indeed, HFF-1 and LN229 cells express similar levels of β1 integrin, but only LN229 cells die in response to sGal-3 (Supplementary Fig. S4A and S4B). Of note, β1 integrin is of higher molecular weight in cancer cells, suggesting a potential role for posttranslational modifications.

Figure 4.

β1 integrin is required for sGal-3–mediated cell death and shows preferential activation and interaction with sGal-3 in cancer cells because of altered glycosylation. A, Integrin β1 knockdown protects cancer cells from sGal-3–activated cell death (left). LN229 cells were pretreated with either control (si-con) or two independent β1 siRNAs for 72 hours, before being seeded at 5,000 cells/well in 96-well plates. Twenty-four hours later, cells were treated with 200 μL of 1× control or sGal-3 CM. Cell viability was examined 72 hours later by SRB assay in triplicates. **, P < 0.01; unpaired t test. Western blot showing efficient siRNA knockdown of β1 protects cells from sGal-3 induction of PARP cleavage, a marker of late-stage apoptosis (right). B, Coimmunoprecipitation experiments show increased binding of sGal-3 to cell surface β1 integrin on LN229 cells (L) versus HFF-1 cells (H). Cells were cultured for 3 hours at room temperature with sGal-3 CM, then lysed, and immunoprecipitated with anti–Gal-3 and β1 or α5 integrin antibodies. Immunoprecipitated proteins were subjected to Western blotting for Gal-3 and β1 integrin (top). Note that coimmunoprecipitations between sGal-3 and β1 or α5 integrins are only found in LN229 cells. Intensity of Gal-3–β1 integrin and Gal-3–α5 integrin binding was compared between tumor and normal cells by densitometry (bottom). Data are graphed as fold binding induction based on coimmunoprecipitation band intensity between tumor and normal cells (normal cell binding was set at 1). The β1/β1 and Gal-3/Gal-3 coimmunoprecipitations show input levels for each protein in both cell lines. C, Western blot showing that sGal-3 treatment switches β1 integrin into its active conformation and activates PARP cleavage. D, GST–Gal-3 pull-down experiment shows that 24 hours pretreatment with antibodies that switch β1 integrin into its active (12G10) or inactive (P5D2) conformation, respectively, augment or inhibit interaction with Gal-3. E, Western blot showing that β1 integrin activation antibody (12G10) augments PARP cleavage in response to sGal-3 in LN229 cells. F, Crystal Violet cell viability assay showing that 24 hours pretreatment with antibodies that mediate β1 integrin switch toward active or inactive conformations induces or prevents sGal-3–mediated killing in LN229 cells, but has no effect in HFF-1 cells. Quantification from triplicates (***, P < 0.001 unpaired t test; left). Representative images (right).

Figure 4.

β1 integrin is required for sGal-3–mediated cell death and shows preferential activation and interaction with sGal-3 in cancer cells because of altered glycosylation. A, Integrin β1 knockdown protects cancer cells from sGal-3–activated cell death (left). LN229 cells were pretreated with either control (si-con) or two independent β1 siRNAs for 72 hours, before being seeded at 5,000 cells/well in 96-well plates. Twenty-four hours later, cells were treated with 200 μL of 1× control or sGal-3 CM. Cell viability was examined 72 hours later by SRB assay in triplicates. **, P < 0.01; unpaired t test. Western blot showing efficient siRNA knockdown of β1 protects cells from sGal-3 induction of PARP cleavage, a marker of late-stage apoptosis (right). B, Coimmunoprecipitation experiments show increased binding of sGal-3 to cell surface β1 integrin on LN229 cells (L) versus HFF-1 cells (H). Cells were cultured for 3 hours at room temperature with sGal-3 CM, then lysed, and immunoprecipitated with anti–Gal-3 and β1 or α5 integrin antibodies. Immunoprecipitated proteins were subjected to Western blotting for Gal-3 and β1 integrin (top). Note that coimmunoprecipitations between sGal-3 and β1 or α5 integrins are only found in LN229 cells. Intensity of Gal-3–β1 integrin and Gal-3–α5 integrin binding was compared between tumor and normal cells by densitometry (bottom). Data are graphed as fold binding induction based on coimmunoprecipitation band intensity between tumor and normal cells (normal cell binding was set at 1). The β1/β1 and Gal-3/Gal-3 coimmunoprecipitations show input levels for each protein in both cell lines. C, Western blot showing that sGal-3 treatment switches β1 integrin into its active conformation and activates PARP cleavage. D, GST–Gal-3 pull-down experiment shows that 24 hours pretreatment with antibodies that switch β1 integrin into its active (12G10) or inactive (P5D2) conformation, respectively, augment or inhibit interaction with Gal-3. E, Western blot showing that β1 integrin activation antibody (12G10) augments PARP cleavage in response to sGal-3 in LN229 cells. F, Crystal Violet cell viability assay showing that 24 hours pretreatment with antibodies that mediate β1 integrin switch toward active or inactive conformations induces or prevents sGal-3–mediated killing in LN229 cells, but has no effect in HFF-1 cells. Quantification from triplicates (***, P < 0.001 unpaired t test; left). Representative images (right).

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sGal-3–β1 integrin interaction is increased in tumor cells

To further assess the interaction of sGal-3 with β1 integrin and examine whether integrin β1 exhibits higher affinity for sGal-3 in tumor versus normal cells, we performed coimmunoprecipitation assays. We treated cultured cells with sGal-3, washed off unbound lectin, lysed cells, and performed coimmunoprecipitations to assess sGal-3−β1 interactions at the cell surface. We focused on α5β1 integrin, because of its elevated expression in glioblastoma compared with adjacent normal brain tissue (28). Cell surface interactions between sGal-3 and α5β1 integrin were evidenced in LN229 glioma, but not HFF-1 fibroblasts (Fig. 4B), even though both cell types express comparable levels of α5β1 and bind similar amounts of exogenous sGal-3 at the cell surface (Supplementary Fig. S4G and S4H). Combined, these experiments show that sGal-3′s affinity for cell surface α5/β1 integrins is increased in tumor cells, and upon binding induces proapoptotic signaling.

sGal-3 switches β1 integrin into its active conformation, and β1 activation status regulates sGal-3–mediated tumor cell killing

The activation status of integrins is dependent upon conformational changes in their extracellular domains, which regulates their ligand affinity (29). We first examined whether sGal-3 treatment could alter β1 integrin conformation, and found that sGal-3 treatment led to a rapid β1 integrin conformation switch into its active form, which was accompanied by induction of PARP cleavage (Fig. 4C). To determine whether conformation of β1 integrin complexes per se influences its binding to Gal-3, we used neutralizing (P5D2) and activating (12G10) anti-β1 integrin antibodies (Fig. 4D). Activating antibody increased β1 integrin affinity for Gal-3 as demonstrated by a GST–Gal-3 pull-down experiment, while blocking antibody abrogated β1 integrin–Gal-3 interaction. Congruently, antibody-mediated activation of β1 integrin complex further sensitized tumor cells to sGal-3–mediated PARP cleavage and concomitant death, while a blocking antibody reduced killing (Fig. 4E and F). These results show that sGal-3 interaction and death-inducing signaling is dependent upon the activation state of the β1 integrin complex and suggests that sGal-3 can actively switch β1 toward its active state.

sGal-3 mediates tumor cell killing through a calpain/GSK3β-dependent signaling cascade

Next, we investigated which signaling cascade was involved in sGal-3–triggered apoptosis. By examining downstream mediators of integrin signaling, we found that sGal-3 rapidly (within 30 minutes) reduced the level of the inactive form of GSK3β (phosphorylated at Ser 9) in an integrin-linked kinase (ILK)-independent fashion (Fig. 5A, lanes 4, 5 and 9, 10). To verify ILK inhibitor (Cpd22) activity, we examined phosphorylation of Akt (Ser 473), one of ILK's known substrates (30), and observed inhibition (Fig. 5A, lane 2 vs. 7). These data show that sGal-3 induces both ILK-dependent and -independent integrin signaling. The ILK/Akt axis was not involved in sGal-3–mediated killing as ILK and Akt inhibitors failed to prevent cell death (Supplementary Fig. S5A and S5B).

Figure 5.

sGal-3 induces calcium influx and triggers a calpain-dependent proapoptotic signaling cascade. A, Western blot showing that sGal-3 CM (500 ng/mL, 80 minutes) reduces GSK3β serine 9 phosphorylation and increases Akt serine 473 phosphorylation in LN229 cells. ILK inhibitor, cpd22 (0.5 μmol/L, 12 hours pretreatment/cotreatment), blocked Akt phosphorylation, but had no effect on GSK3β. B, Western blot showing that sGal-3 (500 ng/mL, 12 hours) binding to LN229 cells induces a proapoptotic response by calpain-dependent cleavage of GSK3β, Bax, and Bad. Caspase-8–dependent Bid was not affected. Calpain inhibitor III (10 μmol/L cotreatment, 12 hours) blocked the cleavage. C, Time course of calpain activation in LN229 cells by sGal-3 (500 ng/mL) using a Calpain GLO assay. Calpain activation was absent in calpain inhibitor III (10 μmol/L cotreatment, 12 hours) treated cells and in nontumor cells (HFF-1). N = 3 (triplicates). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test). D, sGal-3–mediated activation of calpain (GLO assay) in LN229 cells is blocked by pretreatment (30 minutes) with lactose [β-D-Galp-β(1-4)-D-Glc; 25 mmol/L], but not melibiose [D-Gal-α(1-6)-D-Glc; 25 mmol/L]. N = 3 (triplicates). ***, P < 0.001 (unpaired t test). E, Calpain GLO assay showing that sGal-3 CM (500 ng/mL), but not control CM supplemented with rGal-3 (500 ng/mL) activates calpain in LN229 cells. N = 3 (triplicates). ***, P < 0.001 (unpaired t test). F, Calcium colorimetric assay showing that sGal-3 treatment induces cytosolic calcium accumulation in LN229 cells. Verapamil (50 μmol/L, 24-hour pretreatment), a calcium channel blocker, and lactose-pretreated sGal-3 neutralized this effect. N = 3 (triplicates). **, P < 0.01 (unpaired t test).

Figure 5.

sGal-3 induces calcium influx and triggers a calpain-dependent proapoptotic signaling cascade. A, Western blot showing that sGal-3 CM (500 ng/mL, 80 minutes) reduces GSK3β serine 9 phosphorylation and increases Akt serine 473 phosphorylation in LN229 cells. ILK inhibitor, cpd22 (0.5 μmol/L, 12 hours pretreatment/cotreatment), blocked Akt phosphorylation, but had no effect on GSK3β. B, Western blot showing that sGal-3 (500 ng/mL, 12 hours) binding to LN229 cells induces a proapoptotic response by calpain-dependent cleavage of GSK3β, Bax, and Bad. Caspase-8–dependent Bid was not affected. Calpain inhibitor III (10 μmol/L cotreatment, 12 hours) blocked the cleavage. C, Time course of calpain activation in LN229 cells by sGal-3 (500 ng/mL) using a Calpain GLO assay. Calpain activation was absent in calpain inhibitor III (10 μmol/L cotreatment, 12 hours) treated cells and in nontumor cells (HFF-1). N = 3 (triplicates). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired t test). D, sGal-3–mediated activation of calpain (GLO assay) in LN229 cells is blocked by pretreatment (30 minutes) with lactose [β-D-Galp-β(1-4)-D-Glc; 25 mmol/L], but not melibiose [D-Gal-α(1-6)-D-Glc; 25 mmol/L]. N = 3 (triplicates). ***, P < 0.001 (unpaired t test). E, Calpain GLO assay showing that sGal-3 CM (500 ng/mL), but not control CM supplemented with rGal-3 (500 ng/mL) activates calpain in LN229 cells. N = 3 (triplicates). ***, P < 0.001 (unpaired t test). F, Calcium colorimetric assay showing that sGal-3 treatment induces cytosolic calcium accumulation in LN229 cells. Verapamil (50 μmol/L, 24-hour pretreatment), a calcium channel blocker, and lactose-pretreated sGal-3 neutralized this effect. N = 3 (triplicates). **, P < 0.01 (unpaired t test).

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Interestingly, phospho-GSK3β downregulation was accompanied by ILK-independent cleavage of total GSK3α/β (Fig. 5A, lanes 4 and 9), an effect which dramatically increased over time (Fig. 5B). Such cleavage is reminiscent of calpain-mediated cleavage of the N-terminus of GSK3β (31), therefore we tested calpain involvement in sGal-3–mediated cell killing. Calpain inhibitor (MDL28170) strongly reduced GSK3α/β cleavage and restored phospho-GSK3β levels (Fig. 5B). A calpain assay confirmed that sGal-3 treatment rapidly increased calpain activity in tumor cells but not in control fibroblasts (Fig. 5C), while a pretreatment with lactose (competitive Gal-3 ligand) prevented calpain activation (Fig. 5D). The observed calpain activation was specific to sGal-3, as rGal-3 failed to achieve it (Fig. 5E). Importantly, calpain activation was necessary for sGal-3–mediated apoptosis, as calpain inhibition decreased levels of both cleaved Bax and PARP (Fig. 5B) and prevented tumor cell death (Supplementary Fig. S5).

Elevated intracellular calcium level is responsible for calpain activation (32), and integrin ligation can increase calcium channel influx (33). To analyze whether sGal-3 could trigger Ca2+ transport, we performed a calcium colorimetric assay in LN229 cells and found sGal-3 rapidly increased cytosolic calcium accumulation (Fig. 5F). Pretreatment with lactose or a calcium channel blocker (verapamil) neutralized this effect. Furthermore, verapamil treatment abrogated sGal-3–mediated cell death (Supplementary Fig. S5).

In aggregate, these data show that the mechanism underlying sGal-3–mediated tumor cell death sequentially involves integrin activation, calcium channel–facilitated Ca2+ influx, Ca2+-mediated calpain activation, and induction of the proapoptotic GSK3β/Bax signaling cascade following calpain-mediated cleavage.

N-linked glycosylation is required for sGal-3–mediated killing

Considering that aberrant N-glycosylation is associated with malignant transformation (10), and integrins are major carriers of N-glycans (34), we examined whether aberrant N-glycosylation of β1 integrin in tumor cells contributes to their susceptibility to sGal-3. Tumor cell treatment with kifunensine, an N-glycosylation inhibitor (NGI) that blocks α-mannosidases important in N-glycan biosynthesis and formation of complex N-glycans, reduced the molecular weight of β1 integrin, and abrogated GST–Gal-3/β1 interaction (Fig. 6A), while treatment with benzyl-2-α-GalNAc, an O-glycosylation inhibitor (OGI) at 2 mmol/L concentration, affected neither Gal-3/β1 integrin interaction nor β1 integrin molecular weight. Furthermore, kifunensine protected tumor cells from sGal-3–mediated killing, whereas benzyl-2-α-GalNAc did not (Fig. 6B). These results show that complex N-glycans, recognized by galectins, are necessary for sGal-3/β1 integrin interaction and sGal-3–mediated apoptosis.

Figure 6.

Alteration in MGAT5 expression affects complex N-glycan formation on β1 integrin, sGal-3–β1 integrin interaction, and sGal-3–mediated cell killing. A, GST–Gal-3 pull-down assay showing that NGI kifunensine (100 μmol/L), an α-mannosidase inhibitor that blocks N-glycan processing, abrogated GST–Gal-3–β1 integrin interaction. Loss of N-glycans reduces size of β1 integrin (see input). Treatment with OGI benzyl-N-acetyl-α-D-galactosamide (α-benzyl-GalNAc; 2 mmol/L) had no effect. B, Crystal Violet cell viability assay showing kifunensine pretreatment (24 hours) prevented sGal-3–mediated cell death in LN229 cells. Cells were pretreated with NGI or OGI for 24 hours, then 1× control (Ctrl) or sGal-3 CM was added, and cells incubated for another 72 hours. N = 3 (in triplicates). **, P < 0.01 (unpaired t test). C, PHA-L pull-down assay shows MGAT5 overexpression (+) increases, while MGAT5 knockdown (−) decreases PHA-L binding to β1 integrin. Cells were transiently transfected with control plasmid (Ctrl.) or expression vectors for MGAT5 (+) or shRNA for MGAT5 (−) and cell extracts were analyzed after 48 hours. PHA-L agarose beads were used to specifically pull down proteins carrying N-glycan branches synthesized by MGAT5, followed by Western blot analysis for β1 integrin (left). RT-PCR analysis (middle) and Western blot (right) show increase/decrease in MGAT5 mRNA/protein levels. D, Coimmunoprecipitation experiments showing increased/decreased binding of PHA-L to cell surface β1 integrins with overexpression (+) or knockdown (−) of MGAT5. Cells were transiently transfected as in C, then incubated with purified PHA-L (2.5 μg/mL) for 2 hours at room temperature, and cell extracts prepared. Cell surface proteins bound to PHA-L were then immunoprecipitated with anti–PHA-L antibodies (2.5 μg/mL) and protein G agarose beads followed by β1 integrin Western blot analysis (Ctrl., +, −) as above. E, GST–Gal-3 pull-down assay shows that alteration in MGAT5 expression modulates Gal-3 interaction with β1 integrin. Cells were transiently transfected as in C, then cell extracts were incubated with GST–Gal-3 beads, and bound β1 integrin detected by Western blot analysis (Ctrl., +, −) as above. F, Crystal Violet cell viability assay showing modulation of MGAT5 expression alters cell sensitivity to sGal-3–mediated killing. Note that increased MGAT5 expression sensitizes HFF-1 cells to sGal-3 killing. Cells were transiently transfected as in C, then treated with sGal-3 CM for 72 hours. N = 3 (in triplicates); *, P < 0.05; **, P < 0.01 (unpaired t test). G, GLO assay showing increase in MGAT5 expression alters the cell sensitivity to sGal-3–induced apoptosis. Cells were transiently transfected as in C, then treated with sGal-3 for 6 to 12 hours, and caspase-3/7 activation measured. Note that increased MGAT5 expression sensitizes HFF-1 cells to sGal-3–induced caspase-3/7 activation, while MGAT5 knockdown reduces cell death in LN229 cells. Fold induction is based on ratio of sGal-3/control luciferase units. Fold ratio at 0 hour is 1 (n = 2). H, Western blot showing that transient transfection of MGAT5 renders 293 cells susceptible to sGal-3–mediated PARP cleavage.

Figure 6.

Alteration in MGAT5 expression affects complex N-glycan formation on β1 integrin, sGal-3–β1 integrin interaction, and sGal-3–mediated cell killing. A, GST–Gal-3 pull-down assay showing that NGI kifunensine (100 μmol/L), an α-mannosidase inhibitor that blocks N-glycan processing, abrogated GST–Gal-3–β1 integrin interaction. Loss of N-glycans reduces size of β1 integrin (see input). Treatment with OGI benzyl-N-acetyl-α-D-galactosamide (α-benzyl-GalNAc; 2 mmol/L) had no effect. B, Crystal Violet cell viability assay showing kifunensine pretreatment (24 hours) prevented sGal-3–mediated cell death in LN229 cells. Cells were pretreated with NGI or OGI for 24 hours, then 1× control (Ctrl) or sGal-3 CM was added, and cells incubated for another 72 hours. N = 3 (in triplicates). **, P < 0.01 (unpaired t test). C, PHA-L pull-down assay shows MGAT5 overexpression (+) increases, while MGAT5 knockdown (−) decreases PHA-L binding to β1 integrin. Cells were transiently transfected with control plasmid (Ctrl.) or expression vectors for MGAT5 (+) or shRNA for MGAT5 (−) and cell extracts were analyzed after 48 hours. PHA-L agarose beads were used to specifically pull down proteins carrying N-glycan branches synthesized by MGAT5, followed by Western blot analysis for β1 integrin (left). RT-PCR analysis (middle) and Western blot (right) show increase/decrease in MGAT5 mRNA/protein levels. D, Coimmunoprecipitation experiments showing increased/decreased binding of PHA-L to cell surface β1 integrins with overexpression (+) or knockdown (−) of MGAT5. Cells were transiently transfected as in C, then incubated with purified PHA-L (2.5 μg/mL) for 2 hours at room temperature, and cell extracts prepared. Cell surface proteins bound to PHA-L were then immunoprecipitated with anti–PHA-L antibodies (2.5 μg/mL) and protein G agarose beads followed by β1 integrin Western blot analysis (Ctrl., +, −) as above. E, GST–Gal-3 pull-down assay shows that alteration in MGAT5 expression modulates Gal-3 interaction with β1 integrin. Cells were transiently transfected as in C, then cell extracts were incubated with GST–Gal-3 beads, and bound β1 integrin detected by Western blot analysis (Ctrl., +, −) as above. F, Crystal Violet cell viability assay showing modulation of MGAT5 expression alters cell sensitivity to sGal-3–mediated killing. Note that increased MGAT5 expression sensitizes HFF-1 cells to sGal-3 killing. Cells were transiently transfected as in C, then treated with sGal-3 CM for 72 hours. N = 3 (in triplicates); *, P < 0.05; **, P < 0.01 (unpaired t test). G, GLO assay showing increase in MGAT5 expression alters the cell sensitivity to sGal-3–induced apoptosis. Cells were transiently transfected as in C, then treated with sGal-3 for 6 to 12 hours, and caspase-3/7 activation measured. Note that increased MGAT5 expression sensitizes HFF-1 cells to sGal-3–induced caspase-3/7 activation, while MGAT5 knockdown reduces cell death in LN229 cells. Fold induction is based on ratio of sGal-3/control luciferase units. Fold ratio at 0 hour is 1 (n = 2). H, Western blot showing that transient transfection of MGAT5 renders 293 cells susceptible to sGal-3–mediated PARP cleavage.

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MGAT5 regulates Gal-3/β1 integrin interaction

MGAT5 is the gatekeeper enzyme that controls the synthesis of complex tetra-antennary N-glycans on glycoproteins. It is one of the most important enzymes involved in the regulation of the biosynthesis of glycoprotein oligosaccharides and is believed to underlie aberrant N-glycosylation in cancer (5). MGAT-5 is an N-acetylglucosaminyltransferase that catalyzes the addition of N-acetylglucosamine in β1-6 linkage to the α-linked mannose of biantennary N-linked oligosaccharides present on newly synthesized glycoproteins in the Golgi apparatus (35). Addition of N-acetylglucosamine to α-mannose residues is the limiting step for further branch elongation into poly-N-acetyl-lactosamines (oligosaccharides comprised of repeating galactose and N-acetylglucosamine residues), which are preferential Gal-3 ligands (36). To determine whether MGAT5 modulation affects Gal-3/β1 integrin interactions, we used overexpression and shRNA neutralization studies. Increased MGAT5 expression augmented the presence of complex N-glycans on β1 integrin of both LN229 and HFF-1 cells as shown in PHA-L pull-down studies of cell extracts (Fig. 6C). PHA-L, is a phytohemagglutinin that has a carbohydrate-binding specificity for the complex N-glycans synthesized by MGAT5. Conversely, MGAT5 knockdown reduced PHA-L binding.

These results were confirmed in a more physiologic setting, where cultured cells were treated with pure PHA-L, washed and lysed, and PHA-L bound to cell surface β1 integrin immunoprecipitated with anti–PHA-L antibodies followed by Western blot for β1 integrin (Fig. 6D). Importantly, alteration in MGAT5 expression only affected the composition of cell surface N-glycans, without altering expression of β1 integrin. We then determined whether this posttranslational modification altered sGal-3 binding and mediated killing (Fig. 6E and F). Increased MGAT5 augmented β1 integrin binding and sGal-3 killing potency in LN229 cells, and remarkably, somewhat sensitized HFF-1 cells to Gal-3–induced cytotoxicity (Fig. 6F). Conversely, reduction in MGAT5 expression reduced LN229 susceptibility to sGal-3–mediated killing. These changes in cytotoxicity were accompanied with alterations in activation of caspase-3/7 (Fig. 6G). Overexpression of MGAT5 rendered HEK293 cells sensitive to sGal-3 killing in an N-glycan–dependent fashion (Fig. 6H; Supplementary Fig. S6A).

Overexpression of MGAT5 in glioma cells led to robust activation of β1 integrin as evidenced by talin binding to the cytoplasmic tail of β1 integrin (Supplementary Fig. S6B); a similar response was observed in fibroblasts despite lower transfection efficiency. Cotransfection of MGAT5 and β1 integrin in 293 cells also augmented β1 integrin activation (Supplementary Fig. S6C). Altogether, these results show that MGAT5 promotes β1 integrin conformational activation and the resultant binding to sGal-3.

Expression of N-glycosyltransferases is increased in tumor cells

Three enzymes are critical in the sequential synthesis of high-affinity glycan ligands for Gal-3, which are comprised of tetra-antennary branches containing poly-N-acetyl-lactosamines. Briefly, MGAT5 creates the GlcNAcβ(1–6)Man branched structure, which is further elaborated by β1,4-galactosyltransferases (B4GALT1-8). Subsequent and repetitive reactions by β1,3-acetylglucosaminyltransferases (B3GNT1-4) and β1,4-galactosyltransferases create the tandem repeats of Gal and GlcNAc that constitute poly-N-acetyl-lactosamines. To determine whether tumor cells express aberrant levels of these glycosyltransferases, we performed RT-PCR to assess their mRNA expression. Indeed, tumor cells expressed significantly higher levels of β3GnT2; β4GalT1, 2, and 5; and MGAT5 (Supplementary Fig. S7A, S7B, and S7D). The mRNA expression levels of MGAT5, β3GnT2, and β4GalT5 genes were also elevated in CD133+ GSCs compared with adult brain NPCs (Supplementary Fig. S7C).

To quantify the glycosyltransferase activities of these enzymes, we probed for the specific glycan structures they synthesize on β1 integrin using branch-specific pull-down assays followed by Western blotting (Supplementary Fig. S7D). MGAT5-specific glycans were pulled down with PHA-L-agarose, β4GalTs-specific glycans with RCA-agarose (37), and for the detection of N-acetyl-lactosamine, the joined product of β3GnT2 and β4GalTs action, we used the Gal-3-GST pull-down system. Tumor cells showed significantly higher amounts of glycan branches than normal cells (Supplementary Fig. S7E). In sum, these results show that tumor cells overexpress multiple glycosyltransferases (Supplementary Fig. S7B), which leads to the coordinated formation of tetra-antennary branches with poly-N-acetyl-lactosamine on β1 integrin (Supplementary Fig. S7D), and explains why tumor cells display enhanced susceptibility to sGal-3–mediated killing.

We have engineered a chimeric form of sGal-3, a major glycan-binding protein, by extending its N-terminus through the conjugation of a noncleaved signal peptide. sGal-3 exhibits approximately 100-fold greater proapoptotic activity toward cancer cells than recombinant wild-type Gal-3. sGal-3 preferentially binds to cancer cells, which harbor aberrant complex N-glycans on their cell surface due to oncogenesis-related upregulation of glycosyltransferases (38). sGal-3 displays selective and potent cytotoxicity toward cancer cells, and induces a novel oncoglycanated-β1 integrin/calpain/caspase-9 proapoptotic signaling axis that is independent of anoikis, which is caspase-8 dependent. sGal-3 is cytotoxic to a variety of genetically heterogeneous cancer cell lines from different organs, even death-resistant cells that have hyperactivation of survival signaling or defective apoptosis pathways (39). Thus, we unveil a new neoglycan-based cancer cell susceptibility to cell death, which can be further exploited for cancer treatment.

Our data shows that β1 integrin expression is necessary but not sufficient for sGal-3–mediated killing. While both normal and tumor cells express variable levels of β1 integrin, cancer cells display aberrantly enhanced glycosylation of β1 integrin and we show this underlies the selective proapoptotic effect of sGal-3 on cancer cells. Alteration in cell surface β1 integrin glycosylation can promote tumor dissemination (11), and we now show that such tumor-specific epitopes can be targeted for cytotoxic therapy. Because β1 integrin has 11 N-glycosylated Asparagine (Asn) residues on its extracellular domain, further work is necessary to determine which of these carry the critical N-glycans responsible for killing selectivity and what novel types of glycan chains are formed. Determining which of the 12 possible αxβ1 integrin dimers can initiate apoptosis in response to sGal-3 also needs further investigation.

Additional work is also warranted to determine why sGal-3 is approximately 60- to 100-fold more potent at tumor cell killing than rGal-3. One possibility is that preparation of rGal-3 from bacteria compromises its binding activity. To address this caveat, we demonstrated efficient Gal-3–β1 integrin interaction in a pull-down experiment, using recombinant GST–Gal-3. While endogenous Gal-3 is secreted through a nonclassical Golgi apparatus–independent secretion mechanism, the N-terminal signal peptide of sGal-3 will direct it toward classical secretion. The involvement of the Golgi apparatus in sGal-3 maturation may lead to posttranslational modifications not present on endogenous Gal-3. To address this possibility, we produced sGal-3 in cells treated with either N- or O-glycosylation inhibitors, but did not see a change in sGal-3 molecular weight. A more plausible explanation for the novel death-inducing activity of sGal-3 is that the conjugated chimeric N-terminal signal peptide alters protein conformation and behavior. Indeed, we showed that it can induce a shift in β1 integrin conformation to the active state. The tPA signal peptide possesses 15 hydrophobic amino acids in α-helical structure, which may facilitate α-helix–mediated protein–protein interaction. Because extracellular Gal-3 can oligomerize through its N-terminus (40, 41), the chimeric signal peptide of sGal-3 could potentially facilitate this process and augment Gal-3–β1 integrin binding, and potentiate downstream signaling via cross-linked integrin complexes. Further work is necessary to address this hypothesis, but it is supported by prior work with Galectin-1, where addition of rigid α-helix or flexible random coil linkers between CRD domains promoted multimerization, lattice formation, and augmented glycan binding (42).

Conceptually, sGal-3 binding to β1 integrin could either activate a proapoptotic signaling cascade downstream of the activated integrin, or alternatively antagonize preexisting ligand-activated β1 integrin-mediated survival signals. The fact that anoikis, a form of apoptosis induced by cell detachment from the extracellular matrix (ECM), is caspase-8–dependent, rather than caspase-9 in our study; and our finding that knockdown of β1 integrin prevents sGal-3–induced apoptosis, argue against integrin survival signaling blockage as a major mechanism. While growing our cells on standard negatively charged polystyrene (no ECM coating), we considered that integrin-mediated survival signaling might be activated by the cells' own secreted ECM components. To test this, we examined the activation status of focal adhesion kinase (FAK), a hallmark of ECM-mediated integrin survival signaling, but found no FAK phosphorylation at Y397 (as a positive control we showed fibronectin induced phospho-FAK; data not shown).

Further investigation into the sGal-3 killing mechanism allowed us to define its critical mediators. sGal-3 binds activated β1 integrin, and thereby rapidly triggers intracellular Ca2+ influx and subsequent calpain activation. Calpain is a member of a family of calcium-dependent cysteine proteases, and can activate GSK3β through N-terminal cleavage (43). Calpain can coordinate apoptosis induction via proteolytic activation of proapoptotic factors at the mitochondria (44). Concordantly, we found that sGal-3 treatment triggered a calpain-dependent proapoptotic cascade, starting from cleavage-mediated activation of GSK3α/β, to downstream Bax, Bad, and PARP cleavage. Calpain activation is calcium dependent, which is regulated by calcium channels (33), and we found that inhibition of calcium channel function successfully interfered with sGal-3/integrin-mediated calpain activation and ensuing apoptotic cell death signals.

Abnormal glycan structures are present in cancer because of altered glycosyltransferase activities (3). Aberrant glycosylation was once thought to be a passenger event in cellular transformation, but is now increasingly recognized as a driver in tumor formation (1). Elevated MGAT5 expression has been reported in various tumor cells and is believed to facilitate tumor growth, angiogenesis, and metastasis, making it a therapeutic target for cancer (45). Our study evidenced overexpression of MGAT5 in a panel of genetically heterogeneous cancer cells and showed this was, at least in part, the basis for their susceptibility to sGal-3–mediated killing. Neutralization of MGAT5 expression by RNA interference induced resistance to sGal-3 in tumor cells, whereas sensitivity to sGal-3–mediated killing could be transferred to normal cells by forced expression of MGAT5. In addition, overexpression of MGAT5 switched β1 integrin into its active confirmation and elevated β1–talin interaction in tumor cells.

Our study further showed that the expression levels of glycosyltransferases (β4GalTs and β3GnT2) that act downstream of MGAT5 in mediating the elongation of tetra-antennary N-glycans into poly-N-acetyl-lactosamine chains were also overexpressed in cancer cells in a coordinated fashion. This led to enhanced N-glycan formation on β1 integrin as detected by glycan-specific plant lectin binding assays. Consistently, inhibition of either β1 integrin expression or N-glycosylation abrogated sGal-3–induced apoptosis. While cancer cell overexpression of individual glycosyltransferases was in the range of 2–2.5 fold, we anticipate that their combined upregulation has a synergistic effect in generating more complex N-glycans on β1 integrin in tumor cells. This likely explains tumor cell hypersensitivity to sGal-3 and suggests a promising therapeutic index for cancer treatment.

Our results shed further light on the glycosylation-dependent proapoptotic role of sGal-3 and its possible exploitation for cancer therapy. We showed cancer cells are more sensitive to sGal-3–induced apoptosis compared with normal cells and sGal-3 strongly inhibited tumor cell growth and prolonged mouse survival in subcutaneous and intracranial mouse models. Local delivery of sGal-3 to the tumor microenvironment had no obvious toxicity and was well tolerated by the animals. Therefore, our results support further investigations into using tPA signal peptide–Gal-3 conjugate as a wide-spectrum anticancer therapeutic. Novel therapeutic agents have been developed by conjugating natural peptides, peptide analogues, and chemical agents for improved selectivity, efficiency, and safety (17, 46). The normal function of N-terminal signal peptides is to guide protein sorting and secretion, but it has been shown that uncleaved signal peptides can modify protein function (47, 48).

In conclusion, our study provides proof-of-principle for the targeting of neosynthesized glycoantigens in cancer and shows this can be achieved through treatment with a novel signal peptide–galectin-3 conjugate, which potently and specifically induces cancer cell death. It further demonstrates the potential of chimeric signal peptide conjugation as a novel approach to dramatically enhance protein therapeutic activity.

C.G. Hadjipanayis is a paid consultant for Synaptive Corp. and NX Development Corp. J.J. Olson is a paid consultant for the American Cancer Society. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S-H. Lee, E.G. Van Meir, F. Khwaja Rehman, A. Zerrouqi, R.D. Cummings

Development of methodology: S-H. Lee, F. Khwaja Rehman, K.C. Tyler, A. Zerrouqi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S-H. Lee, E.G. Van Meir, F. Khwaja Rehman, K.C. Tyler, B. Yu, Z. Zhang, A. Zerrouqi, C.G. Hadjipanayis, J.J. Olson, S. Osuka

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S-H. Lee, E.G. Van Meir, F. Khwaja Rehman, Z. Zhang, R.D. Cummings

Writing, review, and/or revision of the manuscript: E.G. Van Meir, F. Khwaja Rehman, R.D. Cummings

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S-H. Lee, E.G. Van Meir

Study supervision: E.G. Van Meir

Other (generation and characterization of Gal-3 inducible cell lines, performed tumor growth studies): S-H. Lee, A. Zerrouqi

Other [lab manager (technician)]: N.S. Devi

Other (provided N08-74 human glioma stem cells): M. Kaluzova, C.G. Hadjipanayis

We thank Dr. W. Stallcup for GST-Gal-3 expression vectors, Dr. E. Miyoshi for the pCXN2-MGAT5 plasmid, and Dr. M. Pierce for the pSUPER-MGAT5 plasmid. This work was supported by NIH grants R01-CA086335, R01-CA163722, and R01-NS096236 (to E.G. Van Meir); U01-CA168930 (to R.D. Cummings); the Pediatric Brain Tumor Foundation of the US (to E.G. Van Meir); the Goldhirsh Foundation (to E.G. Van Meir); the Genetics and Molecular Biology program of the Laney Graduate School of Emory University (to F. Khwaja Rehman); and the NSF (PRISM; DGE0231900 to F. Khwaja Rehman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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