Published OnlineFirst January 22, 2020; DOI: 10.1158/1078-0432.CCR-18-3280 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY A Chimeric Signal Peptide–Galectin-3 Conjugate Induces Glycosylation-DependentCancerCell–SpecificApoptosis Sok-Hyong Lee1, Fatima Khwaja Rehman1, Kari C. Tyler1, Bing Yu1, Zhaobin Zhang1, Satoru Osuka1, Abdessamad Zerrouqi1, Milota Kaluzova1, Costas G. Hadjipanayis1, Richard D. Cummings2,3, Jeffrey J. Olson1,2, Narra S. Devi1, and Erwin G. Van Meir1,2,4,5,6 ABSTRACT ◥ Purpose: Exploitation of altered glycosylation in cancer is a Results: We found sGal-3 preferentially binds to b1 integrin on major goal for the design of new cancer therapy. Here, we designed the surface of tumor cells due to aberrant N-glycosylation resulting a novel secreted chimeric signal peptide–Galectin-3 conjugate from cancer-associated upregulation of several glycosyltransferases. (sGal-3) and investigated its ability to induce cancer-specific cell This interaction induces potent cancer-specific death by triggering death by targeting aberrantly N-glycosylated cell surface receptors an oncoglycan-b1/calpain/caspase-9 proapoptotic signaling cas- on cancer cells. cade. sGal-3 could reduce the growth of subcutaneous lung cancers Experimental Design: sGal-3 was genetically engineered from and malignant gliomas in brain, leading to increased animal sur- Gal-3 by extending its N-terminus with a noncleavable signal vival. peptide from tissue plasminogen activator. sGal-3 killing ability Conclusions: We demonstrate that sGal-3 kills aberrantly was tested on normal and tumor cells in vitro and its antitumor glycosylated tumor cells and antagonizes tumor growth activity was evaluated in subcutaneous lung cancer and orthotopic through a novel integrin b1–dependent cell-extrinsic apoptotic malignant glioma models. The mechanism of killing was investi- pathway. These findings provide proof-of-principle that aber- gated through assays detecting sGal-3 interaction with specific rant N-oncoglycans represent valid cancer targets and support glycans on the surface of tumor cells and the elicited downstream further translation of the chimeric sGal-3 peptide conjugate for proapoptotic signaling. cancer therapy. Introduction the synthesis of GlcNAcb1,6-Mannose glycan branches, leading to the formation of tetra-antennary N-glycans (5), which carry poly-N- Altered glycosylation of cell surface proteins is a characteristic of acetyl-lactosamine, the preferred ligand for Galectin-3 (Gal-3). Con- cancer and leads to abnormal glycoproteins with N- and O-glycans on sequently, there is a strong interest in exploiting aberrantly glycosy- human tumors (1). Tumor-associated glycosylation occurs through lated tumor cell surface proteins as targets for cancer therapy (3), but two mechanisms: “incomplete synthesis,” which leads to truncated this objective has not been realized to date (6). forms of normal glycan structures (2), and “neo-synthesis,” where Integrins are cell surface adhesion molecules composed of dimers of abnormal cancer-specific glycans are generated through cancer- a and b subunits and their signaling is important for cell proliferation, associated changes in expression of glycosyltransferases (3). Aberrant- survival, cytoskeletal reorganization, and migration. Upon activation, ly enhanced glycosylation contributes to tumorigenesis and tumor integrins undergo rapid, reversible conformational changes, which immune evasion (4). Carcinomas show increased expression of N- promote recruitment of intracellular signaling molecules (7). Modi- acetylglucosaminyltransferase V (MGAT5), an enzyme that catalyzes fication 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 1Department of Neurosurgery, Emory University, Atlanta, Georgia. 2Winship Cancer Institute, Emory University, Atlanta, Georgia. 3Department of Biochem- integrins increases the migration and metastasis potential of several istry, Emory University, Atlanta, Georgia. 4Department of Hematology & Medical cancer types (11). Oncology, School of Medicine, Emory University, Atlanta, Georgia. 5Department Lectins are carbohydrate-binding proteins that recognize distinct of Neurosurgery, School of Medicine, University of Alabama at Birmingham, glycan moieties on glycoproteins or glycolipids through carbohydrate 6 Alabama. O'Neal Comprehensive Cancer Center, University of Alabama at recognition domains (CRD). Gal-3 belongs to the b-galactoside bind- Birmingham, Alabama. ing galectin family, is secreted through a nonclassical secretion path- Note: Supplementary data for this article are available at Clinical Cancer way, and is unique among galectins in that it carries a long collagen-like Research Online (http://clincancerres.aacrjournals.org/). N-terminal domain that permits oligomerization, and for its dual role Current address for F. Khwaja Rehman: University of North Florida, 1 UNF Drive, in apoptosis (12). Intracellular Gal-3 has a well-documented role as Jacksonville, Florida 32224; current address for A. Zerrouqi, Medical University antiapoptotic factor in cytoplasm and at the mitochondrial membrane of Warsaw, Department of Biochemistry, Warsaw, Poland; and current address through carbohydrate-independent mechanisms (13, 14). In contrast, for R.D. Cummings, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. carbohydrate-dependent proapoptotic responses of extracellular Gal-3 have been observed at supraphysiologic concentrations (1–10 mmol/L) Corresponding Author: Erwin G. Van Meir, University of Alabama at Birming- in lymphocytic cells (15), but not in other adherent cell types where ham, WTI 520E, 1720 2nd Ave. South, Birmingham, GA 35294. Phone: 205-975- 0694; E-mail: [email protected] Gal-3 regulates migration through the modulation of cell adhesion molecules and extracellular matrix (16). Clin Cancer Res 2020;26:2711–24 Here, we sought to determine whether the weak proapoptotic doi: 10.1158/1078-0432.CCR-18-3280 activity of extracellular Gal-3 could be enhanced through a peptide Ó2020 American Association for Cancer Research. conjugate approach in its N-terminus, and harnessed for targeting AACRJournals.org | 2711 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst January 22, 2020; DOI: 10.1158/1078-0432.CCR-18-3280 Lee et al. sGal-3 conditioned media (CM) were added to each well. For MGAT5 Translational Relevance overexpression or knockdown studies, the pCXN2-MGAT5 and pSU- Tumor-selective targeting is a critical requirement for suc- PER-MGAT5 expression vectors were used (11, 21). cessful cancer therapeutics. A major difference between normal and cancer cells is the presence of aberrantly enhanced glycans Generation of stably transfected cells – on tumor cells. Yet, successful exploitation of augmented gly- Doxycycline-inducible sGal-3 transfected clones were generated in cosylation in cancer for the design of new cancer therapy has LN229-L16 glioma cells (Tet-on clone derived from LN229; ref. 22) by remained elusive. Here, we show that an engineered chimeric transfecting them with pCMV-Neo and pTRE-sGal3 plasmids (1:10 m form of Galectin-3 can potently induce cancer-specific cell death ratio) and selecting stable clones with 1,000 g/mL of G418. 293 cells by targeting aberrantly enhanced N-glycans on tumor cells. stably expressing MGAT5 were generated as described previously (23). These findings provide proof-of-principle that aberrant N-onco- Production of sGal-3 glycans represent valid cancer targets that can be successfully 293 cells were transiently transfected using GenePORTER reagent targeted for therapeutic gain. (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 À20C or used undiluted (1Â) on target cells in cell aberrantly glycosylated cancer cells. Peptide conjugates have been viability assays. Purification of sGal-3 from CM was performed using a developed to selectively enhance cell surface receptor binding (17). lactosyl-Sepharose column as described previously (24). Preparation Our study demonstrates that aberrant b1-integrin glycosylation can be and purification of His-tag sGal3 was performed as described previ- exploited for tumor-specific targeting with a signal peptide conjugated ously (25); see details in Supplementary Materials and Methods. Gal-3, supporting its further development for therapeutic applications. RT/PCR and Western blot analyses Detailed information for immunoblot preparation is provided in Materials and Methods Supplementary Materials and Methods. Chemicals, cell lines, and primary cells Antibody-mediated activity blocking assays Recombinant human galectin-3, sugars (lactose, sucrose, and meli- Neutralization of b1 integrin on cells was achieved by addition of 5 biose), apoptosis inhibitors [caspase-3 (Ac-DEVD-CHO), caspase-8 mg/mL anti-human b1 integrin inhibitory antibodies (clones P5D2 and (Z-IETD-FMK), and caspase-9 (Z-LEHD-FMK)], glycosylation inhi- AIIB2; The Developmental Studies Hybridoma Bank) or control bitors (kifunensine for N-linked glycosylation and Benzyl 2-aceta- immunoglobulins (normal mouse IgG, Santa Cruz Biotechnology) mido-2-deoxy-a-D-galactopyranoside for O-linked glycosylation),