Galectin-3, a Druggable Vulnerability for KRAS-Addicted Cancers

Abstract

Identifying the molecular basis for cancer cell dependence on oncogenes such as KRAS can provide new opportunities to target these addictions. Here, we identify a novel role for the carbohydrate-binding protein galectin-3 as a lynchpin for KRAS dependence. By directly binding to the cell surface receptor integrin αvβ3, galectin-3 gives rise to KRAS addiction by enabling multiple functions of KRAS in anchorage-independent cells, including formation of macropinosomes that facilitate nutrient uptake and ability to maintain redox balance. Disrupting αvβ3/galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvβ3-expressing KRAS-mutant lung and pancreatic cancer patient-derived xenografts and spontaneous tumors in mice. Our work reveals galectin-3 as a druggable target for KRAS-addicted lung and pancreas cancers, and indicates integrin αvβ3 as a biomarker to identify susceptible tumors.Significance: There is a significant unmet need for therapies targeting KRAS-mutant cancers. Here, we identify integrin αvβ3 as a biomarker to identify mutant KRAS-addicted tumors that are highly sensitive to inhibition of galectin-3, a glycoprotein that binds to integrin αvβ3 to promote KRAS-mediated activation of AKT. Cancer Discov; 7(12); 1464-79. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

Figure 1. Integrin αvβ3 and Galectin-3 drive KRAS addiction for a subset of KRAS mutant lung cancers

A) A subset of KRAS mutant lung cancer cells express integrin αvβ3 B–D) 3D growth capacity was quantified by counting colony formation in soft agar for 14 days. E) Effect of shRNA-mediated knockdowns on 2D or 3D viability was measured using CellTiter-Glo® for 72 hours. F–G) Patient-derived xenograft (PDX) tumors were stained for β3 expression (brown) using immunohistochemistry. Scale bar, 50μm. The effect of KRAS, Galectin-3, or β3 knockdown on 3D growth was evaluated as colony formation in soft agar. H–I) Western blots show phospho-Akt and phospho-Erk. J–K) Embryonic fibroblasts from genetically-engineered mice (wildtype, KRAS G12D, Galectin-3 knockout, or the combination) were compared for KRAS activity using a GST-pull down assay and colony formation in 2D. Scale bar, 250μm. All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001. See also Supplementary Fig. S1.

Figure 2. KRAS-addicted lung cancer cells achieve enhanced nutrient uptake via macropinocytosis

A) Macropinocytosis uptake assay using TMR-dextran as a marker of macropinosomes (red) in lung cancer cells after 4 hours in 3D under serum deprivation, including the macropinocytosis inhibitor, EIPA. Scale bar, 10μm. B) Uptake and proteolytic cleavage de-quenches the DQ-BSA signal (green), indicating uptake of nutrients into functional macropinosomes. Scale bar, 10μm. C) αvβ3-positive A549 cells require expression of KRAS, β3, and Galectin-3 for uptake of TMR-dextran, while this is enhanced in αvβ3-negative H727 cells by ectopic β3 expression. D) For patient-derived xenograft tumors with heterogeneous expression of αvβ3, the DQ-BSA signal indicating macropinocytotic uptake is enhanced in cells positive for β3 expression. Scale bar, 10μm. E) Inhibitors of macropinocytosis (EIPA) vs. clathrin-mediated endocytosis (Chlorpromazine) were tested for their effect on cell viability in 3D. F) Ectopic expression of β3 integrin sensitized H727 cells to the effects of the macropinocytosis inhibitor EIPA. G) Patient-derived xenograft tumors with heterogeneous expression of αvβ3 were sorted by β3 expression and the effects of EIPA on cell viability in 3D was tested using the CellTiter-Glo assay. H) In mouse fibroblasts, expression of oncogenic KRAS and Galectin-3 is required for sensitivity to EIPA. All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001. See also Supplementary Fig. S2.

Figure 3. KRAS-addicted lung cancer cells need αvβ3/Galectin-3 to maintain low ROS levels

A) The effect of KRAS knockdown on mitochondrial ROS levels was visualized as fluorescence staining using MitoSOX Red or 8-oxo-dG. MitoSOX Red staining was quantified by flow cytometry (4 hours). Scale bar, 10μm. B) The effect of oxidative stress (hydrogen peroxide) on viability was compared for cells growing in suspension. C) Ectopic β3 expression protects H727 cells from the effects of oxidative stress. D) By flow cytometry, the β3-high population sorted from patient-derived xenograft PDX-8 show lower levels of mitochondrial stress (MitoSOX) compared with the β3-low population. E) The effect of β3 or Galectin-3 knockdown on ROS levels was evaluated using flow cytometry (MitoSOX). All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Blocking αvβ3/Galectin-3 binding with GCS-100 selectively kills KRAS-addicted lung cancer cells

A–B) A cell-free binding assay shows direct binding between integrin αvβ3 and Galectin-3, and its competitive inhibition by lactose. C) A Galectin-3 inhibitor, GCS-100, disrupts αvβ3/Galectin-3 binding in the cell free assay. D) Flow cytometer analysis for cell surface Galectin-3 expression. E–F) The effect of GCS-100 on 3D viability of KRAS mutant lung cancer cell lines and PDX models is enhanced by αvβ3 expression. G) Embryonic fibroblasts from genetically-engineered mice (wildtype, KRAS G12D, Galectin-3 knockout, or the combination) were compared for sensitivity to GCS-100. All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001.

Figure 5. GCS-100 selectively kills KRAS-addicted lung tumors

A) Mice with established subcutaneous KRAS mutant lung PDX-8 tumors were treated with vehicle (n=9) or GCS-100 (n=10) (20 mg/kg i.p. 3 times per week) for 15 days. Change in tumor volume vs. time (left) and after 15 days of treatment (right). B) Sections from PDX-8 tumors were stained for TUNEL as an indicator of apoptosis, and the percent of TUNEL+ cells quantified using ImageJ. Scale bar, 50μm. C–D) KRAS^LSLG12D mice were infected with Adeno-Cre via intratracheal injection. After 3 months, sections of lung tissue reveal β3-expressing tumors with areas of adenoma, dysplasia, and adenocarcinoma. Scale bar, 50μm. E) Mice were randomized and treated with either vehicle control or GCS-100 for 1 additional month. Representative histological images show lung tumor burden. Scale bar, 2 mm and 500μm for inset. Tumor burden (tumor area as a percentage of total lung area) in KRAS^LSLG12D mice treated with vehicle (n=7) or GCS-100 (n=7) at a dose of 20 mg/kg i.p. 3 times per week. Table shows effect of the drug on tumor histopathology. F) Effect of GCS-100 on apoptosis, evaluated using TUNEL staining by IHC. Scale bar, 50μm. All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001.

Figure 6. GCS-100 shows efficacy only for KRAS mutant αvβ3+ cancer cells

A) Protein expression of integrin β3 is shown for a panel of KRAS mutant pancreatic cancer cell lines. B) Expression of αvβ3 enhances sensitivity to GCS-100 for pancreatic cancer cells in vitro. C–D) Mice with established subcutaneous FG, FG+β3, or PDX pancreatic tumors were treated with vehicle or GCS-100 (20 mg/kg i.p. 3 times per week). Change in tumor volume vs. time (left) and at the endpoint (right). Scale bar, 5mm. E) PDX tumor sections were stained for TUNEL as an indicator of apoptosis and Ki-67 as an indicator of cell proliferation, and the percent of positive cells quantified using ImageJ. Scale bar, 100μm. All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001.

Figure 7. Galectin-3 blockade prevents αvβ3/KRAS complex, reduces nutrient uptake, and enhances ROS levels

A–B) Disrupting Galectin-3 using GCS-100 prevents biochemical association between αvβ3 and KRAS, and blocks phosphorylation of Akt. C–E) p-Akt immunostaining is significantly suppressed in tumors from mice treated systemically with GCS-100. Scale bar, 5μm. F) GCS-100 treatment reduces macropinocytosis in αvβ3-positive lung cancer cells, measured as TMR-dextran uptake and DQ-BSA de-quenching. Scale bar, 10μm. G) Representative images from vehicle or GCS-100-treated PDX tumors incubated ex vivo with DQ-BSA (green). Tumor cells are marked by anti-cytokeratin staining (red) Scale bar, 50 μm. FITC-BSA uptake was quantified using ImageJ. H) Treating αvβ3-positive A549 or H1792 cells with GCS-100 (to inhibit Galectin-3) increases mitochondrial ROS levels (MitoSox signal analyzed by flow cytometry). I) Immunohistochemical analysis of oxidative stress (8-oxo-dG expression) in FG, FG+β3, and PDX-8 tumors treated with vehicle or GCS-100. Scale bars, 20 μm (in E) and 100μm (in F). J) Schematic summarizes the molecular basis for KRAS addiction of KRAS-dependent lung cancer and potential for clinical development of GCS-100. All data represent mean ± standard deviation from at least 3 independent experiments. Statistical significance was determined by Student’s t-test *P<0.05, **P<0.01, ***P<0.001.