Galectin-1 inhibition induces cell apoptosis through dual suppression of CXCR4 and Ras pathways in human malignant peripheral nerve sheath tumors

Abstract

Background: The Ras signaling pathway is commonly dysregulated in human malignant peripheral nerve sheath tumors (MPNSTs). It is well known that galectin-1 (Gal-1) is essential to stabilize membrane Ras and thereby induce the activation of Ras. However, the role of Gal-1 in MPNST progression remains unknown. The aim of this study was to examine whether Gal-1 knockdown could have an effect on the Ras signaling pathway.

Methods: Cell viability, apoptosis assay, and colony formation were performed to examine the effects of inhibition of Gal-1 in MPNST cells. We used a human MPNST xenograft model to assess growth and metastasis inhibitory effects of Gal-1 inhibitor LLS2.

Results: Gal-1 was upregulated in MPNST patients and was highly expressed in MPNST cells. Knockdown of Gal-1 by small interfering (si)RNA in Gal-1 expressing MPNST cells significantly reduces cell proliferation through the suppression of C-X-C chemokine receptor type 4 (CXCR4) and the rat sarcoma viral oncogene homolog (RAS)/extracellular signal-regulated kinase (ERK) pathway, which are important oncogenic signaling in MPNST development. Moreover, Gal-1 knockdown induces apoptosis and inhibits colony formation. LLS2, a novel Gal-1 allosteric small molecule inhibitor, is cytotoxic against MPNST cells and was able to induce apoptosis and suppress colony formation in MPNST cells. LLS2 treatment and Gal-1 knockdown exhibited similar effects on the suppression of CXCR4 and RAS/ERK pathways. More importantly, inhibition of Gal-1 expression or function by treatment with either siRNA or LLS2 resulted in significant tumor responses in an MPNST xenograft model.

Conclusion: Our results identified an oncogenic role of Gal-1 in MPNST and that its inhibitor, LLS2, is a potential therapeutic agent, applied topically or systemically, against MPNST.

Keywords: CXCR4; LLS2; MPNST; RAS; galectin-1.

Fig. 1

Gal-1 expression in human MPNST. (A) NF1, CXCR4, and LGALS1 transcript levels from 2 Oncomine datasets. Henderson, MPNST, 4 patients; Nakayama, MPNST, 3 patients. Data, representative units according to Oncomine output. (B) The expression levels of Gal-1 were evaluated by immunohistochemistry (magnification, x400). Gal-1 expression in 7, 4, and 22 samples of normal nerve tissue, neurofibroma, and MPNST, respectively. Gal-1 expression was scored and displayed by boxplot (dashed line: mean; lines above and below the dashed line, third quartile to the first quartile). ***P < 0.001; 2-tailed Student’s t-test.

Fig. 2

Effect of Gal-1 inhibition in MPNST cells. (A) Immunoblots demonstrate the expression of neurofibromin in normal huSC (positive control), NF96.2, and NF2.2 cells. (B) Suppression of endogenous Gal-1 expression by siRNA in NF1^−/− NF96.2 and NF1^+/− NF2.2 MPNST cells. (C) MPNST cells transfected with 40 nM scramble control siRNA or siRNA against Gal-1; cell survival was determined by MTT. (D) Morphology of MPNST cells 72 h after transfected with 40 nM scramble control siRNA or siRNA against Gal-1. (E) Caspase-3/7 activities in NF96.2 and NF2.2 cells 72 h after transfection with 40 nM siRNA. ***P < 0.001; 2-tailed Student’s t-test. Data shown are mean ± SD.

Fig. 3

Effect of Gal-1 knockdown in NF1^−/− NF96.2 xenograft model. (A) Suppression of endogenous Gal-1 expression by shRNA in NF96.2 cells, and cell proliferation was measured. (B) Colony formation on NF96.2 cells and quantification of colony number larger than 50 μm after 2 weeks. (C) Bioluminescent images show the growth of subcutaneously implanted luciferase-tagged NF96.2 cells (stably transfected with shRNA or Gal-1 shRNA) in nude mice on week 14 (n = 6 mice per group). (D) Quantification of the tumor bioluminescent signal. (E) Weight of the tumor xenografts. **P < 0.01, ***P < 0.001; 2-tailed Student’s t-test. Data shown are mean ± SD.

Fig. 4

Effect of Gal-1 knockdown on Ras pathways in MPNST cells. (A) Co-localization of Gal-1 and Ras. (B) Active Ras pull-down assay. NF96.2 and NF2.2 cells were co-transfected with 1.5 μg shControl or shGal-1, and 1 μg pcDNA or pcDNA-H-Ras(G12V). Seventy-two hours post-transfection, cell lysates were extracted and incubated with Raf-1 RBD agarose. Input: total protein extracted from MPNST cells were not incubated with Raf-1 RBD agarose. Eluted proteins were analyzed by SDS-PAGE and immunoblotted with anti-RAS antibodies. (C) CXCR4 was examined by immuoblotting after 72 h post-transfection with control shRNA or shGal-1, and pcDNA or pcDNA-CXCR4. (D) Immunoblotting of phospho-MEK, MEK, phospho-ERK, and ERK in NF96.2 and NF2.2 transfected with 1 μg control shRNA or shGal-1 for 72 h. (E) NF96.2 and NF2.2 cells were co-transfected with 1 μg control shRNA or shGal-1, and 1 μg pcDNA or pcDNA-H-Ras(G12V) or pcDNA-CXCR4; cell survival and (F) caspase-3/7 activities were measured at 72 h after transfection. *P < 0.05, **P < 0.01, ***P < 0.001; 2-tailed Student’s t-test. Data shown are mean ± SD. Scale bars, 20 μm.

Fig. 5

The therapeutic efficacy of LLS2 against MPNST cells. (A) Cell survival of NF96.2 and NF2.2 MPNST cells treated with indicated concentrations of LLS2 for 72 h. Half-maximal inhibitory concentrations of LLS2 on NF96.2 and NF2.2 are 21.3 μM and 31.2 μM, respectively. (B) Caspase-3/7 activities in NF96.2 and NF2.2 cells after 24 h treated with 0.25% DMSO or 25 µM LLS2 (C) Representative images and quantification of colony formation. (D) Active Ras pull-down assay. MPNST cells were treated with 0.25% DMSO or 25 μM LLS2, and cell lysates were prepared at 24 h, followed by incubating with Raf-1 RBD agarose. Input: total protein extracted from MPNST cells were not incubated with Raf-1 RBD agarose. Eluted proteins were analyzed by SDS-PAGE and immunoblotted with RAS antibodies. (E) Immunoblots of CXCR4, phospho-MEK, MEK, phospho-ERK, and ERK in NF96.2 and NF2.2 treated with vehicle control 0.25 % DMSO or 25 μM LLS2 for 24 h. ***P < 0.001; 2-tailed Student’s t-test. Data shown are mean ± SD.

Fig. 6

LLS2 have antitumor activity in NF1^−/− NF96.2 xenograft model. (A) Xenograft tumors. (B) Volume and (C) weight of the tumor xenografts were measured at 3 weeks after treatment. (D) Timeline of LLS2 treatment protocol; bioluminescent images for vehicle (8.7% alcohol/8.7% cremophor) or LLS2 treated mice were obtained at 12 weeks after initial i.v. injection of NF96.2 cells. **P < 0.01, ***P < 0.001; 2-tailed Student’s t-test, n = 6. Data shown are mean ± SD.