AXL is a potential therapeutic target in dedifferentiated and pleomorphic liposarcomas

Abstract

Background: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined.

Methods: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo.

Results: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo.

Conclusions: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.

Fig. 1

AXL and its ligand, GAS6, are highly expressed in DDLPS and PLS. a AXL expression was assessed by IHC analysis of TMAs containing NF samples and WDLPS, DDLPS, and PLS tumor samples (representative images are shown, 200× magnification). b Western blot analyses of TAM RTKs in panels of DDLPS and PLS cell lines. c GAS6 secretion was measured by ELISA in conditioned media for the indicated DDLPS and PLS cell lines. The data are means with SEMs for triplicate experiments (* = p < 0.05)

Fig. 2

GAS6-mediated AXL activation increases protumorigenic properties of LPS cells in vitro. a DDLPS (Lipo-246 and Lipo-863) or PLS (LiSa2 and PLS-1) cells were incubated with low-serum media for 24 h prior to a 15 min stimulation with 400 ng/mL GAS6 and analyzed via western blotting. b Changes in DDLPS and PLS cell proliferation were measured by MTS assays with or without GAS6 stimulation for 48 h. Percent of cell growth is expressed as a percentage of control. c Modified Boyden chambers were used to assess the effects of GAS6 stimulation on cell migration and invasion. Representative images of each condition are shown (200× magnification). Percent of cell migration and invasion is expressed as a percentage of control. The data in the bar graphs are means with SEMs for triplicate experiments (* = p < 0.05; *** = p < 0.001)

Fig. 3

Transient knockdown of AXL decreases cell proliferation, migration, and invasion. a Representative western blots showing signaling dysregulation in Lipo-246, Lipo-863, LiSa2, and PLS-1 cells that were transfected with either NT or AXL-specific siRNA. b Cell proliferation of AXL-specific siRNA-transfected DDLPS and PLS cell lines was measured by MTS assay after 48 h (96 h post-transfection). Percent of cell growth is expressed as a percentage of control. c Modified Boyden chambers were used to assess DDLPS and PLS cell migration and invasion following transfection with AXL-targeting siRNA. Percent of cell migration and invasion is expressed as a percentage of control. The data in the bar graphs are means with SEMs for triplicate experiments (* = p < 0.05; *** = p < 0.001)

Fig. 4

Stable knockdown of AXL in DDLPS and PLS cells reduces cell proliferation, migration, and invasion. a Representative western blots showing signaling alterations induced with stable knockdown of AXL in DDLPS and PLS cell lines. b MTS assays were performed on DDLPS and PLS cell lines to evaluate cell proliferation after 48 and 96 h in AXL knockdown cells. Percent of cell growth is expressed as a percentage of control. c After stable knockdown of AXL, the ability of DDLPS and PLS cell lines to migrate and invade was evaluated. Percent of cell migration and invasion is expressed as a percentage of control. The data in the bar graphs are means with SEMs for triplicate experiments (* = p < 0.05; *** = p < 0.001)

Fig. 5

Stable AXL knockdown in Lipo-246 cells significantly reduces tumor weight and volume. a AXL knockdown in Lipo-246 cells was evaluated by western blot prior to subcutaneous injection. b Tumor volume was monitored at the indicated time points. c Xenografts were weighed ex vivo following experiment termination. d Representative IHC images of Lipo-246 xenografts expressing NT control constructs or one of two AXL-targeting shRNAs are shown (400× magnification). Hematoxylin-eosin (H&E); cleaved caspase 3 (CC3). (* = p < 0.05; *** = p < 0.001)

Fig. 6

AXL knockdown in Lipo-863 reduces in vivo tumorigenicity. a Prior to subcutaneous injection, Lipo-863 cells were assessed for AXL by immunoblotting. b Xenograft volume was measured three times weekly. c Tumors were harvested and weighed at the end of the experiment. d Representative IHC images of Lipo-863 xenografts expressing NT control constructs or AXL-specific shRNAs are presented (400× magnification). Hematoxylin-eosin (H&E); cleaved caspase 3 (CC3). (* = p < 0.05)