AXL Is a Novel Predictive Factor and Therapeutic Target for Radioactive Iodine Refractory Thyroid Cancer

Abstract

Papillary thyroid carcinomas (PTCs) have an excellent prognosis, but a fraction of them show aggressive behavior, becoming radioiodine (RAI)-resistant and/or metastatic. AXL (Anexelekto) is a tyrosine kinase receptor regulating viability, invasiveness and chemoresistance in various human cancers, including PTCs. Here, we analyze the role of AXL in PTC prognosis and as a marker of RAI refractoriness. Immunohistochemistry was used to assess AXL positivity in a cohort of human PTC samples. Normal and cancerous thyroid cell lines were used in vitro for signaling, survival and RAI uptake evaluations. 38.2% of human PTCs displayed high expression of AXL that positively correlated with RAI-refractoriness and disease persistence or recurrence, especially when combined with v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation. In human PTC samples, AXL expression correlated with V-akt murine thymoma viral oncogene homolog 1 (AKT1) and p65 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation levels. Consistently, AXL stimulation with its ligand growth arrest-specific gene 6 (GAS6) increased AKT1- and p65 NF-kB-phosphorylation and promoted survival of thyroid cancer cell lines in culture. Enforced expression or activation of AXL in normal rat thyroid cells significantly reduced the expression of the sodium/iodide symporter (NIS) and the radioiodine uptake. These data indicate that AXL expression levels could be used as predictor of RAI refractoriness and as a possible novel therapeutic target of RAI resistant PTCs.

Keywords: AXL; disease persistence/recurrence; p65 NF-kB activation; radioactive iodine (RAI)-refractoriness; thyroid cancer.

Figure 1

Anexeleto (AXL) staining in thyroid cancer samples. Representative images of high and low/negative AXL immunohistochemical staining in thyroid cancer samples at 40× magnification, scale bar = 50 μm. High and negative/low staining was defined as described in the Section “Materials and Methods”.

Figure 2

Fluorescence in Situ Hybridization (FISH) analysis of AXL in thyroid cancer samples. Representative FISH patterns in normal and abnormal interphase cells using the AXL/CEN 19 probe (scale bar = 5 μm). (A) Normal AXL gene copies, two red and two green signals (2R2G); (B) High amplification of AXL gene (cluster red signals and 2G); (C) Low amplification of AXL gene (4R2G Ratio > 2); (D) Polysomic FISH patterns (3R4G).

Figure 3

Kaplan-Meier estimation of Disease Free Survival (DFS) in thyroid cancer patients depending on AXL expression. Disease Free Survival curves of patients with papillary thyroid cancer stratified in two groups as negative/low or high AXL expression. Persisting patients were included in DFS analysis and the value 0 of the recurrence time was assigned to them in the analysis.

Figure 4

Kaplan-Meier estimation of Disease Free Survival (DFS) in thyroid cancer patients stratified for AXL expression and BRAF mutational status. Disease Free Survival curves of patients with papillary thyroid cancer stratified as: (i) AXL low + BRAF V600E negative, (ii) AXL low + BRAF V600E positive, (iii) AXL high + BRAF V600E negative, (iv) AXL high + BRAF V600E positive. Persisting patients were included in DFS analysis and the value 0 of the recurrence time was assigned to them in the analysis.

Figure 5

Signaling pathway activation in thyroid cancer samples. Immunohistochemical staining of representative Papillary thyroid carcinoma (PTC) samples with anti-phospho-AKT1, anti-phospho-p65 NF-kB, anti-phospho-ERK1/2 at 40× magnification, scale bar = 50 μm. High and low staining for each protein are defined as described in the Materials and Methods section.

Figure 6

In vitro evaluation of AXL functions. (A) AXL, phospho-AKT1 and phospho-p65 levels evaluated in the indicated thyroid cell lines by Western Blot (WB) analysis. Anti-tubulin antibodies served as control for equal loading. (B) AXL, phospho-AKT1 and phospho-p65 levels in 8505c and TPC-1 cells treated with GAS6 (100 ng/mL) for the indicated time points, evaluated by WB analysis. (C) Levels of AXL, phospho-p65, and p65 in 8505c cell clones stably silenced for AXL (shAXL). Two clones are shown. AXL, phospho-AKT1, AKT1, phospho-p65, and p65 levels in 8505c cells treated or not with GAS6 (100 ng/mL) for the indicated time points in the presence or absence of Bosutinib (25 μM) or siRNAs targeting AXL (siAXL). Anti-tubulin antibodies served as control for equal loading. (D) Percent relative to control of 8505c apoptotic cells, assessed by TUNEL assay, treated or not with GAS6 (100 ng/mL) in the presence or absence of LY294002 (15 μM—AKT1 inhibitor) and JSH23 (5 μM—p65 inhibitor). * p < 0.05 vs. not treated cells. §, p < 0.05 vs. the relative control. (E) PARP1 cleaved levels in 8505c cells treated or not with GAS6 (100 ng/mL) in the presence or absence of LY294002 (15 μM) and JSH23 (5 μM). Anti-tubulin antibodies served as control for equal loading.

Figure 7

NIS modulation by AXL. (A) NIS mRNA expression and protein levels in PC CL3 cells transfected with pCMV6 empty vector, transfected with AXL and treated for 12 h with bosutinib (5 μM) or GAS6 (100 ng/mL). mRNA expression levels expressed as fold change (%) over control (empty vector-transfected). * p < 0.05 vs. the relative empty vector transfected cells. (B) Iodide uptake levels expressed as fold change (%) over control (empty vector-transfected) in PC CL3 cells transfected with AXL and treated with bosutinib 12 h (5 μM) or GAS6 (100 ng/mL). * p < 0.05 vs. the relative empty vector (pCMV6) transfected cells.