Galectin‐3 promotes CXCR2 to augment the stem‐like property of renal cell carcinoma

Abstract Although targeted therapy is usually the first‐line treatment for advanced renal cell carcinoma (RCC), some patients can experience drug resistance. Cancer stem cells are tumour‐initiating cells that play a vital role in drug resistance, metastasis and cancer relapse, while galectins (Gal) participate in tumour progression and drug resistance. However, the exact role of galectins in RCC stemness is yet unknown. In this study, we grew a subpopulation of RCC cells as tumour spheres with higher levels of stemness‐related genes, such as Oct4, Sox2 and Nanog. Among the Gal family, Gal‐3 in particular was highly expressed in RCC tumour spheres. To further investigate Gal‐3's role in the stemness of RCC, lentivirus‐mediated knockdown and overexpression of Gal‐3 in RCC cells were used to examine both in vitro and in vivo tumorigenicity. We further assessed Gal‐3 expression in RCC tissue microarray using immunohistochemistry. Upon suppressing Gal‐3 in parental RCC cells, invasion, colony formation, sphere‐forming ability, drug resistance and stemness‐related gene expression were all significantly decreased. Furthermore, CXCL6, CXCL7 and CXCR2 were down‐regulated in Gal‐3‐knockdown tumour spheres, while CXCR2 overexpression in Gal‐3‐knockdown RCC restored the ability of sphere formation. Gal‐3 overexpression in RCC promoted both in vitro and in vivo tumorigenicity, and its expression was correlated with CXCR2 expression and tumour progression in clinical tissues. RCC patients with higher co‐expressions of Gal‐3 and CXCR2 demonstrated a worse survival rate. These results indicate that highly expressed Gal‐3 may up‐regulate CXCR2 to augment RCC stemness. Gal‐3 may be a prognostic and innovative target of combined therapy for treating RCC.

cell RCC (ccRCC), its overall efficacy rate is restricted by its toxicity. Molecular targeting drugs, including tyrosine kinase and mTOR inhibitors, have been approved for treating advanced RCC. 1,2 Nevertheless, long-lasting treatment responses cannot be achieved, and the overall survival rate is still poor due to drug resistance.
Cancer stem cells (CSCs) may contribute to drug resistance in human solid tumours. 3,4 The frequency of functionally defined CSCs varies among different patients. With self-renewal as one of their hallmarks, CSCs can initiate tumour formation and metastasis. Furthermore, CSCs can be identified by in vitro sphere-forming assays and common cell surface markers, especially CD133 and CD44.
CSCs can express ATP-binding cassette (ABC) transporters to become more resistant to chemotherapy compared to the bulk of a tumour cell mass. 3,4 The tumour microenvironment supports cancer progression and CSC formation through growth factors, cytokines and chemokines. For example, within the tumour microenvironment, endothelial cells produce angiocrine factors and myofibroblasts secrete the stem cell factor, CXCL12 and Wnt to modulate the stemness of CSCs. 5 Another critical component of the tumour microenvironment, galectin can control immune surveillance and aid tumour metastasis. 6 Galectins (Gals) are galactoside-binding lectins that contain conserved carbohydrate-recognition domains (CRDs) to bind β-galactose.
According to their structural features, galectins are classified into the following three categories: prototype, chimera type and tandemrepeat type. The chimera galectin type has only one member, Gal-3, 7 whose expression is required for initiating the transformed phenotype of tumours by interacting with oncogenic Ras. 8 The Gal-3-RAS interaction promotes the RAS anchorage to the plasma membrane, which results in the constitutive activation of phosphatidylinositol 3-kinase and Raf-1. 9 The tumorigenic potential of Gal-3 may also function through binding with β-catenin or transcriptional factors to increase the expressions of cyclin D and c-MYC and augment cell cycle progression. 10 Furthermore, intracellular Gal-3 inhibits cell death induced by cisplatin and paclitaxel, thus contributing to cancer cells' drug resistance and CSC formation. 11,12 Extracellular Gal-3 has in vitro angiogenic activity by inducing the migration of endothelial cells. 13 Increased protein levels of Gal-3 are correlated with the poor survival of various cancers, including leukaemia, lymphomas, breast cancer and thyroid cancer. 6 Gal-3 was overexpressed in RCC patients with distant metastasis. 14 While chemokines and their receptors influence the initiation and progression of tumours in the tumour microenvironment, their role in the Gal-3-promoted CSC formation and drug resistance of RCC remains unclear. 5 In this study, we found that Gal-3 was highly expressed in the CSCs of RCC, as well as the clinical tissues of advanced RCC. Silencing Gal-3 in RCC cells decreased CSC formation, drug resistance and CXCR2, while CXCR2 overexpression in Gal-3-knockdown cells restored the tumorigenesis ability. Our results indicate that highly expressed Gal-3 may enhance the stemness property of RCC by promoting CXCR2.

| Cell lines
We obtained the human RCC cell lines Caki-1 and ACHN (VHL wild type) from the American Type Culture Collection. We purchased the human RCC cell line A-498 (VHL mutation) from Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). These cell lines were cultured as described in a previous study. 15

CXCR2 by lentivirus-mediated system
Lentivirus-mediated silencing and overexpression of Gal-3 and CXCR2 of the RCC cells were performed as described in a previous study. 15 We obtained pLKO.1 plasmid containing shRNA targeting human Gal-3 (shGal-3#1, Clone ID TRCN0000029305; shGal-3#2, Clone ID TRCN0000029307) and CXCR2 (shCXCR2, Clone ID TRCN0000009138) from the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). Full-length DNA encoding Gal-3 and CXCR2 genes were amplified using RT-PCR and cloned to pLAS2w. The primer sequences for cloning the full length of Gal-3 and CXCR2 are listed in Table S1. To knockdown Gal-3 in RCC spheres, A-498-derived primary tumour spheres were dissociated into single cells, re-seeded in a 10% FBS-RPMI medium, infected with shGal-3 or shLuc lentivirus, and then cultured to form secondary spheres (2S).

| RT-qPCR
RT-qPCR was performed as described in a previous study. 15 The specific primers used in the RT-qPCR are presented in Table S2.

| Cell migration and invasion assays
We evaluated tumour cell migration and invasion assays using transwell assay (Costar, 8-μm pore; Corning, NY) as described in a previous study. 15

| Colony formation assay
RCC cells (1 × 10 3 ) were suspended in 0.33% Bacto-agar (Sigma-Aldrich) and then layered over 0.5% Bacto-agar in six-well plates. On day 30, we counted the colonies after fixing them with methanol and staining them with Giemsa.

| Sphere formation
RCC cells were cultured in a tumour sphere medium (Gibco, BRL, Life Technologies) that contained serum-free DMEM/F12 (1:1) medium, 1X B27 supplement, 20 ng/mL human recombinant basic fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF). RCC cells were seeded at 500 cells per 96-well (or 8 × 10 4 per 6-well), and the tumour sphere medium was replaced with fresh medium every 3-4 days. ACHN cells were cultured for 14 days, while A-498 and Caki-1 cells were cultured for 21 days. We used a microscope to count the spheres. Primary spheres (1°sphere) were dissociated to single cells and re-seeded to yield the second generation (2°sphere).

| In vivo tumour growth
We purchased male NOD/SCID mice (NOD.CB17-Prkdcscid/ IcrCrlBltw) from BioLASCO Taiwan Co., Ltd. and maintained them under specific pathogen-free conditions at the Animal Center of National Yang-Ming University, as approved by the university's Institutional Animal Care and Use Committee. To establish a xenograft tumour model, empty vector (ev)-or Gal-3-infected Caki-1 (3 × 10 3 -10 5 /100 μL) monolayer or sphere cells were subcutaneously implanted into the abdominal flanks of six-to eight-week-old male NOD/SCID mice. We measured tumour size with a calliper and calculated it as length × width × height (in mm 3 ) every week.

| Tissue microarray (TMA) and immunohistochemistry (IHC)
We purchased tissue microarray slides from Biomax (US Biomax Inc., Rockville, MD) and performed IHC as described in a previous study. 15 TMA slides were incubated with the Gal-3 or CXCR2

| Statistical analysis
Data are expressed as the mean ± SD. Differences between two groups were determined using Student's t test. We adopted the Sur-vExpress 16 web-based tool to analyse the gene expression of Gal-3 and CXCR2 in ccRCC (accession no. KIRC-TCGA). Survival durations were analysed using the Kaplan-Meier method and compared in the patient groups with the log-rank test. Using Cox survival analysis, we classified a population of ccRCC patients into high-risk and low-risk groups in accordance with their prognostic index. Statistical significance was set at P < 0.05.

| Enrichment of renal CSCs
To determine whether cultured human RCC cell lines contained a population of CSCs, RCC cells were cultured in a defined serumfree selection tumour sphere medium for a few days. The morphology of the RCC cell spheres is shown in Figure 1A. We observed only 9% sphere formation in A-498, 7% in Caki-1 and 11% in ACHN cells ( Figure 1B). The stemness-associated genes were analysed using RT-qPCR, and the results showed that the mRNA levels of Nanog, Sox2, Oct4, CD44, CD133, ABCB1, ABCC1, ABCG2 and Notch1 were significantly increased in RCC tumour spheres compared with parental cells ( Figure 1C). Furthermore, we adopted Western blotting to confirm the protein levels of Nanog, Sox2 and Oct4 in three RCC tumour spheres (

| Galectin-3 was highly expressed in the tumour spheres of RCC cells
Galectins have been reported to promote cancer cells' chemoresistance and CSC formation. 12,17 Therefore, we analysed the galectin levels in renal CSCs using RT-qPCR. Regarding the galectin family, the expression of Gal-2, Gal-3, Gal-4 and Gal-7 was significantly increased in A-498 CSCs compared with parental cells. Of those, Gal-3 demonstrated a more than 30-fold increase in RCC tumour spheres ( Figure 1E). We then utilized other RCC cells to verify whether Gal-3 was also up-regulated in these renal tumour spheres and found that Gal-3 mRNA expression demonstrated a significant sevenfold increase in the tumour spheres of Caki-1 and ACHN cells ( Figure 1F). Western blotting was further adopted to confirm the Gal-3 expression in RCC cells. Compared with parental cells, tumour spheres expressed levels of galectin-3 protein that were twice as high ( Figure 1F).

| Knockdown of galectin-3 in parental RCC cells decreased self-renewal capacity and drug resistance
To determine the role of Gal-3 in cell motility and the sphere-form-

| Galectin-3 maintained the stemness properties of renal CSCs
To further examine the role of Gal-3 in maintaining CSCs, we infected A-498 primary tumour spheres with shGal-3 lentivirus and then cultured them to form secondary spheres. First, we used Western blot to confirm galectin-3 knockdown in secondary spheres (Figure 3A). Furthermore, silencing Gal-3 significantly reduced the sphere formation ( Figure 3B), anchorage-independent growth (Figure 3C), migration and invasion ( Figure 3D) ability of the secondary sphere cells. Therefore, we also observed Gal-3 to participate in the maintenance of the stemness features of renal CSCs.

| Down-regulation of chemokine/cytokine expression in Gal-3-knockdown RCC tumour spheres
To study the molecular mechanism of Gal-3 in the CSCs, we analysed chemokine and chemokine receptor levels using RT-qPCR. As shown in Figure 4A,

| Suppression of CXCR2 led to decreased sphere-forming ability in RCC cells
We silenced CXCR2 in parental A-498 cells to investigate the role of CXCR2 in CSC formation. Compared to cells infected with the control virus expressing shLuc, cells infected with the shCXCR2 virus expressed lower levels of this particular chemokine receptor (Figure 4C). Furthermore, sphere-forming ability was significantly downregulated by 50%-60% in the shCXCR2-infected RCC cells ( Figure 4D).

| Overexpression of CXCR2 in shGal-3-infected RCC cells restored cell motility, colony formation and self-renewal capacity
To explore the role of CXCR2 in the formation of Gal-3-mediated

| Overexpression of galectin-3 in RCC cells promoted sphere-forming capacity and in vitro and in vivo tumorigenicity
We used lentivirus-mediated Gal-3 overexpression in Caki-1 cells to further confirm the role of Gal-3 in renal CSC formation. First, Gal-3 overexpression in RCC cells was confirmed by RT-qPCR and western blot ( Figure 6A). Compared to parental RCC cells, sphere cells with an empty vector expressed higher levels of galectin-3, Oct4, Nanog, Sox2 and CD44 ( Figure 6B). Overexpression of Gal-3 considerably promoted these stemness genes and CXCR2 expression in RCC sphere cells ( Figure 6B). Furthermore, migration, invasion ( Figure 6C), colony formation ( Figure 6D) and sphere-forming ability ( Figure 6E) were all significantly up-regulated in Gal-3-infected RCC sphere cells.
To assess the tumour growth capacity of renal cancer stem cells, we (data not shown). Furthermore, tumours generated by the Gal-3-overexpressed RCC spheres were larger than the RCC spherederived tumours ( Figure 6F). These results suggest that Gal-3 overexpression in RCC sphere cells promote in vivo tumour growth.

| Galectin-3 expression correlated with CXCR2, tumour progression and prognosis in RCC tissues
To investigate the expression of Gal-3 and CXCR2 in human RCC tissues, we performed immunohistochemistry staining on tissue microarrays that contained samples from 75 patients with ccRCC.
We observed higher Gal-3 expression in the advanced stages (III+IV) and grade (poorly differentiated) RCC tissues ( Figure 7A and B).
CXCR2 expression was significantly correlated with tumour differentiation ( Figure 7C) but not RCC stage (data not shown). Furthermore, Gal-3 expression was significantly higher in the CXCR2 high expression group than in the low expression group ( Figure 7D). To further study the correlation between the expression levels of Gal-3/CXCR2 and patient prognosis, we used the online tool SurvExpress 16 to analyse 415 patients with various stages of ccRCC. Using Cox survival analysis, we found that patients with higher co-expressions of Gal-3 and CXCR2 had a significantly worse survival rate ( Figure 7E) and that Gal-3 expression correlated with CXCR2 expression, tumour progression and prognosis in RCC.

| DISCUSSION
The overexpression of Gal-3 is associated with the increased invasiveness of many kinds of tumours. Higher levels of Gal-3 are found in the sera of cancer patients with metastasis. 18 Gal-3 promotes cancer progression through intra-and extra-cellular mechanisms in the tumour microenvironment. Intracellular Gal-3 interacts with RAS and β-catenin to enhance cell transformation and proliferation. [8][9][10] Furthermore, Gal-3 augments tumour stem cell property and drug resistance through its interaction with β-catenin. 12 Several chemokine and chemokine receptor genes, such as CXCR4, CXCR7 and CCL5, 19,20 are the downstream genes of β-catenin. In this study, we found that Gal-3 overexpression may promote CXCR2 to augment the stemness property of RCC. In our previous study, cancer spheres secreted higher levels of Gal-3, while Gal-3 knockdown reduced secretion levels. Recombinant Gal-3 promotes cancer sphere formation. 12 Furthermore, previous studies have demonstrated Gal-3 to interact with epidermal growth factor receptor (EGFR) and transforming growth factor-β receptor (TGFβR). 9 Therefore, extracellular Gal-3 may also stimulate sphere formation in collaboration with EGF or bFGF signalling in the tumour sphere medium.
Existing evidence indicates that drug resistance regulation by Gal-3 may result from intracellular effects on the apoptotic pathways. 11 The anti-apoptotic mechanisms of Gal-3 include: (1) the phosphorylation status of Gal-3, 21 (2) the Gal-3 translocation from the nucleus to the cytoplasm, 22 (3) the regulation of mitochondrial membrane potential, 23 (4) the modulation of survival signalling pathway, 24 and (5) the regulation of the caspase pathway. 25 Furthermore, Gal-3 plays a crucial role in regulating the Wnt/β-catenin signalling pathway. The best evidence so far of the importance of the Wnt pathway to CSCs biology has been reported in myeloid leukaemia, but its contribution has also been reported in the maintenance of the CSCs of melanoma, breast, colon, and lung cancers. 12,26 Therefore, with regard to inhibiting the common upstream regulator of Wnt/β-catenin signalling, Gal-3 may be an effective target for cancer stem cell therapy.
Clear evidence has shown that CXCR2 and its associated ligands play important roles in various types of cancer. Most of the ELR + CXC chemokines that have been described as promoters of tumour bodies reduce lung tumour growth in mice. 30  is a prognostic factor for the overall survival of RCC. CXCL7 promotes RCC cell proliferation both in vitro and in vivo, and a CXCL7/ CXCR2 blockade by antibody or inhibitor reduces tumour growth in mice. 34 Altogether, these findings indicate the importance of CXCR2 in the progression and targeting therapy of RCC. 43 In summary, we have demonstrated that highly expressed Gal-3 can up-regulate CXCR2 to augment the stemness property of RCC.
Gal-3 and CXCR2 expressions were correlated with RCC tumour progression, and Gal-3 expression correlated with CXCR2 expression in RCC tissues. As we found that higher co-expressions of Gal-3 and Patients were divided into two groups (low and high) based on whether their CXCR2 levels were below or above the median value. Galectin-3 expression levels were compared between low and high levels of CXCR2 in ccRCC tissues. *P < 0.05. (E) Kaplan-Meier curves according to galectin-3 and CXCR2 expression levels in patients with ccRCC were conducted using SurvExpress web-based analysis. Censoring samples are shown as "+" marks. Data set, concordance index (CI) and P-value of the log-rank test are shown. Red and green curves denoted high-risk (high expression levels) and low-risk (low expression levels) groups, respectively