The receptor tyrosine kinase Axl is an essential regulator of prostate cancer proliferation and tumor growth and represents a new therapeutic target

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Abstract

Deregulation of the receptor tyrosine kinase Axl has been implicated in the progression of several human cancers. However, the role of Axl in prostate cancer remains poorly understood, and the therapeutic efficacy of Axl targeting remains untested. In this report we identified Axl as a new therapeutic target for prostate cancer. Axl is consistently overexpressed in prostate cancer cell lines and human prostate tumors. Interestingly, the blockage of Axl gene expression strongly inhibits proliferation, migration, invasion and tumor growth. Furthermore, inhibition of Axl expression by small interfering RNA regulates a transcriptional program of genes involved in cell survival, strikingly all connected to the nuclear factor-κB pathway. Additionally, blockage of Axl expression leads to inhibition of Akt, IKKα and IκBα phosphorylation, increasing IκBα expression and stability. Furthermore, induction of Akt phosphorylation by insulin-like growth factor 1 in Axl knockdown cells restores Akt activity and proliferation. Taken together, our results establish an unambiguous role for Axl in prostate cancer tumorigenesis with implications for prostate cancer treatment.

Introduction

Prostate cancer is the most frequent solid cancer in older men in the Western world, representing one of the most frequent causes of cancer deaths, and is now emerging in developing countries as well.1 While androgen ablation therapy, surgery and radiation therapy are effective for the treatment of local prostate cancer, only one drug currently in clinical trials has demonstrated some efficacy against hormone-refractory metastatic disease.

The receptor tyrosine kinase Axl (also known as UFO, ARK and Tyro7) belongs to the TAM (Tyro-3, Axl and Mer) family, whose members are distinguished from each other by a conserved sequence within the kinase domain and adhesion molecule-like domains in the extracellular region. Previous reports have shown Axl to have transforming potential when overexpressed.2, 3 Axl is deregulated in several types of cancer and has been implicated with aggressive phenotype, migration, invasion and tumor progression, suggesting that Axl may be a relevant therapeutic target for cancer.4, 5, 6, 7 Furthermore, its expression has also been shown to predict poor overall patient survival in breast and pancreatic cancers.8, 9

Activation of Axl occurs on binding to the growth arrest-specific gene 6 (Gas6). Gas6 contains an N-terminal γ-carboxy-glutamic acid domain, whose carboxylation is dependent on vitamin K and is essential for its activity and receptor binding.10, 11 Gas6 also binds to other receptors of the TAM family and acts as a growth factor for many non-transformed cell types and Axl-transfected tumor cell lines.12, 13 Rather than conferring a mitogenic signal, Axl has been implicated in cell survival, cellular adhesion and chemotaxis as well as blood vessel function.14, 15

Axl activation has been linked to phosphatidylinositol 3-OH kinase (PI3K) and its downstream targets S6K and Akt,16, 17 MAP kinases, Stat and nuclear factor (NF)-κB signal-transductions pathways.

Axl expression has been correlated with metastatic potential. Adequate evidence supports the notion that Axl acts as an oncoprotein when upregulated. However, its mechanisms of action remain elusive.18 As Axl impinges on multiple aspects of tumor progression, it is critical to decipher its mechanism of Axl action in order to design therapeutic strategies to interfere with this pathway. We have explored the involvement and relevance of Axl in prostate cancer in order to test its efficacy as a therapeutic target. We demonstrate here that Axl is upregulated in prostate cancer cell lines and clinical samples. Furthermore, we implicate Axl as a crucial regulator in proliferation, migration and invasion of prostate cancer cells and tumor formation in vivo. Importantly, we also identify the NF-κB pathway as a target for Axl signaling that contributes to cell survival and tumorigenesis. Thus, Axl appears to have a crucial role in prostate cancer development and metastasis, demonstrating its importance as a potential new target for prostate cancer therapy.

Results

Differential expression of Axl/Gas6 system in prostate cancer

Axl is deregulated in several types of cancers and has been implicated in tumor progression. To determine whether Axl is abnormally expressed in prostate cancer, we tested a variety of prostate cancer cell lines as well as primary prostate epithelial cells for Axl and Gas6 (Figures 1a and b).

Figure 1
figure1

Regulation status of Axl in prostate cancer cell lines. Reverse transcription–PCR analysis of Axl (a) and Gas6 (b) in prostate cancer cell lines. Total RNA was collected from DU145, PC-3, LNCaP, CL1, CW22 and CW19 cells. Normalization of each sample was carried out by measuring the amount of human glyceraldehyde-3-phosphate dehydrogenase complementary DNA. (c) Western blot analysis of Axl and Gas6 expression in prostate cancer cell lines. (d) Phosphorylation status of Axl. Protein extracts were immunoprecipitated using anti-Axl antibody, and the presence of phosphorylated Axl was probed by antiphosphotyrosine antibody.

Androgen-insensitive PC-3 and DU145 prostate cancer cells expressed significantly higher levels of Axl and Gas6 mRNA than androgen-sensitive LNCaP, CW22 and CW19 cells (Figures 1a and b). We also evaluated Axl expression in a clonal cell line, CL1, generated upon conversion of the androgen-sensitive LNCaP cells to an androgen-insensitive state by the removal of androgen in the media. As previously described, LNCaP cells were devoid of Axl mRNA (Figure 1a). However, CL1 cells showed significantly enhanced levels of Axl expression, and CL1 and LNCaP cells expressed similar levels of Gas6 mRNA (Figure 1b). As Axl activation occurs after binding to Gas6 in a γ-carboxylation of glutamic acid residue-dependent event,10, 11 we evaluated γ-glutamyl carboxylase expression levels in prostate cancer cell lines. We observed that all tested cells express γ-glutamyl carboxylase at similar levels (data not shown).

As protein and mRNA expression do not necessarily correlate, we analyzed Axl and Gas6 protein levels in whole-cell extracts. As seen in Figure 1c, PC-3, DU145 and CL1 cells express high levels of Axl protein, correlating with the reverse transcription–PCR data. Gas6 protein levels also correlated with the mRNA data (Figure 1c).

In order to evaluate whether Axl tyrosine kinase is activated in these cell lines, we determined the Axl phosphorylation status as a prerequisite for kinase activation. Western blot analysis with an antiphosphotyrosine antibody of immunoprecipitated Axl protein demonstrated that Axl is tyrosine phosphorylated in PC-3, DU145 and CL-1 cells, but not in LNCaP and CW19 cells (Figure 1d). β-Tubulin expression in cell lysate was used as a loading control for immunoprecipitation of Axl (Supplementary Figure 1). These data indicate that Axl is constitutively active in androgen-insensitive cells and both genes, Axl and Gas6, are co-expressed, which is a crucial step for Axl phosphorylation and activation. The results strongly support our hypothesis that the Axl signaling pathway has a critical role in prostate cancer.

Axl is upregulated in human prostate cancer tissues

Axl overexpression has been associated with invasiveness, metastasis and angiogenesis in various cancer cell types, and is implicated in the prognosis of cancer patients.14, 15, 19, 20, 21 In order to evaluate Axl and Gas6 expression levels in human prostate cancer tissue, we performed real-time PCR using the Origene TissueScan Prostate Cancer Tissue Arrays. Ninety-six complementary DNAs were normalized against β-actin, and the expression levels of Axl were evaluated by real-time PCR. Among the 96 tissue samples, 15 are normal prostate tissue, 69 are adenocarcinoma of prostate (stages I, II, III and IV), 10 benign prostate hyperplasia and 2 carcinoma of the bladder. As seen in Table 1, Axl is upregulated in 50.72% of adenocarcinomas when compared with normal prostate (fold induction varying from 2 to 1500). Gas6 mRNA is equally expressed in prostate cancer tissue when compared with normal prostate (data not shown). These data provide strong evidence that Axl is a critical player in prostate cancer development and progression.

Table 1 Real-time PCR analysis of Axl gene expression in human prostate cancer tissue

Blockage of Axl expression inhibits proliferation, migration and invasion in prostate cancer cell lines

Gas6/Axl signaling has been shown to modulate cell growth and invasiveness promoting survival of pulmonary endothelial and neuronal cells.20, 21 To establish the relevance of Axl in prostate cancer proliferation, migration and invasion, we used a small interfering RNA (siRNA) approach to inhibit endogenous Axl expression. Lentivirus-siRNA vectors specific for Axl as well as a lentivirus vector against green fluorescent protein (GFP; control) were used to infect PC-3 and DU145 cells resulting in 90% reduction of Axl expression (Supplementary Figure S2). Inhibition of Axl dramatically reduced proliferation of DU145 and PC-3 cells (Figure 2a). Additionally, Axl knockdown strongly reduces migration and invasion abilities of DU145 cells by 83% (P<0.0001) and 71.95% (P<0.0001), respectively, after 24-h incubation when compared with DU145 infected with LV-siRNAGFP (Figure 2b). Moreover, wound healing assays corroborated our observations, as knockdown of Axl reduced migration of DU145 cells by 52.83% (Figures 2c and d). These results indicate that Axl is an important regulator of prostate cancer cell proliferation, migration and invasion.

Figure 2
figure2

Blockage of Axl inhibits proliferation, invasion and migration in prostate cancer cell lines. DU145 and PC-3 were infected with LV-siRNAAxl and LV-siRNAGFP as control. (a) Proliferation assay after 48 and 72 h post-infection. Data shown are mean±s.d. of triplicate independent experiments. (b) Migration and invasion assays were measured 48 h post-infection. Invading cells were fixed and stained, and 3–5 random microscopic fields were counted. Values shown are mean±s.d. from a representative experiment. (c) Wound healing assay. Wounds were measured in three different positions at 0, 8 and 24 h and mean distance of control set as 100%. (d) Graph representation of wound healing assay.

Axl has an important role in prostate cancer tumor growth in vivo

To determine whether blockage of Axl has an effect on tumor formation in vivo, PC-3 cells infected with LV-siRNAGFP or LV-siRNAAxl, as well as uninfected cells, were orthotopically implanted into the prostate of MF-1 nude mice. Two months later, mice were examined for tumor formation, tumor weight and IL-6 expression in the serum. In contrast to the control cells, blockage of Axl reduces tumor growth (Figure 3a) and IL-6 expression (Figure 3b) by 79.25% (P<0.0001) and 94.05% (P<0.0001), respectively, in mice implanted with PC-3 Axl−/− when compared with mice implanted with PC-3GFP−/− or non-infected PC-3. Moreover, we did not observe any metastasis to lymph nodes in mice implanted with PC-3Axl−/− when compared with mice implanted with PC-3GFP−/− or non-infected PC-3 (data not shown).

Figure 3
figure3

Inhibition of Axl inhibits tumor formation in MF-1 nude mice and IL-6 secretion. PC-3 cells (2 × 106) infected with LV-siRNAAxl or LV-siRNAGFP or uninfected cells were implanted orthotopically into the prostate of MF1 mice. The size of the tumors (a), tumor weight and IL-6 expression (b) were measured 2 months after implantation. Values are represented as mean±s.d. of five individuals and * represents P<0.0001. Wt, wild type.

NF-κB pathway is regulated by Axl in prostate cancer

To elucidate the Axl target genes associated with the biological phenotypes, RNAs from PC-3 cells infected with different Axl-specific siRNA sequences were hybridized to Affymetrix HT U133AAofAv2 GeneChips. Systems biology analysis of the genes differentially expressed upon Axl knockdown identified the NF-κB signaling pathway as a major target for Axl as NF-κB formed a major focus hub with high statistical significance within a network of Axl-regulated genes (Supplementary Figure S3). The NF-κB/IκB pathway is directly implicated in tumorigenesis, cancer cell survival and metastasis.22, 23, 24

To further corroborate our previous results, a κB consensus sequence-luciferase vector, GADD45α and IL-6 promoter-luciferase constructs as well as the parental pXP2 vector were transfected into DU145 cells infected with LV-siRNAGFP as control and LV-siRNAAxl. We demonstrated that inhibition of Axl reduced κB–luciferase activity by 62% compared with the parental pXP2 vector (Figure 4a). Furthermore, Axl knockdown reduced the IL-6 promoter activity by 73% and a 2.5-fold transcriptional activation of the GADD45α promoter as compared with the control cells (Figure 4b).

Figure 4
figure4

Transcriptional activity of NF-κB in prostate cancer cells. DU145 cells infected with LV-siRNAAxl and LV-siRNAGFP. Cells were transfected with the κB–luciferase (κB–luc), full-length IL-6 promoter luciferase (pXP2–IL6), GADD45α promoter luciferase (pXP2–GADD45α) constructs or with the parental vector (pXP2). Luciferase activity in the lysates was determined 16 h later. Data shown are mean±s.d. of triplicates of one representative transfection normalized by measuring the amount of β-galactosidase gene expression. Transcriptional activity of the κB–luc construct (a) and the pXP2–IL6 and pXP2–GADD45α constructs (b). Data are represented as mean of fold activation of the parental vector as indicated on the left. (c) Re-expression of NF-κBp65 restores proliferation in Axl knockdown cells. DU145Axl−/− and GFP−/− cells were transfected with pCI-p65 and pCI vector, and proliferation was measured 48-h post transfection. Data shown are mean±s.d. of triplicate independent experiments. (d) Blockage of NF-κB pathway inhibits proliferation in prostate cancer cells. DU145 cells were treated with 5 nM 6-amino quinazoline, 50 μM isohelenin, 50 μM SC-514 or 200 μM wedelolactone using dimethylsulfoxide (DMSO) as a control. Proliferation was detected 24-h post treatment. (e) Blockage of Axl and NF-κB inhibits proliferation of prostate cancer cells. DU145Axl−/− and GFP−/− cells were treated with NF-κB inhibitors. Proliferation was measured 24-h post treatment. Data shown are mean±s.d. of triplicate independent experiments for each treatment.

NF-κB has been shown to regulate genes that have important roles in cancer progression such as IL-1 and IL-6,25, 26 CXCL5 (refs 27 and 28) and GADD45β. To further analyze the effects of Axl knockdown on NF-κB-regulated genes, we evaluated the mRNA expression levels of the IL-8, IL-1, GADD45β and CXCL5 genes by real-time PCR. As seen in Supplementary Figure S4, all four genes were downregulated in Axl knockdown cells corroborating our notion that Axl regulates NF-κB activity in prostate cancer cells.

To confirm the association between the NF-κB pathway and Axl, we performed an electrophoretic mobility shift assay using whole-cell extracts from DU145Axl+/+ (positive control), DU145Axl−/− cells and Axl−/− cells transfected with Axl and NF-κBp65 expression vectors and LNCaP cells in which NF-κB is not activated (negative control). Our electrophoretic mobility shift assay data clearly indicated that NF-κB is not activated in DU145Axl−/− cells, but this can be reversed by expression of Axl or NF-κBp65 (Supplementary Figure S5).

In order to evaluate the functional consequences of Axl regulation of the NF-κB pathway, we evaluated the effect of overexpression of NF-κBp65 on proliferation, in Axl knockdown cells. DU145Axl−/− and GFP−/− cells were transfected with pCIp65 plasmid or pCI control plasmid and proliferation was evaluated 48-h post transfection. As observed in Figure 4c, overexpression of NF-κBp65 protein restores proliferation to DU145Axl−/− cells when compared with control.

Pharmacological inhibitors of the NF-B pathway inhibit proliferation in prostate cancer cells

Our group has previously shown that adenoviral expression of IκBα, a NF-κB inhibitor, induces apoptosis, blocks proliferation in cancer cells and inhibits tumor formation.29 We, therefore, moved toward a more clinically relevant model and used pharmacological inhibitors of the NF-κB pathway to determine the functional consequences of NF-κB inhibition in Axl knockdown cells.

DU145 cells were incubated with 6-amino quinazoline and isohelenin, potent inhibitors of NF-κB transcriptional activation,30, 31 SC-514, a reversible and highly selective inhibitor of IKK-2 and wedelolactone, a irreversible inhibitor of IKKα and β-kinase activity32 or dimethylsulfoxide (control) for 24 h. Under the conditions tested, only SC-514 and 6-amino quinazoline strongly inhibited proliferation (Figure 4d) demonstrating that pharmacological NF-κB inhibitors are potent inhibitors of cell proliferation.

Subsequently, we evaluated whether NF-κB inhibitors potentiate the effect of Axl knockdown on prostate cancer proliferation. DU145Axl−/− and GFP−/− cells were incubated with 6-amino quinazoline and SC-514 or dimethylsulfoxide (control) for 48 h. As seen in Figure 4e, inhibition of Axl gene expression reduces proliferation by twofold (P=0.0009), while 6-amino quinazoline and SC-514 reduced proliferation of cancer cells by five- and twofold, respectively (P<0.0001 and P<0.0001). Importantly, NF-κB inhibitors potentialized the effect of Axl knockdown on proliferation and apoptosis. 6-Amino quinazoline and SC-514 treatments in Axl−/− prostate cancer cell inhibits proliferation up to 10-fold (P=0.001, 0.0077, respectively) when compared with Axl−/− cells treated with dimethylsulfoxide.

Axl promotes prostate cancer progression by inducing the NF-κB pathway through activation of Akt and IKKα

In order to gain further insights into Axl regulation of the NF-κB pathway, we decided to evaluate key molecular players that have been implicated in the activation of the NF-κB pathway. A close interaction of Axl and the Akt/PI3K pathway has been demonstrated.17 The mitogenic effect due to Axl activation has been shown to require PI3K and its downstream target Akt.17 Activated Akt then triggers downstream signals involved in cell survival and growth.33 Furthermore, activation of Akt has been shown to regulate the NF-κB pathway by phosphorylation and activation of the IKK complex leading to phosphorylation of IκBα.34

To confirm the correlation between Axl expression, Akt phosphorylation and NF-κB activation, we first evaluated the activation status of Akt and IKKα in Axl knockdown cell lines. As observed in Figure 5a, blockage of Axl expression leads to inhibition of Akt phosphorylation, circumvents phosphorylation of IKKα and consequentially reduces phosphorylation of IκBα. As this process avoids the degradation of IκBα by the proteasome, we further analyzed the impact of Axl knockdown on IκBα ubiquitination and degradation. DU145GFP−/− and DU145Axl−/− cells were transfected with a myc-tagged ubiquitin construct (pCDNAmyc–Ub), an Axl expression vector (pCDNAAxl-flag) and a combination of both in the presence or absence of proteasome inhibitor MG132. Western blot analysis of immunoprecipitated IκBα protein reveals that ubiquitination of IκBα is increased in DU145GFP−/− cells when compared with DU145Axl−/− (Figure 5b). Furthermore, re-expression of Axl in DU145Axl−/− cells increases ubiquitination of IκBα (Figure 5b) and Akt and IKKα phosphorylation (Figure 5c).

Figure 5
figure5

Axl-mediated Akt activation is essential for NF-κB activity and proliferation of prostate cancer cells. (a) Western blot analysis of protein extracts obtained from DU145Axl−/− and GFP−/− cells probed for phosphorylation status of Akt, IKKα and IκBα, and total protein expression of Axl, Akt, IKKα, IκBα and GAPDH. (b) Blockage of Axl inhibits ubiquitination of IκBα. DU145 Axl−/− and GFP−/− cells were transfected with pCDNA Myc–Ub and pCDNA Flag–Axl expression vector or parental vector and treated with proteasome inhibitor (MG132) for 4 h. Proteins were immunoprecipitated using anti-IκBα antibody. Ubiquitinated IκBα was detected by using anti-myc antibody. Re-expression of Axl protein or IGF1 treatment restores Akt phosphorylation and proliferation. DU145Axl−/− and GFP−/− cells were transfected with pCDNA Flag-Axl or parental control plasmid or treated with IGF1 (50 ng/ml) for 2 h or with wortmannin (1 μM) for 30 min. Protein expression or proliferation was analyzed 24-h post transfection or treatment. Relative expression levels were calculated using Visionworks software (UVP), in which the intensity of ubiquitinated proteins were normalized by the intensity of IκBα. Normalized value obtained from GFP−/− non-transfected cells was considered as control and set as 1. Relative expression compared to control was determined. (c) Western blot using anti-Axl, anti-phospho-Akt, anti-Akt, anti-phospho-IKKα, anti-IKKα and anti-GAPDH antibodies. (d) Western blot using anti-phospho-Akt, anti-Akt and anti-GAPDH antibodies. (e) Proliferation assay. Data shown are mean±s.d. of triplicate independent experiments for each condition.

To fully elucidate the functional relevance of Axl regulation of Akt activation in cancer proliferation, we also evaluated the Akt phosphorylation status of DU145Axl−/− cells treated with insulin-like growth factor 1 (IGF1). IGF1 is known to induce Akt activation by acting through the insulin receptor pathway.35

Compared with the control, Akt and IKKα phosphorylation in DU145Axl−/− cells treated with IGF1 was significantly increased (Figure 5d). Additionally, IGF1 treatment as well as transfection of Axl expression vector restores proliferation in DU145Axl−/− cells, which can be inhibited by wortaminnin treatment (Figure 5e), suggesting that induction of proliferation, mediated by Axl, is directly related to Akt activation. These results further imply that Axl regulation of NF-κB is for a large part dependent on activation of the Akt–IKKα pathway and contributes to cancer cell proliferation.

Inhibition of Axl inhibits IL-6 protein secretion and blocks Stat3 activation in prostate cancer cells

Our in vivo experiment demonstrated that inhibition of Axl reduces IL-6 expression in our mouse model. Aberrant constitutive IL-6 gene expression has been implicated in prostate cancer progression and has been directly linked to prostate cancer mortality.25 Furthermore, we have previously shown that endogenous IL-6 gene expression in prostate cancer cells is at least partially mediated via constitutive activation of NF-κB and AP-1, and blockade of these transcription factors leads to inhibition of Stat3 phosphorylation.25

To evaluate whether IL-6 protein secretion is dependent on Axl–Akt–NF-κB activation, we analyzed IL-6 protein levels in tissue culture supernatants obtained from DU145GFP−/−, DU145Axl−/− and DU145Axl−/− cells transfected with the Axl expression vector or treated with IGF1. IL-6-specific enzyme-linked immunosorbent assay analysis revealed that DU145GFP−/− cells secrete significantly higher levels of IL-6 (38.5 pg/ml) when compared with DU145Axl−/− cells (7.17 pg/ml), while re-introduction of Axl expression in Axl−/− cells as well as IGF1 treatment restores IL-6 protein secretion to 26.8 and 27.5 pg/ml, respectively (Figure 6a).

Figure 6
figure6

Inhibition by Axl blocks IL-6 secretion and constitutive activation of Stat3 in prostate cancer cells. DU145Axl−/− and GFP−/− cells were transfected with Axl expression vector or parental vector or treated with IGF1 (50 ng/ml) for 2 h. (a) IL-6 measurement 24-h post transfection or treatment. (b) Immunoblot analysis using anti-phospho-Stat3, anti-Stat3 and anti-GAPDH antibodies. (c) Schematic representation of the Axl pathway in cancer.

IL-6 utilizes Janus kinase–signal transducers and activators of transcription (JAK-Stat) as major mediators of signal transduction.36 To further delineate the effect Axl expression has on the IL-6 pathway, we evaluated the phosphorylation status of Stat3 in DU145GFP−/− cells and DU145Axl−/− cells by immunoblotting with an anti-phospho-Stat3Tyr705 antibody. Cells were either transfected with Axl expression vector or treated with IGF1. IL-6 levels were compared with untransfected and untreated controls. We observed that inhibition of Axl and downregulation of IL-6 gene expression blocks Stat3 phosphorylation in DU145Axl−/− cells when compared with DU145GFP−/− cells. Moreover, re-introduction of Axl expression in Axl−/− cells as well as IGF1 treatment restores Stat3 phosphorylation levels (Figure 6b). These results most vividly implicate Axl as an important regulator of the IL-6–Stat3 signaling pathway.

Discussion

Axl and members of the TAM subfamily (Axl, Sky and Mer) have been shown to be deregulated in many types of cancers.6, 12, 18, 20, 37 Furthermore, Axl has been shown to have an important role in cancer metastasis as its blockage reduces metastasis in breast and lung cancer models.4, 5, 6, 19

Only a few reports have evaluated the regulation of Axl in prostate cancer,38, 39, 40 and its importance for cancer progression remains elusive as its mechanisms of action remain poorly understood. Jacob et al.,40 using real-time PCR, demonstrated that Axl is differently expressed in prostate cancer cell lines when compared with normal cells. Here we provide a detailed analysis of the role of Axl in prostate cancer and demonstrate that Axl mRNA and protein levels are dysregulated mainly in androgen-insensitive prostate cancer cells. Additionally, our data clearly indicate Axl as a crucial regulator for prostate cancer progression and metastasis. Using a panel of clinical samples, we determined that Axl is upregulated in 50.72% of adenocarcinoma of the prostate in stages I, II and III when compared with normal tissue. Metastatic cancer cells are characterized by acquisition of motility, allowing growth at distant organs. Actually, Axl has been shown to be implicated in cancer migration and invasion and has been reported as an essential regulator for the epithelial-to-mesenchymal transition in breast cancer metastasis9 and in prostate cancer. Axl activation was shown to induce mitogenic activity.38 In this regard, our findings that blockage of Axl expression significantly reduces migration, invasion and proliferation of prostate cancer cells and inhibits tumor growth clearly implicate upregulation of Axl in tumorigenesis and support the notion that Axl is an oncogene.

Our observations that Axl is deregulated in prostate cancer and its inhibition results in decreased migration and invasion capabilities reinforce previous data described for different types of cancers.7, 9, 19, 21, 39, 41 Together, our data clearly indicate that Axl contributes to the aggressive phenotype of prostate cancer and tumor growth.

Dissection of the biological pathways targeted by Axl will also provide ample new entry points for drug therapy and may be useful to screen new compounds that are more effective and specific in targeting Axl. In fibroblasts and endothelial cells, Axl phosphorylation activates survival signaling, including the NF-κB transcription factor.42, 43 Our microarray analysis reveals that Axl regulates several survival pathways, strikingly, the NF-κB signaling pathway in prostate cancer. The NF-κB/IκB pathway has been directly implicated in cancer cell survival and metastasis. Constitutive activation of NF-κB is frequently observed in various cancer types, including prostate cancer, and is a critical step for cancer cells to escape apoptosis. Our data also show that inhibition of Axl expression leads to the inactivation of NF-κB by inhibition of IKKα activity and consequently abrogation of IκBα phosphorylation, ubiquitination and degradation, avoiding translocation of NF-κB into the nucleus. Furthermore, inhibition of NF-κB has been shown to induce apoptosis and block tumor growth.29 Moreover, downstream target genes of the NF-κB signaling cascade, such as IL-8,44 CXCL5,45 IL-146 and GADD45β gene,29, 47 were found to be tightly regulated in Axl knockdown prostate cancer cells. Taken together, our results establish the NF-κB pathway as a major target for Axl in prostate cancer and support the need for the exploration of new approaches to target Axl.

As cancers are very heterogeneous and escape therapy due to several mechanisms, a two-pronged approach would be significantly more effective. Indeed, combining blockage of Axl expression with an NF-κB inhibitor led to a synergistic inhibition of proliferation in prostate cancer cells.

Other molecular targets affected by Axl activation may include PI3K/AKT. The PI3K signaling pathway has been shown to be a downstream effector of Axl in cancer cells.8, 13, 48 Indeed, Akt activation was described to correlate with higher levels of Axl expression found in androgen-insensitive prostate cancer cell lines.38 Activation of PI3K/Akt has been shown to stimulate the NF-κB pathway, enhancing tumor cell invasion in breast and ovarian cancers.7, 9, 48

In this context, our data support previous findings that Axl regulation of the NF-κB pathway is dependent on Akt activation.13 Actually, inhibition of Axl leads to a decrease in Akt and IKKα phosphorylation and activity, and consequentially an increase in the stability of the IκBα-NF-κB complex in the cytoplasm. Furthermore, restoration of Akt and IKKα activity by IGF1 treatment increases proliferation of Axl−/− cells, clearly implicating Akt in Axl induction of cell survival. Additionally, we demonstrate for the first time a correlation between Axl expression and the IL-6 pathway.

The IL-6 pathway has been implicated in prostate cancer.25, 36 IL-6 levels in blood independently predict poor outcome in patients with localized tumors.36 IL-6 expression is at least partially mediated via constitutive activation of NF-κB. Binding of IL-6 to its receptor, activates JAK kinases, leading to activation of Stat-3.49 In this study, we demonstrate for the first time a connection between Axl and IL-6 expression and Stat-3 activation. Blockage of Axl leads to the inhibition of secretion of IL-6, which in turn reduces the autocrine IL-6 pathway activation reducing proliferation of androgen-insensitive prostate cancer cells via inhibition of STAT-3 activation. The Axl/Akt/NF-κB cascade may cooperatively work to achieve the maximal antiapoptotic effect in cancer cells partially by inducing IL-6 secretion (Figure 6c).

In conclusion, our results establish Axl as an essential and critical mediator of cell survival in prostate cancer cells via activation of the Akt–NF-κB signaling pathway. Moreover, we establish Axl as a new therapeutic entry point that may give rise to novel therapeutic modalities in the fight against prostate cancer.

Materials and methods

Cell lines and reagents

The human prostate cancer cell lines DU145, PC-3, LNCaP, CWR22 and CWR19 were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) on regular basis and authenticated by the cell bank by DNA profile (STR) and cytogenetic analysis; CL1, derived from LNCaP cells were kindly provided by Dr Sun Paik, University of California, Los Angeles. Cells were cultured as described.29, 50

Reagents

NF-κB inhibitors, 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (6-amino quinazoline), isohelenin, wortannin, SC-514 and wedelolactone and proteasome inhibitor MG132 and wortaminnin were obtained from Calbiochem (San Diego, CA, USA). Final concentrations were 5 nM (6-amino quinazoline), 50 μM (SC-514), 50 μM (isohelenin) and 200 μM (wedelolactone), 10 μM (MG132) and 1 μM (wortannin), and 0.1% dimethylsulfoxide.

Real-time PCR

Total RNA was harvested using QIAshredder (Qiagen, Valencia, CA, USA) and the RNeasy mini kit (Qiagen). Real-time PCR in clinical samples was performed using the Origene TissueScan Prostate Cancer Tissue Array I and II (OriGene Technologies, Rockville, MD, USA) according to the manufacturer's protocol. Primers used for real-time PCR and details are described in Supplementary Data.

siRNA lentiviral vectors

Mission shRNA Lentiviral Transduction Particles were obtained from Sigma-Aldrich (St Louis, MO, USA). An additional lentivirus encoding siRNA against Axl was cloned using Advantage 2 PCR kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and the virus was generated according to methodology described previously.29 siRNA sequences are described in Supplementary data.

Production of lentiviral vectors

Vesicular stomatitis virus-G envelope protein-pseudotyped lentiviruses were prepared and purified as described before.29, 50

Immunoprecipitation and western blot analysis

Immunoprecipitation and western blot analysis were performed as described.29, 50 The following primary antibodies were used: Axl, p-Stat3, Stat3, p-Akt, Akt, IKKα, p-IKKα, GAPDH and phosphotyrosine (Cell Signaling Technology, Beverly, MA, USA) as well as Gas6, p-IκBα, IκBα, ubiquitin and tubulin (Santa-Cruz Biotech, Santa Cruz, CA, USA). For immunoprecipitation assays, 500 μg were immunoprecipitated using anti-Axl or anti-IκBα antibodies coupled with protein-G agarose.

Proliferation and apoptosis assays

Proliferation and apoptosis assays were performed using Cell Proliferation Kit I (MTT; Roche, Basel, Switzerland) and Apoptotic Cell Death Detection ELISA (Roche), respectively, according to the manufacturer's protocol.

Invasion and migration assays

Cell migration and invasion assays were performed, as previously described,25 using a modified transwell chamber migration assay and invasion assay matrigel-coated membrane (BD Biosciences, Bedford, MA, USA).

Orthotopic implantation of PC-3 tumor cells

Eight-week-old male MF-1 nude mice were bred at the animal facility from the University of Cape Town and housed in a pathogen-free environment. PC-3 cells (2 × 106) infected with LV-siRNAGFP and LV-siRNAAxl were used for orthotopic implantation as described before.29, 50 At the end of the experiment (8 weeks), animals were killed; tumors were carefully dissected and weighed. Lymph nodes were analyzed to determine metastases. IL-6 level was measured by enzyme-linked immunosorbent assay. Phlebotomy was done by accessing the retroorbital venous plexus to obtain 150 μl of blood from each mouse.

IL-6 detection

IL-6 was assayed as described25 using ELISA assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

Electrophoretic mobility shift assay

DNA binding reactions and electrophoretic mobility shift assay assays were performed, as described previously,25 using whole-cell extracts from DU145, DU14Axl−/− and LNCaP prostate cancer cells. Experimental details and probe sequence are described in Supplementary data.

Plasmids and transfection assays

The κB–luciferase, full-length IL-6 and GADD45α promoter constructs and pCDNAFlag-Axl are described before.25, 29 pcDNAMyc-Ub was kindly donated by Dr Marcelo Gomes (Universidade de São Paulo, Brazil). Transfection was performed using Lipofectamine plus reagent (Invitrogen) as described.29, 50

Luciferase assay

Luciferase assay was performed as described before29, 50 using the Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's protocol.

Microarray analysis

PC-3 cells were infected with two different siRNAs against Axl or GFP siRNA lentivirus at multiplicity of infection 10. Twenty-four hours post-infection, the media were replaced and cells incubated for an additional 48 h. RNA was collected using QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen) and converted into cRNA according to the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA). Experiments were performed in duplicates. cRNAs were hybridized to the HT U133AAofAv2 array plate (Affymetrix), washed and scanned according to the manufacturer's instructions (Supplementary Information). Scanned array images were analyzed by dChip, where model-based gene expression values were obtained using a smoothing-spline normalization method as described.29, 50 All date sets have been deposited in the Gene Expression Omnbus, www.ncbi.nlm.nih.gov/geo.

Statistical analysis

Statistical analysis was done using Student's t-test.

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Acknowledgements

JDP is recipient of the ICGEB post-doctoral fellowship. JFV is recipient of CAPES international fellowship. This study was supported by the Department of Defense grant PC051217 (LFZ), NIH grants 1RO1 CA85467 (TAL) and the Prostate Cancer Foundation (TAL).

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Correspondence to L F Zerbini.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Oncogene website

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Paccez, J., Vasques, G., Correa, R. et al. The receptor tyrosine kinase Axl is an essential regulator of prostate cancer proliferation and tumor growth and represents a new therapeutic target. Oncogene 32, 689–698 (2013) doi:10.1038/onc.2012.89

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Keywords

  • tyrosine kinase receptor
  • Axl
  • prostate cancer
  • proliferation
  • NF-κB
  • IL-6

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