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. Author manuscript; available in PMC: 2013 Dec 1.
Published in final edited form as: Cancer Res. 2012 Oct 4;72(23):6209–6216. doi: 10.1158/0008-5472.CAN-12-0337

Augmentation of therapeutic responses in melanoma by inhibition of IRAK-1,-4

Ratika Srivastava 1, Degui Geng 1, Yingjia Liu 1, Liqin Zheng 2, Zhaoyang Li 1, Mary Ann Joseph 1, Colleen McKenna 1, Navneeta Bansal 1, Augusto Ochoa 2, Eduardo Davila 1,3
PMCID: PMC3677596  NIHMSID: NIHMS412033  PMID: 23041547

Abstract

Toll-like receptors (TLR) are expressed by a variety of cancers, including melanoma, but their functional contributions in cancer cells are uncertain. To approach this question, we evaluated the effects of stimulating or inhibiting the TLR/IL-1 receptor-associated kinases IRAK-1 and IRAK-4 in melanoma cells where their functions are largely unexplored. TLRs and TLR-related proteins were variably expressed in melanoma cell lines, with 42% expressing activated phospho-IRAK-1 constitutively and 85% expressing high levels of phospho-IRAK-4 in the absence of TLR stimulation. Immunohistochemical evaluation of melanoma tumor biopsies (n=242) revealed two distinct patient populations, one which expressed p-IRAK-4 levels similar to normal skin (55%) and one with significantly higher levels than normal skin (45%). Levels of p-IRAK-4 levels did not correlate with clinical stage, gender or age, but attenuating IRAK-1,-4 signaling with pharmacological inhibitors or siRNA enhanced cell death in vitro in combination with vinblastine. Moreover, in a xenograft mouse model of melanoma, the combined pharmacological treatment delayed tumor growth and prolonged survival compared to subjects receiving single agent therapy. We propose p-IRAK-4 as a novel inflammation and pro-survival marker in melanoma with the potential to serve as a therapeutic target to enhance chemotherapeutic responses.

Keywords: IL-1 receptor associated kinase (IRAK), biomarker, melanoma, progression, chemotherapy sensitization

Introduction

The incidence of melanoma has been on the rise in the US and worldwide over the last 30 years and has the fastest rising cancer incidence in the US(1–3). Melanoma and is the 5th/6th most common cancer in men and women, respectively (1–3). The median survival of patients with advanced disease is approximately 6 months and the survival rate at 5 years is 6% and for 45% for Stage III patients(1;2). Treatment failure is largely attributed to melanoma’s resistance to all existing forms of cancer therapies.

Recent reports indicate that Toll-like receptors (TLR) signaling within non-immune cells, including several types of human cancers, can contribute to cancer progression(4–7). TLRs recognize infectious microorganisms as well as endogenous signals released by dying or stressed cells. The engagement of all known TLRs, except TLR3, initiates IL-1 receptor-associated kinase (IRAK) signaling(8–10). IL-1, IL-18 and IL-33 can also activate IRAK signaling. IRAK-4 kinase activity is regulated by autophosphorylation (Ser346, Thr342, and Thr34) (8–11) which in turn can activate IRAK-1. IRAK-4,-1 activation results in the downstream activation of various kinases and transcription factors including JNK, AP-1, NF-κB, and p38 MAPK, leading to the production of a mixture of chemokines and pro-inflammatory cytokines including TNF-α, IL-1, IL-6, and IL-8(12). IRAK signaling can also induce the expression of several proteins involved in cell survival and division(13).

In the current study, we examined the TLR and TLR signaling-related protein expression profile on various melanoma cell lines as well as the expression of the activated (phosphorylated) form of IRAK-4 on patient biopsies. Cytokine production and cell survival in response to stimulating or inhibiting IRAK-1,-4 in melanomas were examined in vitro. In vivo, the therapeutic efficacy of combinatorial treatment with IRAK-1,-4 inhibitor plus chemotherapy was investigated in mice with an established human melanoma tumor.

Methods and materials

Melanoma cell lines

Human melanoma cell lines were obtained from American Type Culture Collection within two years of manuscript submission. Melanoma cell lines were initially expanded and cryopreserved within one month of receipt. Cells were typically used for 6 months at which time a fresh vial of cryopreserved cells was used. Malme-3M cells were maintained in Iscove’s Modified Dulbecco’s Medium, SK-MEL-2, WM115, C32, and RPMI-7951 in Eagle’s Minimum Essential Medium, A375 in Dulbecco’s Modified Eagle’s Medium, and G361 in McCoy’s 5a Medium Modified. All media were purchased from Invitrogen Life Technologies (Grand Island, NY) and supplemented with FBS, penicillin and streptomycin according to culture media recommended by ATCC.

Western blot and immunohistochemical staining

Total cell extracts were prepared from melanoma cell. Proteins (20–30μg/lane from cell lines) were resolved in Tris-glycine sodium dodecyl sulfate (SDS) gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked for four hours with 5% milk in phosphate-buffered saline and 0.05% Tween-20, followed by incubation with antibodies against TLR1 (N-23), phosphorylated (p)-IRAK-1 (Ser 376), total IRAK-1 (H-273) (Santa Cruz Biotech, Santa Cruz, CA, USA); TLR2, TLR4, TLR7, TLR9, TRIF, IRAK-M, IRAK-4, Tollip, GAPDH, (14C10) and β-actin (Cell signaling, Danvers, MA, USA); TLR3/CD283, TLR5 (IMGENEX, San Diego, CA, USA); TLR6 (3D10H11), TLR8 (44C143), TLR10, MyD88 (Abcam, Cambridge, MA, USA), Poly (ADP-ribose) polymerase (PARP), caspase-3 (Cell Signaling), overnight at 4°C and, subsequently, incubated with horseradish peroxidase-conjugated secondary antibody, and detected using enhanced chemiluminescence (ECL Plus; Amersham Pharmacia Biotech). Melanoma tissue arrays were purchased from US Biomax, Inc (Rockville, MD). For immunohistochemical staining samples were stained with mouse monoclonal anti-human phosphor-IRAK-4 antibody (Abnova; dilution, 3.5μg/ml) or normal mouse serum. Tissue histology sections (5-μm-thick) were deparaffinized, hydrated, heated in boiling 10 mmol/L sodium citrate (pH 6.0), for antigen retrieval, for 30 min and washed in Tris buffer. Peroxide blocking was done with 3% H2O2 in methanol at room temperature for 15 min, followed by 10% fetal bovine serum in TBS-t for 30 min at room temperature. Primary antibody incubation was done overnight at 4°C. Secondary antibody incubation with horse anti-mouse (1:200; Vector Laboratories, Burlingame, CA) was done for 1 hr, followed by application of diaminobenzidine chromogen for 5 min. The slides were then counterstained with haematoxylin and topped with a coverslip. IHC specimens were analyzed using “quick score” system in which the intensity of the immunohistochemical reaction was scored by multiplying the intensity 1 (weak), 2 (moderate), and 3 (strong) and the proportion of cells staining positively 0 (<10%), 1 (10–25%), 2 (26–50%), 3 (51–75%) or 4 (>75%). In some experiments, A375 cells were transfected via electroporation (Amaxa, Program X-001, Lonza buffer L) with siRNA-hIRAK-1 and siRNA-hIRAK-4 (5μg/ml; InVivogen).

A375 melanoma xenograft model

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ IL2RG (NSG) mice (Jackson Laboratories, Bar Harbor, Maine) were subcutaneously (s.c.) injected with 2.5×106 A375 melanoma cells delivered in PBS. The Institutional Animal Care and Use Committee approved use of mice. When tumors reached a size of approximately 50mm2 mice were injected intratumorally (i.t.) with 35 mg/kg of IRAK-1, -4 inhibitor or an equal volume of vehicle (DMSO). Mice were injected via intraperitoneal injection (i.p.) with vinblastine (0.25 mg/kg) every 2 days for 5 days starting on the same day that mice received with IRAK-1,-4 inhibitor. Tumor sizes were analyzed using a mixed model approach for repeated measurements and mouse survival data were analyzed with the exact log-rank test.

TLR agonists, IRAK inhibitor, chemotherapies and flow cytometry

The TLR1-TLR2 ligand tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys (Pam3CysK4) was purchased from Invivogen (San Diego, CA, USA). In some experiments we used the IRAK-1/4 inhibitor (EMD Millipore, Darmstadt, Germany), which is a cell-permeable benzimidazole compound that selectively inhibits IRAK-1 and -4 and exhibits little activity against a panel of 27 other kinases. In some experiments cells were treated with the indicated concentrations of IRAK-1,-4 inhibitor and vinblastine, cisplatin, and 5-fluorouracil (Sigma-Aldrich, St. Louise, MO), and apoptosis was quantitated by flow cytometry after staining cells with FITC-labeled annexin-V (BD Pharmingen, San Jose, CA) and propidium iodide (PI; Sigma-Aldrich).

Results and Discussion

TLR expression profiles on melanoma cell lines

Previous reports have shown the presence of TLR mRNA transcripts and a limited number of TLRs by flow cytometry on human melanomas (4;6). We examined in greater detail the expression profile of TLR1 through TLR10 and several TLR-related signaling proteins on various melanoma cell lines by Western blot. With the exception of C32 and Malme-3M, all melanoma cells expressed relatively high but variable levels of TLR1 (Fig. 1A). Appreciable levels of TLR2 and TLR3 were detected in SK-Mel-2 and A375. TLR3 was also moderately expressed on RPMI-7951 and G361 cells. Most cell lines expressed variable levels of TLR4 and TLR7. TLR5 was strongly expressed on SK-Mel, WM115 and A375, moderately expressed on G361 and Malm-3M, but weakly detected on C32 and RPMI-7951. TLR8 levels were low on SK-Mel-2 and A375. TLR9 was strongly expressed on SK-Mel-2 and WM115 and moderately expressed on Malm-3M and G361. TLR10 expression was weak and variable on the different cell lines. All cell lines expressed the TLR adapter molecules MyD88 and TRIF.

Figure 1. TLR and TLR signaling-related protein expression profiles on melanoma cell lines.

Figure 1

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(A) The expression levels of TLR1-TLR10 and various TLR-related proteins were examined by western blot in samples from human melanoma cell lines. These data are representative of more than three independent experiments each demonstrating similar trends. (B) The TLR1-TLR2 ligand Pam3CysK4 (2.5μg/ml) was added to A375 cells for 48 hrs. Cytokine and chemokine production was determined using a Milliplex cytokine/chemokine array. (C) IRAK-1,-4 inhibitor or vehicle alone (DMSO) was added to melanoma cells for 48 hrs. The level of phosphorylated and non-phosphorylated p65 subunit of NF-κB was determined by western blot. (D) Cytokine and chemokine levels in the supernatant A375 cell culture were examined using a Milliplex cytokine/chemokine array 48 hours after adding IRAK-1,-4 inhibitor or DMSO. These data are representative of at least three experiments, each demonstrating similar trends. ANOVA; **P < 0.001, *P < 0.05.

IRAK-1 and IRAK-4 play a central role in TLR-mediated signaling. All of the melanoma lines expressed high levels of IRAK-4 and variable levels of IRAK-1 (Fig. 1A). Interestingly, total IRAK-1 as well as the activated form of IRAK-1 (phosphorylated at serine 376; p-IRAK-1) was strongly expressed in Malme-3M, SK-MEL-2, and A375, specifically in the absence of exogenous TLR agonists. Similarly, variable levels of p-IRAK-4 (at threonine 345) were detected in melanoma cells (Fig. 1A). We also examined whether TLR stimulation could augment p-IRAK levels in cells that expressed IRAK or induce p-IRAK in cells deficient in this protein. However, p-IRAK levels in A375 cells, which express relatively high p-IRAK-1 and p-IRAK-4, remained unchanged following TLR1-TLR2 stimulation suggesting that phosphorylated levels may already be at or near the maximum, Supplementary Figure 5B. In G361 cells, which express low levels of p-IRAK-1 (and which express TLR5), neither TLR1-TLR2 agonist (Pam3CysK4) nor TLR5 (Flagellin) stimulation increased or induced p-IRAK-4 or p-IRAK-4 expression levels. Inexplicably, however, the TLR5 agonist flagellin reduced total and p-IRAK-4 levels in both cell lines.

This is the first report demonstrating the expression of constitutively phosphorylated IRAK-1 and IRAK-4 on human cutaneous melanoma cells. These data also represent a comprehensive protein expression profile of TLRs and TLR-signaling proteins on melanoma cells and highlight the differences in the expression of these proteins in different melanoma lines. It is worth noting however, that western blot was used to detect total TLR proteins levels versus flow cytometry which detects surface TLRs.

Cytokine/chemokine production by melanoma cells following activation or inhibition of IRAK-1,-4

The stimulation of TLR-MyD88 or IL-1/18/33–MyD88 activates IRAK-1,-4 resulting in the expression of various chemokines and cytokines involved in cell survival and division as well as factors capable of promoting tumor growth such as angiogenenic and inflammatory cytokines. We compared the cytokine/chemokine profile between A375 cells stimulated with the TLR1-TLR2 agonist Pam3CysK4 and untreated cells. TLR stimulation significantly augmented the production levels of various factors including those associated with angiogenesis such as vascular endothelial growth factor (VEGF), the melanoma growth factor chemokine ligand-1 (CXCL1) and IL-8 which promote cell survival and proliferation (Fig. 1B; p<0.05; ANOVA) (14–16). The levels of granulocyte-macrophage colony stimulating factor (GM-CSF) and IP-10 were also increased following addition of the TLR1-TLR2 agonist (Fig. 1B; p<0.001; ANOVA). TLR1-TLR2 also increased MCP-1 and IL-6 levels but appeared to reduce fractalkine concentrations. To further confirm that the TLR-IRAK signaling pathway was intact in melanoma cells and that changes in cytokines/chemokines are a result of activating this pathway, we transiently overexpressed IRAK-1 in G361 melanoma cells and compared changes in cytokine/chemokine levels with control G361 cells. Overexpressing IRAK-1 increased the levels of various cytokines/chemokines including VEGF, CXCL1, G-CSF and IL-12p40. IRAK-1 expression also induced the expression of IP-10, G-CSF and PDGF-AA but had no effect on EGF production, as shown in Supplementary Figure 1. Collectively, these data indicate that melanoma cells express a functional TLR-IRAK signaling pathway and that the activation of this pathway might play a role in promoting cell survival or proliferation in part through the production and chemokines/cytokines.

On the basis that melanoma cells exhibited increased levels of phosphorylated IRAK-1 and IRAK-4 and IRAK signaling results in the activation of various transcription factors, we examined the outcome of inhibiting IRAK signaling in melanoma cells. Melanoma cells cultured in the presence of an IRAK-1,-4 inhibitor showed marked reduction of phosphorylated NF-κB (p-NF-κB) in all four melanoma cell lines tested as compared with cells treated with vehicle alone (DMSO), Figure 1C. Furthermore, IRAK-1,-4 inhibition reduced the production of VEGF over 90% and diminished CXCL1, monocyte chemotactic protein-1 (MCP1), platelet-derived growth factor alpha (PDGF-A) and fibroblast growth factor (FGF-2) levels in A375 cells (Fig 1D, p<0.05). The fact that that IRAK-1,-4 inhibition reduced the levels of these molecules in the absence of exogenous TLR agonists, suggests that IRAK-1,-4 contributes greatly to cell function through the production of various factors. It is worth highlighting that the addition of IRAK-1,-4 inhibitors also decreased the effects of TLR1-TLR2 agonist (Supplementary Figure 2), further confirming that changes in chemokines/cytokines occurred in a TLR-MyD88-IRAK fashion.

Collectively, these findings support the contention that the activation of IRAK-1,-4 signaling on melanoma cells in vivo might contribute to cancer progression by inducing the expression of various chemokines and cytokines beneficial to cancer cell survival, division, and/or angiogenesis. Furthermore, the inhibition of IRAK-1,-4 drastically reduced the production of several cytokines/chemokines, highlighting the possibility that IRAK-1,-4 signaling plays a central role in cytokine/chemokine production even in absence of exogenous TLR agonists. However, these data do not exclude the possibility that TLRs recognize endogenous TLR agonists or that other cytokines produced in response to IRAK signaling might further potentiate this signaling pathway.

Combining IRAK inhibition with certain chemotherapies augments melanoma cell apoptosis in vitro and in vivo

Melanoma cells become resistant to a variety of chemotherapies by altering their survival signaling pathways during cancer progression(17). Various studies have documented the prosurvival effects that TLR signaling have on different cell types(13). Considering the impact that inhibiting IRAK-1,-4 had on NF-κB activation and chemokine/cytokine production, we examined whether IRAK-1,-4 inhibitor might be used as a means to augment the toxic effects of certain chemotherapies which alone are not very effective. The melanoma cell lines A375 and Malme-3M were cultured in the presence of IRAK-1,-4 inhibitor and various concentrations of vinblastine, 5′fluorauracil and cisplatin. The data in Figure 2A demonstrate increased sensitivity of both cell lines to vinblastine (left panels). The addition of 5′-fluorouracil and IRAK-1,-4 inhibitor also appeared to enhance the apoptosis of Malme3M cells but had no effect on A375 cells (Fig. 2A, middle panels). IRAK-1,-4 inhibition did not increase cisplatin’s cytotoxicity of either cell line (Fig. 2A, right panels). We examined in greater detail the role that IRAK had in mediating the chemoprotective effects. A375 cells engineered to knockdown IRAK-1 expression were treated with vinblastine, and apoptosis was measured 48 hours later. psi-RNA reduced IRAK protein levels over 95%; a representative image is shown in Figure 2B. Reducing IRAK-1 or IRAK-4 expression levels augmented the toxic effects of vinblastine, as compared with cells transfected with the control plasmid Figure 2B (right panel). Cells co-expressing psi-RNA-hIRAK-1 and -4 exhibited a higher percentage of death than cells expressing either of these vectors alone, Figure 2B (right panel). It is worth noting that IRAK inhibitor alone or cells expressing psiRNA-IRAK moderately increased melanoma apoptosis (in the absence of vinblastine), suggesting that IRAK signaling plays an important role in cell survival.

Figure 2. IRAK-1,-4 inhibition augments vinblastine-mediated apoptosis of melanoma cells.

Figure 2

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(A) A375 and Malme-3M melanoma cells were cultured with or without 2.5μM of IRAK-1,-4 inhibitor in the presence varying concentrations of vinblastine, cisplatin or 5′Fluorouracil. Forty-eight hours later apoptosis was evaluated by staining cells with annexin-V and propidium iodide and analyzed by flow cytometry. The result from one of five experiments is shown. (B) A375 cells were transfected via electroporation with siRNA-IRAK-1, -4. IRAK-1 protein levels were examined by western blot. A375-control, A375-psiRNA-hIRAK-1, A375-psiRNA-hIRAK-4 or cells expressing both psiRNA-hIRAK-1 and -4 were cultured in the presence of vinblastine (100nM) for 48 hours and apoptosis was examined by flow cytometry. (C) A375 melanoma cells were cultured in the presence of vehicle alone (DMSO) or various concentrations of IRAK-1,-4. Forty-eight hours later cell lysates were used to analyze the expression of levels of cleaved PARP or caspase-3 by Western blots. (D) A375 melanoma cells were cultured with or without 2.5μM of IRAK-1,-4 and in the presence or absence of vinblastine for 48 hrs. PARP and caspase-3 levels were determined by western blot.

IRAK-1,-4 inhibition resulted in reduced levels of activated NF-κB (Figure 1C). Therefore, we examined whether IRAK–NF-κB signaling is linked to chemoresistance by treating A375 melanoma cells with and without NF-κB inhibitor and in the absence and presence of vinblastine. Interestingly, NF-κB inhibition did not alter A375’s sensitivity to vinblastine (Supplementary Fig 3A; p=0.14). Furthermore, because the toxic effects of doxorubicin have been shown to involve NF-κB signaling, we also examined the effects of inhibiting NF-κB in A375 melanoma cells(18). In sharp contrast to the combinatorial effects of NF-κB inhibition plus vinblastine, NF-κB inhibition increased the toxic effects of doxorubicin on A375 Supplementary Figure 3A. These data suggest that although TLR-IRAK signaling can activate NF-κB and inhibiting IRAK signaling can reduce p-NF-kB levels, the chemoprotective effects observed appear to be mediated via a mechanism independent of NF-κB. Because IRAK signaling results in the activation of various transcription factors including c-jun/Fos, Elk-1, and CREB, we postulate that perhaps IRAK signaling is linked to chemoresistance via the activation of one of these factors. The possibility that these transcription factors could impart melanoma cells a prosurvival signal is substantiated by several studies highlighting that these transcription factors play a critical role in melanoma progression(19–22).

Alternatively, cytokines/chemokines produced in response to TLR-IRAK signaling might also contribute to the chemoprotective effects observed. Therefore, supernatant from TLR1-TLR2-stimulated or unstimulated A375 melanoma cells was added to the C32 melanoma cells in the presence or absence of vinblastine. Another group of cells were treated with TLR1-TLR2 ligand and apoptosis was examined by flow cytometry. The Supplementary Figure 3B demonstrate that supernatant from TLR-stimulated A375 cells moderately reduced vinblastine-induced apoptosis as compared with untreated cells (p<0.05; ANOVA), whereas TLR1-TLR2 ligand had little effect. Furthermore, neither IL-1 or CXCL1 appeared to be the cytokines responsible for the observed chemoprotective effects, Supplementary Figure 3C. These data suggest that cytokines/chemokines produced in response to TLR-IRAK stimulation can contribute to cell survival; however, other IRAK-mediated factors appear to play a more important role. It is also worth noting that different melanoma cell lines might be more or less responsive to factors produced in response to TLR-IRAK stimulation based on the expression levels of different cytokine/chemokine receptors.

As an independent biochemical assay to confirm that A375 melanoma cells triggered apoptosis, we also conducted Western blot analysis using antibodies specific for caspase-3 and PARP. Active caspase-3, which is cleaved to yield catalytically active subunits, was detected following the addition of 10μM IRAK-1,-4 inhibitor (Fig. 2C). Enhanced accumulation of cleaved PARP, which is targeted for caspase-dependent proteolysis, was also observed in IRAK-1,-4 inhibitor–treated cells. Increased levels of cleaved PARP and caspase-3 occurred in a concentration-dependent manner (Fig. 2C). As shown in Figure 2D, the combination of vinblastine and IRAK-1,-4 inhibitor also increased the levels of cleaved PARP and caspase-3. In contrast, treatment with vinblastine or IRAK-1,-4 inhibitor alone had little effect on the levels of these molecules. We also sought to determine whether inhibitor–treated cells exhibited changes in the levels of apoptosis-related molecules. We compared the expression of various apoptosis-related genes in A375 melanoma cell line treated with DMSO vehicle (control), IRAK inhibitor, and vinblastine and observed a gene profile that favored apoptosis in the presence of inhibitor alone or vinblastine (Supplementary Figure 4). Taken together with data in presented Figure 2, these gene array data confirm that inhibiting the IRAK pathway in melanoma cells is important for their survival and indicate that interfering with this pathway can sensitize melanoma cells or at lease function in concert with certain chemotherapies to enhance their toxic effects. These data also demonstrate that some chemotherapeutic drugs, such as vinblastine, that have been deemed ineffective might be rendered therapeutically useful when combined with a TLR/IRAK inhibitor.

The anti-tumor effects of combinatorial therapy using vinblastine and IRAK-1,-4 inhibitor were tested in vivo. A375 cells were subcutaneously (s.c.) injected into NSG mice and grown to 30–50mm2. Mice were injected intraperitoneally with vinblastine or intratumorally with IRAK-1,-4 inhibitor (35mg/kg) or with vinblastine or IRAK-1,-4 inhibitor alone. Mice receiving IRAK-1,-4 inhibitors plus vinblastine showed a marked reduction in tumor growth and improved mouse survival (median survival: 38 days) as compared with mice receiving vehicle (DMSO) plus vinblastine (median survival: 22 days) or mice receiving IRAK-1,-4 inhibitor (median survival: 19 days), Figures 3A and 3B. Mice receiving single therapy of IRAK-1,-4 inhibitor or vinblastine showed modest tumor growth delay but similar survival as compared with control mice (median survival: 17 days). Despite a tumor growth delay, mice receiving IRAK-1,-4 inhibitors plus vinblastine succumbed to tumor challenge 40 days after initiation of treatment. Nonetheless, these results emphasize the anti-tumor effects of combinatorial therapy and highlight the need for further optimization. Identifying novel signaling pathways that can improve the efficacy of chemotherapeutic drugs to treat melanoma patients is critical, considering that advanced melanoma is highly resistant to all known chemotherapies(17). The synergistic effects of IRAK-1,-4 inhibitor and vinblastine may reside in the mechanism of action by vinblastine. Whereas, vinblastine kills cells by suppressing microtubule dynamics, cisplatin and 5′fluorouracil damage DNA by inducing DNA (and protein) crosslinking and by irreversibly inhibiting thymidylate synthase, respectively. Ongoing studies by our group are focused on identifying additional chemotherapeutic drugs that are enhanced when used in combination with IRAK-1,-4 inhibitor.

Figure 3. Treatment with IRAK-1,-4 inhibitor enhances the therapeutic effects of vinblastine in mice with an established human melanoma tumor.<.

Figure 3

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br>NSG mice (n=5) were injected subcutaneously with A375 melanoma cells. When tumors reached a size of approximately 50mm2 mice remained untreated or injected intraperitoneally with vinblastine (0.25 mg/kg; administered every 2 days for 10 days), or peritumorally with IRAK-1,-4 inhibitor (35mg/kg), or vinblastine plus IRAK-1,-4 inhibitor. Tumor sizes were calculated by measuring perpendicular by longitudinal diameter; *, P < 0.05. Mouse survival data were analyzed using the exact log-rank test; *, P < 0.05, **P < 0.001.

Altogether, these findings corroborate a link between IRAK-1,-4 signaling in melanoma cells and survival and suggest a phenotype supportive of melanoma progression in cancer patients. The importance of IRAK-1/IRAK-4 signaling and cell survival is further highlighted in immune cells in which TLR engagement enhances the expression of various anti-apoptotic genes(13).

Phosphorylated IRAK-4 in melanoma biopsies

Phosphorylated IRAK-4 expression was analyzed by immunohistochemistry in normal skin and in melanoma tissue derived from patients at various clinical stages. While little staining was observed in normal skin, p-IRAK-4 was highly expressed in melanoma samples (representative staining are shown in Fig. 4A). We sought to determine the association between p-IRAK-4 expression and the clinical stage. As shown in Figure 4B, p-IRAK-4 levels were not linked to melanoma stage nor was there a correlation between p-IRAK-4 levels and metastasis. Unexpectedly, however, quantification of the staining revealed two distinct groups in melanoma samples in all clinical stages; one group of melanoma samples expressed p-IRAK-4 levels similar to those on normal skin, and another group expressed significantly higher p-IRAK-4 levels (Fig. 4B; p<0.005; ANOVA). Samples from stage I through IV melanoma also showed a distinct division between high and low/no expression of phosphorylated IRAK-4 (Fig. 4B; p<0.05; ANOVA). Of the 242 melanoma samples analyzed, nearly half expressed elevated levels of p-IRAK-4. It is worth noting that the elevated levels of p-IRAK-4 in melanoma patients did not correlate with patient age or gender (Fig. 4C).

Figure 4. Expression of p-IRAK-4 in melanoma cells biopsy specimens.

Figure 4

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(A) Biopsies obtained from melanoma patients at different clinical stages were stained for phosphorylated IRAK-4 or S100 and examined by immunohistochemistry. Mouse serum was used as a control to examine non-specific antibody staining. Representative staining from patients at different stages of cancer or normal skin are shown (x20 field sections). (B and C) Quantification of p-IRAK-4 staining as a function of clinical stage, age, and gender. The number in parentheses below the clinical stage is the number of patient samples analyzed. The dotted line in C (left panel) represents the trend line from all samples and the bar line in the right panel is the average p-IRAK-4 value generated from samples obtained from female and male patients.

Currently, there is no effective cure for patients with advanced melanoma. This is due in large part to their high resistance to chemotherapeutic drugs. Ongoing studies by our group are focused on identifying the association between total and phosphorylated IRAK-1 and IRAK-4 expression levels with clinical outcome including response to treatment and survival. The immunohistochemical data presented here support are in line with the western blot data showing that a subset (42%; 3 of 7) melanoma cell lines express p-IRAK. Ongoing studies are focused on determining the use of total and/or p-IRAK-1,-4 as biomarkers for prediction of therapy resistance and patient survival. It is important to note that recent studies support our contention that IRAK signaling contributes to cancer progression. Boukerche et al. found that IRAK-1 gene transcript levels in melanoma patients were associated with highly metastatic melanoma individuals wheras, Li et al. observed that re-expression of miR-146a in pancreatic cancer lines downregulated IRAK-1 expression as well as NF-κB activity which blocked pancreatic cancer cell invasiveness and metastasis (23;24).

The studies presented here highlight the possibility of identifying new chemotherapeutic agents that might be currently in use to treat other cancers or other disorders but might be repurposed against melanoma when joined with IRAK inhibitors. Although new drugs such as Vemurafenib (BRAF enzyme inhibitor), Ipilimumab (anti-CTLA-4), BMS-936558 (anti-PD1), and BMS-663513 (anti-CD137)(25) demonstrate antitumor activity, the effects are quite moderate, prolonging survival 2 to 6 months. Additionally, patients eventually develop a resistance to Vemurafenib. For this reason Plexxikon & Roche are currently testing vemurafenib in combination with other signaling pathway inhibitors such as MEK inhibitors in an attempt to prevent the development of tumor chemoresistance. Unfortunately, no other therapies to date have proven effective at improving overall survival of these patients(3;26;27), and the development of new therapeutic agents has not kept up with the increased melanoma incidence(28). We propose that understanding how IRAK inhibition sensitizes melanoma to chemotherapies (or augments their toxic effect) might provide novel insights and allow the discovery of more effective therapies to treat melanoma patients.

Supplementary Material

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Acknowledgments

Grant Support: National Cancer Institute 1R01CA140917-01, NIH Center for Biomedical Research Center Excellence grants 1P20 RR021970, and University of Maryland, Marlene and Stewart Greenebaum Cancer Center.

Footnotes

None of the authors have conflicts of interests with regard to the manuscript submitted for review.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS412033-supplement-1.pptx (3.6MB, pptx)
2
NIHMS412033-supplement-2.pptx (46.8KB, pptx)
3
NIHMS412033-supplement-3.pptx (65.1KB, pptx)
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NIHMS412033-supplement-4.pptx (63KB, pptx)
5
NIHMS412033-supplement-5.pptx (38.6MB, pptx)

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