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. 2022 Dec 17;13(1):14. doi: 10.1007/s13205-022-03420-y

Suppression of TLR signaling by IRAK-1 and -4 dual inhibitor decreases TPF-resistance-induced pro-oncogenic effects in HNSCC

Humayara Khan 1, Sachchida Nand Pandey 2, Abhishek Mishra 3, Ratika Srivastava 1,4,✉
PMCID: PMC9759608  PMID: 36540413

Abstract

Combination of docetaxel, cisplatin and 5-FU, known as TPF, is an FDA-approved treatment for head and neck squamous cell carcinoma (HNSCC). Acquired chemo-resistance to TPF, a primary reason for non-responsiveness to the treatment and relapse of tumor is a major concern for treatment failure, especially in elder patients. In this study, we investigated the role of Interleukin-1 receptor-associated kinases (IRAK) mediated Toll-like receptor (TLR)—signaling in chemo-resistance using a cell line-based in-vitro TPF-resistant HNSCC model of laryngeal origin. TPF chemo-resistant state showed over-expression and phosphorylation of the active downstream kinases IRAK-1 and IRAK-4 along with enhanced proliferative potential, survival, stemness and metastatic capability as compared to the parent cell line. Pharmacological inhibition of IRAK-1 and -4 had a cytostatic effect on chemo-resistant cells and re-sensitized them to chemotherapy. The treatment also decreased the pro-oncogenic effects of the chemo-resistant cells. Our study provides insights into the pro-oncogenic role of amplified IRAK-1 and-4 mediated TLR signaling in TPF-resistant HNSCC. Pharmacological inhibition of IRAK-1 and-4 signaling is a promising therapeutic strategy for TPF-resistant HNSCC. It can also be used as a combination therapy or a chemo-drug sparing regimen in HNSCC.

Keywords: HNSCC, TPF-resistance, TLR, IRAK, Combination therapy, Pro-oncogenic effects

Introduction

Head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer worldwide with 450,000 deaths per year (Sung et al. 2021). Common risk factors for HNSCC are tobacco intake, excessive alcohol consumption and human papillomavirus (HPV) infection (Muhaxheri et al. 2015). The treatment modalities for HNSCC are surgery and radiotherapy, combined with chemotherapy, and immunotherapy especially for advanced HNSCC (Cognetti et al. 2008). Conventional chemo-drugs used for the treatment of advanced HNSCC are docetaxel, paclitaxel, cisplatin, carboplatin, methotrexate and 5-fluorouracil (5-FU) (Elliott et al. 2017). Combining the various drug modalities is being used for better efficacy and reduced toxicity in many cancers. A combination of chemo-drugs docetaxel, cisplatin and 5-FU, referred to as the TPF triplet regimen has been approved by the Food and Drugs Administration (FDA) for HNSCC treatment in 2006 (Vermorken et al. 2007; Lorch et al. 2011). Pembrolizumab (anti-PD1 antibody) or cetuximab (anti-EGFR antibody) in combination with cisplatin/5-FU is another combination therapy approved by FDA for recurrent/metastatic HNSCC in 2019 and 2011, respectively (Cohen et al. 2019, 2013).

TPF is widely given as induction therapy also in HNSCC. It shows improved progression-free survival (PFS) and organ preservation compared to single-agent and double-agent treatment (Kim et al. 2016; Zhong et al. 2018). However, high toxicity, poor tolerability and non-responsiveness in almost 60% of Stage III, IV and elderly patients are a major concern with its wide usage (Sher et al. 2016; Ilie et al. 2012). The reason for non-responsiveness is acquired chemo-drug resistance which results in loco-regional relapse, metastasis and a low-survival rate of advanced stage patients. It is important to understand the molecular mechanism of chemo-resistance. It will not only allow the discovery of new better targeted, multimode therapies but can also be helpful in the identification of TPF-resistant biomarkers.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) present on immune cells as well as non-immune cells including various cancer cells. They can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) as ligands. Chemotherapeutic stress in cancer leads to the release of DAMPs which activates the TLR signaling. It leads to activation of two downstream kinases, Interleukin-1 receptor-associated kinase-4 (IRAK-4) and Interleukin-1 receptor-associated kinase-1 (IRAK-1) through phosphorylation (Kawasaki et al. 2014; Cao et al. 1996).

TLR signaling is reported to regulate cell proliferation, survival and tumor progression in cancers such as gastric cancer, ovarian cancer, lung cancer and breast cancer (Sato et al. 2009). Over-expression and activation of IRAK-1 and-4 in solid tumors such as triple-negative breast cancer (TNBC) (Wee et al. 2015), hepatocellular carcinoma (HCC) (Cheng et al. 2018), melanoma (Srivastava et al. 2012), pancreatic ductal adenocarcinoma (PDAC) (Zhang et al. 2017) and colorectal carcinoma (CRC) (Li et al. 2019) have been reported to promote tumor proliferation, survival, migration and inflammation along with poor prognosis and survival of patients. Chemotherapy resistance is shown to induce IRAK-1 phosphorylation in TNBC and HCC, and IRAK-4 phosphorylation in PDAC and CRC, further promoting aggressive tumor growth. Pharmacological inhibition of IRAK-1 and-4 suppressed tumor progression in chemotherapy-sensitive and resistant solid tumors further sensitizing them to chemotherapy (Wee et al. 2015; Cheng et al. 2018; Srivastava et al. 2012; Zhang et al. 2017; Li et al. 2019). Over-expression of IRAK-1 has been reported to promote the survival of HNSCC cell lines which could be reversed by genetic and pharmacological inhibition of IRAK-1 (Adams et al. 2015). IRAK-1 regulates metastasis of nasopharyngeal carcinoma (NPC) cells. Genetic and pharmacological inhibition of IRAK-1 suppressed metastasis of NPC cells in in vitro and in vivo models (Meng et al. 2020). IRAK-1 phosphorylation is reported to be up-regulated in paclitaxel-resistant NPC cells, and targeting IRAK-1 re-sensitized NPC cells to paclitaxel (Liu et al. 2021). However, the role of IRAK-1 and -4 mediated TLR signaling in TPF chemo-therapy resistance in HNSCC has not yet been investigated.

In this study, we developed an in vitro HNSCC model to mimic chemo-resistance to TPF treatment. The model was used to assess the role of IRAK-mediated TLR signaling in the development of chemo-resistance along with other pro-oncogenic properties. The study also evaluated the therapeutic potential of blocking TLR signaling in combating chemo-resistance through pharmacological inhibition of IRAKs.

Materials and methods

Bioinformatics-based analysis of IRAK1 and IRAK4 expression and survival

Head and neck squamous cell carcinoma-related data available on TCGA research network was mined regarding IRAKs. UALCAN (http://ualcan.path.uab.edu/index.html) is a user-friendly website for the analysis of large scale-cancer genomics (Chandrashekar et al. 2022). A pan cancer analysis of IRAK-1 and IRAK-4 expression, along with its expression analysis in normal adjacent tissues and primary HNSCC tumors in TCGA database was carried out. Overall survival was also analyzed in relation to IRAK-1 and IRAK-4 expression. For IRAK-1 and IRAK-4 expression analysis, a dataset of 130 patients were in high expression group and 389 patients in low expression group were analyzed.

Cell line and reagents

The human laryngeal carcinoma cell line HEp-2 was purchased from NCCS, Pune, India. The cell line was maintained in DMEM (Hyclone, GE) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) in a humidified incubator at 37℃ with 5% CO2. Docetaxel (Zydus Cadila Pharmaceuticals, India), 5-FU (Zydus Cadila Pharmaceuticals, India) and Cisplatin (Celplat) were procured commercially. IRAK-1 and-4 dual inhibitor solution (Cat# 407602, Sigma-Aldrich, St. Louis, MO, USA) was used as a pharmacological small molecule inhibitor. For flow cytometry and western blotting experiments, following antibodies namely anti-CD44 PE (Cat# 12-0441082), anti-Ki-67 FITC (Cat# 11-5699-42), anti-vimentin PE (Cat# MA1-19656), anti-Nanog DyLight 488 (Cat# MA1-017-D488), anti-ALDH1 (Cat# MA5-29023) and anti-p-IRAK-1 (Cat# PA5-38633) and anti-E-cadherin APC (Cat# A15717) from Invitrogen, CA, USA, were used. Anti-IRAK-1 (Cat# 4504), anti-IRAK-4 (Cat# 4363), anti-p-IRAK-4 (Cat# 11,927) and anti-rabbit IgG (H + L) F(ab′)2 Fragment-PE (Cat# 8885) were purchased from Cell Signaling Technology (CST), Beverly, MA, USA. Goat anti-rabbit IgG HRP (Cat# SE134) and anti-GAPDH (Cat# ITT07021) were purchased from ImmunoTag, G-Biosciences, USA, respectively. Goat anti-mouse IgG HRP (Cat# 62114068001A) was purchased from Bangalore Genei, India.

Drug sensitivity assay

Drug sensitivity of the HEp-2 cells was evaluated by the resazurin dye-based assay. Briefly, 1000 cells were seeded per well in a 96 well plate and incubated overnight. Cells were treated with docetaxel, cisplatin, 5-FU and IRAK-1 and-4 dual inhibitor at various concentrations and incubated for 72 h. Subsequently, cells were incubated with 1X Resazurin dye solution (100 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 4 h and fluorescence was measured at 530 nm excitation/590 nm emission on a Synergy HT microplate reader (BioTek Instruments, USA). Percent suppression in cell proliferation was determined by the equation: (average fluorescence of treated cells/average fluorescence of control cells) ×100. A dose–response curve was generated to calculate the IC50 (half-maximal inhibitory concentration), IC25, IC12.5, IC6.25 and IC3.125 values. To determine the effect of combination treatment on the proliferation of chemo-resistant cells, cells were treated with increasing concentrations of chemo-drugs along with IC25 of IRAK-1 and-4 dual inhibitor simultaneously for 72 h. The percent suppression in cell proliferation was measured as described above.

Development of triple-chemo-resistant cell line

A triple chemo-drug-resistant cell line was prepared by subjecting Parent HEp-2 cells to a combination of docetaxel, cisplatin and 5-FU in a dose incremental approach using the method by Govindan et al. (2015) with slight modifications. Cells were first treated with a combination of IC3.125 of all three chemo-drugs (IC3.125 of docetaxel: 0.030 nM, cisplatin: 0.665 μM, 5-FU: 0.004 mM) followed by IC6.25 of all three chemo-drugs (IC6.25 of docetaxel: 0.056 nM, cisplatin: 0.826 μM, 5-FU: 0.010 mM) for 72 h each in two separate cycles. Cells were incubated in a drug-free medium for 72 h in between the two chemo-drugs exposure cycles.

Treatment of cells

Briefly, 10,000 chemo-resistant cells were seeded per well in a 12 well plate and incubated overnight. To evaluate the effect of single-drug treatment on the pro-oncogenic effects, cells were treated with IC25 of IRAK-1 and-4 dual inhibitor or IC12.5 of the individual chemo-drugs. To evaluate the effect combination-drug treatment on the pro-oncogenic effects, cells were treated with a combination of IC12.5 of the specific chemo-drug along with IC25 of IRAK-1 and-4 dual inhibitor together. Cells were further incubated for 72 h at 37 °C with 5% CO2. Post-incubation, cells were harvested and expression of different markers were analyzed.

Gene expression profiling

Total RNA was isolated from 5 × 106 cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Synthesis of c-DNA was performed from 1 µg of RNA using the PrimeScript 1st strand cDNA synthesis kit (Takara, Japan) according to the manufacturer’s instructions. qPCR was performed using TB Green Premix Ex Taq II (Tli RNase H Plus) (Takara, Japan) using specific primers for Bcl-2, Bcl-xL and MMP-2 on a CFX96 Touch™ Real-time PCR detection system (BioRad Laboratories, Inc, USA). GAPDH was used as an internal control.

Flow cytometry analysis

Cells were stained with fluorochrome-conjugated antibodies against CD44, E-cadherin and vimentin. Ki-67 and Nanog were stained by fixing the cells with 2% paraformaldehyde for 10 min, permeabilized with 100% cold methanol and incubated with fluorochrome-conjugated antibodies against the respective markers. Staining for IRAK-1, IRAK-4, p-IRAK-1 and p-IRAK-4 were performed using eBioscience™, USA-based Foxp3/Transcription Factor Staining Buffer Set (Cat# 00-5523-00) according to the manufacturer’s instructions. The cells were washed and resuspended in FACS buffer. Samples analysis was carried out on a FACS Calibur flow cytometer (BD Biosciences, USA).

Western blotting

Cell lysates were prepared using the RIPA buffer supplemented with protease phosphatase inhibitor cocktail (Cat# PPC1010) (Sigma-Aldrich, USA). 50 µg of lysate was resolved on 12% SDS-PAGE and transferred on polyvinylidene difluoride (PVDF) membranes (Amersham, GE Healthcare, UK). Membranes were incubated in blocking buffer (3% BSA in TBST) for 1.5 h at RT and probed with primary antibodies against ALDH1 and GAPDH overnight at 4℃. Membranes were probed with HRP-conjugated anti-mouse and anti-rabbit secondary antibodies for 1.5 h at RT, developed with Clarity™ Western ECL substrate (BioRad Laboratories, Inc, USA) and detected using the ChemiDoc™ Touch imaging system (BioRad Laboratories, Inc, USA).

IL-6 ELISA

IL-6 cytokine was estimated in cell culture supernatants using the Human IL-6 Antibody Pair Kit (Cat# CHC1263, Invitrogen, CA, USA) according to the manufacturer’s instructions.

Statistical analysis

Experiments were performed three times independently and the data are expressed as mean ± S.D. GraphPad Prism software 8.0 was used for statistical analysis. The student’s t test was used for comparison of two datasets and analysis of variance (ANOVA) was used for comparison of multiple datasets. P value of less than 0.05 was considered statistically significant.

Results

TCGA data analysis of IRAK-1 and IRAK-4 expression indicates poor overall-survival of patients in HNSCC

We used the UALCAN database to compare the mRNA expression of IRAK-1 and IRAK-4 in HNSCC with other tumors in the TCGA database and normal adjacent tissues. IRAK-1 was overexpressed in HNSCC (Fig. 1a), although expression of IRAK-4 in HNSCC was moderate in a pan-cancer view of TCGA tumors (Fig. 1b). In comparison with the normal adjacent tissues, IRAK-1 expression was significantly higher but IRAK-4 expression remained unchanged in primary tumor samples of HNSCC (Fig. 1c). We next investigated the prognostic potential of IRAK-1 and IRAK-4 in HNSCC. High IRAK-1 expressing group demonstrated a significantly reduced overall survival (OS) of patients (median survival: 980 days) compared to low/medium IRAK-1 expressing group (median survival: 1762 days) (Fig. 1d). No significant difference was observed in the OS of high and low/medium IRAK-4 expressing group of patients (Fig. 1d) although altogether expression of both IRAK-1 and IRAK-4 were associated with reduced OS of HNSCC.

Fig. 1.

Fig. 1

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Database analysis of expression and prognostic value of IRAK-1 and IRAK-4 in HNSCC patients a Pan-cancer view of IRAK-1 mRNA expression across TCGA tumors. b Pan-cancer view of IRAK-4 mRNA expression across TCGA tumors. c Box-plots demonstrating mRNA expression analysis of (i) IRAK-1 and (ii) IRAK-4, in normal adjacent tissues and primary tumor of HNSCC patients d Kaplan–Meier survival curve estimates demonstrating overall-survival for patients with high and low (i) IRAK-1 and (ii) IRAK-4 expression.

Source: UALCAN browser

Validation of the triple chemo-resistant HEp-2 cell line

A triple chemo-resistant cell line was generated by subjecting HEp-2 to docetaxel, cisplatin and 5-FU following the procedure described in “Materials and methods”. Morphologically, chemo-resistant HEp-2 exhibited slightly enlarged nuclei and cell size compared to the parent HEp-2 (Fig. 2a). The forward scatter (FSC) vs. side scatter (SSC) profile of these cells was compared with parent line by flow cytometry. Chemo-resistant HEp-2 displayed relatively high SSC signals compared to parent HEp-2 indicating increased granularity in the chemo-resistant cells (Fig. 2b).

Fig. 2.

Fig. 2

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Validation of development of chemo-resistant HEp-2 cell line a Morphological assessment displayed enlarged nuclei (indicated by arrows) and cell size of (ii) chemo-resistant HEp-2 compared to (i) parent HEp-2. Representative phase-contrast micrographs at original magnification, 40X. b Flow cytometric analysis of FSC vs. SSC profile of cells demonstrating high SSC of (ii) chemo-resistant HEp-2 compared to (i) parent HEp-2 shown in representative scatter plots. c Increased proliferation of chemo-resistant HEp-2 compared to parent HEp-2 treated with (i) docetaxel, (ii) cisplatin and (iii) 5-FU determined by resazurin assay and presented as percent cell proliferation curves (n = 3)

Further, parent and chemo-resistant HEp-2 were subjected to various concentrations of docetaxel, cisplatin and 5-FU and the anti-proliferative effects of the chemo-drugs on the cells were assessed by resazurin assay (Fig. 2c). The average IC50 of the chemo-drugs derived from the dose–response curves are listed in Table 1. Approximately 1678, 4 and 12-fold increase in the IC50 of docetaxel, cisplatin and 5-FU, respectively in chemo-resistant HEp-2 compared to parent HEp-2 validated the development of chemo-resistant HEp-2 line.

Table 1.

IC50 of chemo-drugs on parent and chemo-resistant HEp-2

Chemo-drug IC50 of chemo-drugs on parent HEp-2 (n = 3) IC50 of chemo-drugs on chemo-resistant HEp-2 (n = 3) Fold Change (increase in IC50 value on chemo-resistant HEp-2 with respect to parent HEp-2)
Docetaxel 0.864 ± 0.42 nM 1450 ± 0.7 nM 1678
Cisplatin 13.05 ± 3.31 μM 53.04 ± 4.88 μM 4
5-FU 0.237 ± 0.062 mM 2.8 ± 0.8 mM 12
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TPF-resistant HEp-2 have higher proliferation, survival potential and cancer stem cells

The proliferation potential of both parent and TPF chemo-resistant cell lines was further assessed by measuring the expression of Ki-67 in the cells by flow cytometry. Chemo-resistant HEp-2 demonstrated an increased Ki-67 expression compared to parent HEp-2 (Fig. 3a). Expression of anti-apoptotic proteins Bcl-2 and Bcl-xL were found 6.3 and 4.4-fold up-regulated at mRNA level in chemo-resistant HEp-2 as compared to parent HEp-2, respectively (Fig. 3b). Cancer stem cells (CSCs) were enumerated using CSCs markers CD44, Nanog and ALDH1. An increase in the proportion of CD44 and Nanog expressing cells was observed in chemo-resistant HEp-2 compared to parent HEp-2 (Fig. 3c). The mean fluorescence intensity (MFI) of CD44 was also higher in chemo-resistant HEp-2 as compared to parent HEp-2 (Fig. 3d). By western blot analysis, marginal over-expression of ALDH1 was observed in chemo-resistant HEp-2 compared to parent HEp-2 (Fig. 3e). Higher expression of all the three markers suggested the presence of more CSCs in TPF-resistant HEp-2 and higher stemness as compared to the parent.

Fig. 3.

Fig. 3

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Chemo-resistant HEp-2 exhibits increased proliferative potential, survival and stemness compared to parent HEp-2. a Statistical analysis demonstrating an increase in the percentage of Ki-67 + cells estimated by flow cytometry. b qRT-PCR analysis of Bcl-2 and Bcl-xL mRNA expression indicating over-expression in chemo-resistant HEp-2 compared to parent HEp-2. c Flow cytometric analysis demonstrating over-expression of CD44 and Nanog in chemo-resistant HEp-2 compared to parent HEp-2. (i) Representative flow cytometric histogram images for CD44 and Nanog expression; (ii) statistical analysis of the percentage of CD44+ and Nanog + cells. d Statistical analysis of MFI expression demonstrating increased CD44 expression in chemo-resistant HEp-2 compared to parent HEp-2. e (i) Western blotting analysis indicates over-expression of ALDH1 in chemo-resistant HEp-2 compared to parent HEp-2. (ii) Relative expression of ALDH1 after normalization with GAPDH was analyzed using Image J (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

TPF-resistance attributes metastatic potential to chemo-resistant HEp-2

Since enhanced stemness is also associated with increased occurrence of metastasis (Li et al. 2014; Wang et al. 2013; Shibue et al. 2017), HNSCC-associated metastasis promoting cytokine and chemokine namely IL-6 and MMP-2 were estimated (Yadav et al. 2011; Stanciu et al. 2017). TPF-resistant HEp-2 produced a significantly higher amount of IL-6 as compared to parent HEp-2 as estimated by ELISA assay (Fig. 4a). An approximately ten-fold increase in mRNA expression of MMP-2 was observed in chemo-resistant HEp-2 as compared to parent HEp-2 (Fig. 4b). By flow cytometric analysis, a 50% decrease in E-cadherin + cells and an 11.7% increase in vimentin + cells were observed in chemo-resistant HEp-2 compared to parent HEp-2 (Fig. 4c). Data suggests that TPF-resistant HEp-2 has higher metastatic potential than parent one.

Fig. 4.

Fig. 4

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Chemo-resistant HEp-2 attains higher metastatic potential than parent HEp-2. a Significantly higher IL-6 cytokine production by chemo-resistant HEp-2 as compared to parent HEp-2. b qRT-PCR analysis of MMP-2 mRNA expression indicates their increased levels in chemo-resistant HEp-2 compared to parent HEp-2. c EMT upon chemo-resistance of HEp-2 demonstrated by flow cytometric analysis of E-cadherin and vimentin expression. (i) Representative flow cytometric histogram images and (ii) statistical analysis of the percentage of E-cadherin+ and vimentin + cells showed reduced E-cadherin and increased vimentin expression in chemo-resistant HEp-2 compared to parent HEp-2 (*p < 0.05, **p < 0.01, ****p < 0.0001)

TLR signaling is activated in chemo-resistant HEp-2 and enhances their proliferation

To know whether TLR signaling is constitutively on or not, total and phosphorylated forms of downstream kinases, IRAK-1 and IRAK-4 were estimated by flow cytometry. The proportion of IRAK-1+ and p-IRAK-1+ cells were significantly higher in chemo-resistant HEp-2 as compared to parent HEp-2 by 16.5% and 19.78%, respectively. An increase in IRAK-4+ cells and p-IRAK-4+ cells in chemo-resistant HEp-2 compared to parent HEp-2 by 25.4% and 21.04%, respectively, were observed. MFI of total IRAKs expression and their phosphorylated forms were also significantly higher in chemo-resistant HEp-2 compared to parent HEp-2 (Fig. 5a).

Fig. 5.

Fig. 5

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Chemo-resistant HEp-2 exhibits significantly higher activation state of IRAK-1 and -4 kinases than parent HEp-2. a (i) Representative flow cytometric histogram images for IRAK-1, p-IRAK-1, IRAK-4 and p-IRAK-4 expression. Statistical analysis of the (ii) percentage of positive cells and (iii) MFI expression of IRAK-1, p-IRAK-1, IRAK-4 and p-IRAK-4 showing higher expression in chemo-resistant HEp-2 compared to parent HEp-2. b Chemical structure of IRAK-1 and-4 dual inhibitor (Source: Sigmaaldrich.com). c IRAK-1 and-4 dual inhibitor treatment demonstrating concentration-dependent inhibition of cell proliferation of parent and chemo-resistant HEp-2 assessed by resazurin assay and presented as percent cell proliferation curves (n = 3). (****p < 0.0001.)

To further inhibit the TLR signaling, we used commercially available IRAK-1 and-4 dual inhibitor in this study. IRAK-1 and-4 dual inhibitor, chemically known as 1-(2-(4-Morpholinyl) ethyl)-2-(3-nitrobenzoylamino) benzimidazole, selectively inhibits the kinase activity of IRAK-1 and IRAK-4 (Agliano et al. 2020). The chemical structure of IRAK-1 and-4 dual inhibitor is presented in Fig. 5b.

A concentration-dependent inhibition in cell proliferation of both parent and chemo-resistant HEp-2 was observed upon treatment with IRAK-1 and-4 dual inhibitor (Fig. 5c). A five-fold increase in the IC50 of the IRAK-1 and-4 dual inhibitor was observed on chemo-resistant cells compared to parent cells (Table 2). This could be due to the over-expression of both IRAK-1 &IRAK-4 in the resistant cells. The resistant cells were further treated with a suboptimal dose, IC25 of IRAK-1 and-4 dual inhibitor, calculated as 15.2 ± 0.351 µM from the dose–response curve on chemo-resistant cells.

Table 2.

IC50 value of IRAK-1 and-4 dual inhibitor on parent and chemo-resistant HEp-2

Parent HEp-2 (n = 3) Chemo-resistant HEp-2 (n = 3) Fold change (increase in IC50 value on chemo-resistant HEp-2 wrt Parent HEp-2 cell line)
IRAK-1 & -4 dual inhibitor 21.58 ± 1.77 µM 121.31 ± 22.53 µM 5
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IRAK-1 and-4 mediated pharmacological inhibition of TLR signaling re-sensitizes chemo-resistant HEp-2 to chemotherapy

The therapeutic potential of IRAK-1 and-4 dual inhibitor both as a single-agent and in combination with individual chemo-drugs was assessed on the proliferation of the chemo-resistant cells. The cells were treated with a combination of the chemo-drugs and IRAK-1 and-4 dual inhibitor as described in “Drug sensitivity assay”. Combination treatment of IRAK-1 and-4 dual inhibitor with the chemo-drugs inhibited the cell proliferation in a concentration-dependent manner (Fig. 6a). Significant reduction in the IC50 of docetaxel, cisplatin and 5-FU was observed upon combination treatment (Table 3).

Fig. 6.

Fig. 6

Fig. 6

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Treatment with IRAK-1 and-4 dual inhibitor re-sensitizes chemo-resistant HEp-2 to chemo-therapy and suppresses pro-oncogenic attributes more effectively. a Inhibition in cell proliferation of chemo-resistant HEp-2 on combining IRAK-1 and-4 dual inhibitor with chemo-drugs (i) docetaxel, (ii) cisplatin, and (iii) 5-FU, determined by resazurin assay and presented as percent cell proliferation curves (n = 3). b Statistical analysis of the percentage of Ki-67+ cells showing a decrease in Ki-67 + cells upon treatment with IRAK-1 and-4 dual inhibitor alone and in combination with individual chemo-drugs analyzed by flow cytometry. c Statistical analysis showed a decrease in (i) Bcl-2 and (ii) Bcl-xL mRNA expression upon treatment with IRAK-1 and-4 dual inhibitor alone and in combination with individual chemo-drugs analyzed by qRT-PCR. d Decrease in Nanog upon treatment of IRAK-1 and-4 dual inhibitor alone and in combination with individual chemo-drugs analyzed by flow cytometry. (i) representative flow cytometric histogram images demonstrating the effect of treatments on Nanog + cells; (ii) statistical analysis of the percentage of Nanog + cells. e (i) Suppression in MMP-2 mRNA expression and (ii) reduction in IL-6 production upon combination treatment analyzed by qRT-PCR and ELISA, respectively. f Decrease in vimentin upon treatment of IRAK-1 and-4 dual inhibitor alone and in combination with individual chemo-drugs analyzed by flow cytometry. (i) Representative flow cytometric histograms demonstrating the effect of treatments on vimentin expression; (ii) statistical analysis of the percentage of vimentin + cells. g Statistical analysis of MFI expression of vimentin upon combination treatment (ns not-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

Table 3.

IC50 of combination treatment of IRAK-1 and-4 dual inhibitor and chemo-drugs on chemo-resistant HEp-2

Chemo-drug IC50of chemo-drug alone (n = 3) IC50of chemo-drug + IRAK-1 and-4 dual inhibitor combination treatment (n = 3) Fold change (decrease in IC50 upon combination treatment with respect to single chemo-drug treatment)
Docetaxel 1450 ± 0.7 nM 7.65 ± 0.68 nM 190
Cisplatin 53.04 ± 4.88 μM 25.2 ± 5 μM 2
5-FU 2.8 ± 0.8 mM 0.45 ± 0.42 mM 6
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The therapeutic potential of IRAK-1 and-4 dual inhibitor both as a single-agent and in combination with individual chemo-drugs was assessed through various pro-oncogenic markers. The cells were treated with IC12.5 of chemo-drugs (Docetaxel: 0.32 ± 0.16 nM, Cisplatin: 5.47 ± 1.2 μM, 5-FU:0.73 ± 0.5 mM) with and without IC25 of IRAK-1 and-4 dual inhibitor as described in “Treatment of cells”. In TPF-resistant HEp-2, an increase in Ki-67 + cells was observed upon cisplatin and docetaxel treatment, while 5-FU suppressed the levels of Ki-67+ cells. Single-agent treatment with IRAK-1 and-4 dual inhibitor alone significantly suppressed the Ki-67+ cells. Further, combining IRAK-1 and-4 dual inhibitor with the chemo-drugs docetaxel and cisplatin significantly decreased the chemotherapy-induced expression of Ki-67+ cells (Fig. 6b).

IRAK-1 and-4 dual inhibitor alone could significantly suppress the Bcl-2 and Bcl-xL levels in chemo-resistant cells. Interestingly, upon treatment with chemo-drugs especially docetaxel, TPF-resistant HEp-2 cells strongly induced expression of Bcl-2 and Bcl-xL. Combining IRAK-1 and-4 dual inhibitor with the chemo-drugs showed significant suppression in the mRNA expression of Bcl-2 and Bcl-xL (Fig. 6c).

A decrease in Nanog + cells upon treatment with IRAK-1 and-4 dual inhibitor alone was observed in chemo-resistant HEp-2. Chemo-resistant HEp-2 treated with the three chemo-drugs separately demonstrated an increase in Nanog + cells. Further, combining IRAK-1 and-4 dual inhibitor with the chemo-drugs significantly decreased the levels of Nanog + cells in chemo-resistant HEp-2 (Fig. 6d).

IRAK-1 and-4 dual inhibitor treatment on chemo-resistant HEp-2 significantly reduced mRNA expression of MMP-2 and drastically reduced the IL-6 production from 3000 to 1000 pg/mL. An over-expression in mRNA of MMP-2 was observed upon rechallenging chemo-resistant HEp-2 with individual chemo-drugs. Combining IRAK-1 and-4 dual inhibitor with these chemo-drugs significantly suppressed MMP-2 mRNA transcript levels in chemo-resistant HEp-2. On the other hand, IL-6 production by chemo-resistant HEp-2 was reduced upon re-exposure to individual chemo-drugs. Docetaxel alone reduced the production of IL-6 by one-third. Combining IRAK-1 and-4 dual inhibitor with docetaxel and cisplatin further significantly reduced the production of IL-6 in chemo-resistant HEp-2 (Fig. 6e).

A significant increase in the proportion of vimentin expressing cells was observed in chemo-resistant HEp-2 upon treatment with the chemo-drugs. IRAK-1 and-4 dual inhibitor alone and in combination with the chemo-drugs, reduced the percentage of vimentin + cells (Fig. 6f). Although a significant decrease in MFI expression of vimentin was observed in chemo-resistant HEp-2 upon treatment with IRAK-1 and-4 dual inhibitor alone and in combination with cisplatin, overall expression levels of E-cadherin remained unchanged (Fig. 6g).

Discussion

TPF is a more commonly used combination treatment regimen for HNSCC, generally preferred for larynx preservation (Ferrari et al. 2020). There are many concerns associated with it, including the development of resistance and non-responsiveness. Along with that, TPF tolerability is another major concern. It can be considered as a choice of treatment only in good general health conditions, with no co-morbidities and early stages of cancer. Elderly patients have considerable adverse effects including excessive body weight loss and other standard ailments. These limitations restrict the wider use of TPF in spite of it being an effective therapy (Ilie et al. 2012; Fayette et al. 2016). Hence, there is a need to evolve newer therapies or adjuvant therapies to overcome the above-listed issues. The very first step, in this direction, is to understand the mechanism through which the cancer cells acquire resistance in order to exploit them for therapy purposes. For the issue of tolerance in elderly patients, which makeup a major proportion of the total patient population, reducing the dose of chemo-drug without compromising their efficacy can be a promising approach. This can be achieved by complementing the low-dose chemo-drug/s with adjuvant therapy.

A comprehensive investigation of the clinical significance of IRAK-1 and IRAK-4 on HNSCC progression and survival was performed using TCGA data. Pan cancer analysis of TCGA tumors data demonstrated high expression of IRAK-1 whereas a moderate expression of IRAK-4 in HNSCC. IRAK-1 was significantly overexpressed in HNSCC tumors (n = 520) compared to normal adjacent tissues. IRAK-1 and IRAK-4 expression were associated with reduced overall survival of patients, where expression of high IRAK-1 alone correlated with worse overall survival of HNSCC patients. The data suggest that IRAK-1 and IRAK-4 can act as predictive and prognostic biomarkers for HNSCC along with playing a potential role in the progression of the cancer. In 2015, Adams et al., reported overexpression of IRAK-1 in 15% of HNSCC biopsy samples (n = 279) available in the TCGA database.

In the present study, to mimic the TPF chemo-resistant state, chemo-resistant cell line was developed by exposing parent laryngeal cancer line HEp-2 to escalating doses of the standard chemo-drugs. Fold increase in the inhibitory concentration of each chemo-drug for the resistant line as compared to the parent verified the acquired resistance. The resistant line showed slightly enlarged nuclei along with high granularity suggesting that they are more secretory in nature than the parent line. Chemo-resistant cell line showed higher proliferative potential and expressed higher levels of anti-apoptotic markers, Bcl-2 and Bcl-xL. Chemo-resistant cells also had a significantly higher proportion of cancer stem cells as quantitated by CD44, Nanog and ALDH1 expression. Expansion of cancer stem cells with increased proliferation and survival attributes can be the major cause of loss of cytotoxic effects of the chemo-drugs.

IL-6 is one of the major pro-inflammatory cytokines shown to be associated with EMT and metastasis (Abaurrea et al. 2021). It has been used as a biomarker in various HNSCC-associated clinical studies. IL-6 is associated with treatment non-responsiveness in HNSCC. Patients with increased levels had a poor prognosis and low overall survival rates (Gao et al. 2016; Uz et al. 2021). Chemo-resistant cells produced a significantly higher amount of IL-6 than the parent line creating a highly pro-inflammatory tumor microenvironment with a greater potential to promote metastasis than the parent line.

MMPs with the potential to degrade the extracellular matrix and various cell adhesion-related molecules act as a primary effector to initiate the process of metastasis and invasion. MMP-2 is shown to be one of the most prominent MMPs involved in this process (Foda et al. 2001; Xu et al. 2005). MMP-2 expression in HNSCC has been clinically shown to be associated with metastasis (Stanciu et al. 2017). Our data showed higher MMP-2 production by the chemo-resistant line as compared to parent one suggesting chemo-resistance beyond a time can also well contribute to metastasis as well as lead to poor prognosis. To confirm this aspect experimentally, we studied the EMT state of the two lines by evaluating the E-Cadherin and Vimentin expression. The chemo-resistant line lost E-cadherin expression and gained vimentin expression suggesting that they have higher metastatic potential. EMT has been clinically well associated with poor PFS as well as OS in HNSCC (van der Heijden et al. 2020).

Our findings provide strong evidence that under chronic exposure of TPF, patients with non-responsiveness can have an aggressive form of cancer with higher stemness and metastatic potential and a worsen disease. Such a condition is eventually attained in most of the patients treated with TPF beyond a certain time post-therapy.

There are emerging evidences suggesting constitutive TLR activation is prevalent in the majority of cancers, including head and neck cancer, melanoma, colon cancer, lymphoma, breast cancer, gastric cancer, leukemia, and Hodgkin’s disease (Kaczanowska et al. 2013). Endogenous TLR ligands, i.e., DAMPs can be well generated upon exposure to chemo-drugs due to stress and cell death (Krysko et al. 2012). They can activate TLR signaling in cancer cells leading to inflammation (Pandolfi et al. 2016) and other associated pro-oncogenic attributes described above.

To confirm with TLR signaling state in cancer cells, expression and phosphorylation state of downstream kinases known as IRAK-1 and IRAK-4 were studied. The reason to choose IRAKs among various downstream signaling molecules involved in TLR signaling is mainly because they converge at most of the TLR types irrespective of their cellular localization and ligand specificities (El-Zayat et al. 2019). Changes in IRAKs expression level as well as phosphorylation state reflect activation of TLR signaling pathway. The number of cells expressing IRAK-1 and IRAK-4 was found significantly high in the chemo-resistant line as compared to parent. Also, the cells with the phosphorylated form of the IRAKs were in a higher proportion in chemo-resistant cells. Upon comparing the mean fluorescence intensity of the IRAKs and their phosphorylated forms, it was well observed that most of the IRAKs were existing in their phosphorylated state, suggesting that TLR signaling is constitutively ongoing in both parent and chemo-resistant line. The magnitude of TLR signaling was higher in the chemo-resistant line as compared to parent.

Identifying sensitive molecular biomarkers associated with TPF resistance is very important for the prognosis of treatment resistance as well as offering personalized and precise treatments to patients. IRAKs and their phosphorylated forms can serve that purpose by being biomarkers in TPF resistance upon clinical validation in a significant cohort of stratified patients.

Various studies have tested IRAK as a drug target for cancer including breast (Wee et al. 2015), melanoma (Srivastava et al. 2012), HNSCC (Adams et al. 2015) and thyroid cancer (Kawamura et al. 2021) as well and found promising results. There are a limited number of reports suggesting therapeutic usage of IRAK inhibitors in combating chemo-resistance. In line with the same, we explored the potential of IRAK inhibitors as a treatment option for TPF chemo-resistant laryngeal cancer in-vitro. IRAK-1 and -4 dual inhibitor showed moderate cytotoxic effects.

Another potential usage of IRAK-1 and-4-based kinase inhibitor is as adjuvant therapy. We tested their potential as re-sensitization agents for the TPF chemo-drugs in chemo-resistant state. Upon combining the IRAK inhibitor with individual chemo-drugs, the IC50 of the chemo-drugs reduced significantly. This can not only be therapeutically exploited as a single chemo-drug regimen-based therapy post-TPF-resistant state but can significantly bring down the required dose of chemo-drug drastically and thus avoid various toxicity and tolerability-related adverse clinical events in elderly patients.

IRAK inhibitor augmented combined chemotherapy treatment not only improved the cytotoxicity but also significantly reduced the associated aggressive oncogenic attributes of chemo-resistant state including their proliferating potential, survival, stemness, inflammatory state, EMT and metastatic potential as assessed by standard markers experimentally. Although, combining IRAK-1 and-4 dual inhibitor with all chemo-drugs demonstrated beneficial outcomes, combination therapy of IRAK-1 and-4 dual inhibitor with chemo-drug cisplatin showed the maximum effect followed by 5-FU.

This study establishes the role of TLR signaling as a key contributor to chemo-resistance development and associated aggressive pro-tumorigenic state. Constitutive TLR signaling can be a result of an abundance of various DAMPs being present in TME providing an advantage to cancer cells and allowing them to transform in a resistant form. TLR pathway-related downstream kinases known as IRAK-1 and IRAK-4 can be clinically explored as a biomarker of TPF resistance. Blocking TLR signaling through IRAKs holds promising treatment options as a combination therapy with a single chemo-drug. Combination therapy has the potential to overcome many adverse clinical outcomes of TPF therapy which is widely being practiced in most of advanced stage patients. Data need to be validated in more than one cell line as well as in-vivo pre-clinical and clinical settings to validate and exploit IRAK inhibition-based combination therapy for TPF-resistant HNSCC.

Conclusion

Despite toxicity being associated with it, chemotherapy is one of the most viable therapeutic options for almost all of the HNSCC patients. Developing resistance to conventional chemotherapy beyond a certain time and toxicity is quite common phenomenon observed in large proportion of patients. Reducing the dose of chemo-drugs without compromising its efficacy, can delay the development of chemo-resistance as well as associated pro-oncogenic effects to a considerable extent. It may also result in better overall survival of patients.

Using the in-vitro cell line-based model, our study provides evidence that the Toll-Like Receptor signaling is enhanced in the chemo-resistant HNSCC state. The intermediate signaling kinases, IRAK-1 and IRAK-4 expression as well their phosphorylated forms were abundant in the resistant HNSCC line than its parent lineage. Our findings were further substantiated by the TCGA data-based analysis. We propose that determination of IRAK-1/4 expression and phosphorylation state can be used as a biomarker to predict chemoresistance in HNSCC patients. Further, our study suggests that using IRAK-1 and-4 dual inhibitor as an immunomodulatory therapeutic drug along with chemo-drug can augment the effect of the chemo-drugs further. This approach can other way also allow the usage of reduced doses of chemo-drugs, resulting in lower toxicity. This study establishes the proof of concept of using the inhibitor for the treatment of TPF-resistant HNSCC. The data needs to be validated in other HNSCC cell lines, appropriate pre-clinical animal model/s as well as in clinical settings to establish IRAK as biomarker and therapeutic target for TPF-resistant HNSCC.

Acknowledgements

This research was funded by a grant from Gujarat State Biotechnology Mission (GSBTM) under the Financial Assistance Programme (Project ID-1393) to Ratika Srivastava. Humayara Khan was supported by research scholarships from The Maharaja Sayajirao University of Baroda, GSBTM and SHODH, Education Department, Govt. of Gujarat (Ref. ID-201901720067). Authors acknowledge The Department of Microbiology and Biotechnology Centre, The Maharaja Sayajirao University of Baroda, Gujarat, India and The Seeding labs, USA for infrastructure and instrumentation facilities.

Abbreviations

HNSCC

Head and neck squamous cell carcinoma

HPV

Human papillomavirus

5-FU

5-Fluorouracil

PD1

Programmed cell death 1

EGFR

Epidermal growth factor receptor

FDA

Food and drugs administration

PFS

Progression-free survival

TLR

Toll-like receptor

PAMPs

Pathogen-associated molecular patterns

DAMPs

Damage-associated molecular patterns

IRAK

Interleukin-1 receptor-associated kinase

TNBC

Triple-negative breast cancer

HCC

Hepatocellular carcinoma

PDAC

Pancreatic ductal adenocarcinoma

CRC

Colorectal carcinoma

NPC

Nasopharyngeal carcinoma

IC

Inhibitory concentration

Bcl-2

B cell lymphoma-2

Bcl-xL

B cell lymphoma extra large

MMP-2

Matrix metalloproteinase-2

ALDH1

Aldehyde dehydrogenase-1

IL-6

Interleukin-6

ANOVA

Analysis of variance

S.D.

Standard deviation

FSC

Forward scatter

SSC

Side scatter

CSCs

Cancer stem cells

MFI

Mean fluorescence intensity

EMT

Epithelial-to-mesenchymal transition

Author contributions

RS conceptualization, funding acquisition, methodology, visualization, project administration, data curation, resources, supervision, validation, writing-original draft, writing-review and editing. SNP and AM TCGA data mining analysis. HK investigation, validation, writing-original draft.

Data Availability

Data will be available on request to corresponding author.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Research involving human participants and animals

Not applicable.

Informed consent

Not applicable.

Contributor Information

Humayara Khan, Email: humayra1005@gmail.com.

Sachchida Nand Pandey, Email: snpandey@mpuh.org, Email: sachidanandpandey@gmail.com.

Abhishek Mishra, Email: abhimishra1103@gmail.com.

Ratika Srivastava, Email: srivastava.ratika@gmail.com, ratika.dbt@email.bbau.ac.in.

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Data Availability Statement

Data will be available on request to corresponding author.

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