β2-AR blockade potentiates MEK1/2 inhibitor effect on HNSCC by regulating the Nrf2-mediated defense mechanism

Abstract

The β2-Adrenergic receptor (β2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, β2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the β2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the β2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of β2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target.

Fig. 1. β2-AR and MEK1/2 inhibitors increase cell death by apoptosis, in a synergistic way.

a Viability assay on UMSCC 103 treated with ICI at 24 h and 48 h. The β2-AR inhibitor IC50 was ~40 μM at 24 h and ~25 μM at 48 h. b The values are represented in the combination index plot in function of ICI/U0126 tested concentrations. The drugs show a significant synergism in two combinations (10 μM ICI + 10 μM U0126 and 25 μM ICI + 10 μM U0126). c Annexin V/PI flow cytometry analysis to determine apoptosis in ICI and U0126-treated cells. Drug combination promotes a strong apoptosis effect, on UMSCC 103, compared to the single treatments. Annexin V/PI assays are also shown in the histograms. (**P ≤ 0.01).

Fig. 2. β2-AR and MEK1/2 inhibitors impair MAPK and PI3K pathways.

a Immunoblot for ERK/ERK^T202/Y2014, Nf-kB/Nf-kB^S536, and p38/p38^Y182 (below band densitometries); β2-AR blocking increases U0126 inhibition of ERK phosphorylation at a short time, the same trend is observed in Nf-kB. P38 phosphorylation is strongly reduced only over ICI treatment. b Immunoblot for mTOR/mTOR^S2448 and Akt/AKT^S473 (below band densitometries); the Akt ratio between phosphorylated and non-phosphorylated forms decreases over ICI treatment, while U0126 has no effect; on the other hand, it increases ICI inhibition of mTOR phosphorylation (*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001).

Fig. 3. β2-AR inhibition induces oxidative stress blocking NRF2 nuclear translocation.

a–b IF and Flow cytometry with CellROX (for oxidative stress determination), 24 h posttreatment (menadione is used as a positive control; N-acetylcysteine (NAC) is used as ROS scavenger). ICI treatment strongly increases ROS production, instead of U0126 which has a milder effect. The Flow Cytometry experiments have been performed in triplicates and the statistical analysis confirmed the significance of the results (**P ≤ 0.01).

Fig. 4. NRF2 perturbation.

a IF of NRF2 localization at 90 min. ICI treatment promotes the cytoplasmatic block of NRF2 b Immunoblot for cytoplasmatic and nuclear NRF2. ICI treatment promotes the cytoplasmatic block of NRF2; otherwise, U0126 has a low opposite effect after 30 min. c SOD activity assay. ICI reduces SOD activity in a dose-dependent manner (*P ≤ 0.05). MEK 1/2 inhibitor mildly increases it, ICI reverts its effect (**P ≤ 0.01, ***P ≤ 0.001). d qRT PCR of NRF2 downstream. ICI treatment reduced the expression of all NRF2 downstream genes (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Fig. 5. β2-AR inhibition induces autophagy.

a IF and flow cytometry for LysoTracker. ICI treatment increases the number and size of lysosomes on UMSCC 103, after 24 h. Flow cytometry histograms for the LysoTracker are shown in the graphs. b Immunoblot for LC3B and p62; band densitometry in ICI and U0126 treated cells, 24 h after treatment. LC3BII increases in ICI alone and ICI/U0126 samples while p62 decreases. c Viability assay on UMSCC103 treated with ICI and U0126 plus 3-MA. 3-MA strongly increases the cytotoxicity of the drug combination (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). d IF for LC3B, ICI treatment increased the number of autophagosomes.

Fig. 6. β2-AR expression is correlated with MAPK, PI3K, and ROS metabolism molecular effectors.

a In silico TCGA data set analysis on HNSC patients showed a strong correlation between NFE2L2 expression and survival rate. Lower not significant mTOR levels are also reported. b Scatter plot presenting the normalized genes expression within β2-AR + expression different disease stages. The same genes were evaluated for disease recurrence and here only MTOR (panel c) has shown a significantly lower expression in patients with disease recurrence. P-values below 0.05 were considered to be significant Analyses were performed using GraphPad Prism 8.

Fig. 7. Inhibition of β₂-AR block Nrf-2, p38, and NF-kB activity and potentiate MEK inhibitor effect on cancer.

Graphical representation of pathways cross-talk involving β2-AR inhibition.