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. 2022 Apr 23;22(1):447.
doi: 10.1186/s12885-022-09511-6.

AXL regulates neuregulin1 expression leading to cetuximab resistance in head and neck cancer

Affiliations

AXL regulates neuregulin1 expression leading to cetuximab resistance in head and neck cancer

Mari Iida et al. BMC Cancer. .

Abstract

Background: The receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) is overexpressed and an important therapeutic target in Head and Neck cancer (HNC). Cetuximab is currently the only EGFR-targeting agent approved by the FDA for treatment of HNC; however, intrinsic and acquired resistance to cetuximab is a major problem in the clinic. Our lab previously reported that AXL leads to cetuximab resistance via activation of HER3. In this study, we investigate the connection between AXL, HER3, and neuregulin1 (NRG1) gene expression with a focus on understanding how their interdependent signaling promotes resistance to cetuximab in HNC.

Methods: Plasmid or siRNA transfections and cell-based assays were conducted to test cetuximab sensitivity. Quantitative PCR and immunoblot analysis were used to analyze gene and protein expression levels. Seven HNC patient-derived xenografts (PDXs) were evaluated for protein expression levels.

Results: We found that HER3 expression was necessary but not sufficient for cetuximab resistance without AXL expression. Our results demonstrated that addition of the HER3 ligand NRG1 to cetuximab-sensitive HNC cells leads to cetuximab resistance. Further, AXL-overexpressing cells regulate NRG1 at the level of transcription, thereby promoting cetuximab resistance. Immunoblot analysis revealed that NRG1 expression was relatively high in cetuximab-resistant HNC PDXs compared to cetuximab-sensitive HNC PDXs. Finally, genetic inhibition of NRG1 resensitized AXL-overexpressing cells to cetuximab.

Conclusions: The results of this study indicate that AXL may signal through HER3 via NRG1 to promote cetuximab resistance and that targeting of NRG1 could have significant clinical implications for HNC therapeutic approaches.

Keywords: AXL; Cetuximab; HER3; Head and neck cancer; Resistance; neuregulin1.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Fig. 1
Fig. 1
AXL leads to cetuximab resistance and increased HER3 activity. A: HN30 cells were treated with 100 nM of IgG, 100 nM of cetuximab (Ctx), or combination of Ctx and Gas6 (200 ng/uL) for 72 h, and cell viability was determined by crystal violet assay. Mean values, SEs, and statistical analyses are representative of two independent experiments. N = 3, **P < 0.01. Whole cell lysates were harvested and fractionated via SDS-PAGE, followed by immunoblotting for the indicated proteins. GAPDH was used as a loading control. B: Cell viability in AXL overexpressed cells was measured via crystal violet assay after 72 h of treatment with cetuximab. Mean values, SEs, and statistical analyses are representative of three independent experiments. N = 3, **P < 0.01. Whole cell lysates were harvested and fractionated via SDS-PAGE, followed by immunoblotting for the indicated proteins. α-Tubulin and GAPDH were used as loading controls
Fig. 2
Fig. 2
HER3 is necessary for AXL to mediate cetuximab resistance. A HN30-AXL and PCI37A-AXL cells were plated and treated with 30 nM of HER3 siRNA (siHER3) or 30 nM non-target siRNA (siNT). The next day, cells were treated with 100 nM of IgG or 100 nM cetuximab (Ctx) for 72 h. Cell proliferation was measured after drug treatment by CCK8 assay. Mean values, SEs, and statistical analyses are representative of three independent experiments. N = 5–10, **P < 0.01. Whole cell lysates were collected 24 h after treatment, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. GAPDH was used as a loading control. B UMSCC1 and UMSCC6 HNSCC cells were plated and treated with 30 nM of siAXL or 30 nM siNT. The next day, cells were treated with 100 nM of IgG or 100 nM Ctx for 72 h. Cell viability was measured after drug treatment by crystal violet assay. Mean values, SEs, and statistical analyses are representative of three independent experiments. N = 3, **P < 0.01. Whole cell lysates were collected at 24 h after treatment, fractionated by SDS-PAGE, and immunoblotted for the indicated proteins. GAPDH was used as a loading control
Fig. 3
Fig. 3
HER3 overexpression alone is insufficient for cetuximab resistance. A: HN30 and PCI37A cells stably overexpressing HER3 or the pcDNA6.0 vector were treated with 100 nM of cetuximab (Ctx) for 72 h before performing crystal violet assay for HN30 cells and CCK8 assays for PCI37A cells. Whole cell lysate was harvested at 24 h after treatment and subjected to immunoblot analysis following fractionation via SDS-PAGE. GAPDH or α-Tubulin was used as a loading control. Mean values, SEs, and statistical analyses are representative of two or three independent experiments. N = 3–6. NS: not significant
Fig. 4
Fig. 4
Exogenous expression of NRG1 leads to cetuximab resistance. A HN30 and PCI37A cells were plated and treated with 100 nM of cetuximab (Ctx), 100 ng/mL of NRG1, or the combination of Ctx and NRG1 for 72 h. Cell proliferation was determined by CCK8 assay. Mean values, SEs, and statistical analyses are representative of two independent experiments. N = 6, **P < 0.01. Whole cell lysates were harvested at 24 h after treatment and fractionated via SDS-PAGE, followed by immunoblotting for the indicated proteins. GAPDH was used as a loading control. B HN30 and PCI37A cells were transfected with 30 nM siHER3 or 30 nM siNT for 24 h before treatment with Ctx (100 nM) or NRG1 (100 ng/ml) for an additional 72 h. Cell viability or cell proliferation was determined by crystal violet assay for HN30 cells and CCK8 assay for PCI37A cells. Mean values, SEs, and statistical analyses are representative of two or three independent experiments. N = 3–10, **P < 0.01. Whole cell lysate was harvested at 24 h after treatment and subjected to immunoblot analysis following fractionation via SDS-PAGE. GAPDH was used as a loading control
Fig. 5
Fig. 5
AXL regulates NRG1. A The expression levels of NRG1 in HN30-Vector, HN30-AXL PCI37A-Vector and PCI37A-AXL cells were determined by qPCR and immunoblot analysis. α-Tubulin was used as a loading control. Mean values, SEs, and statistical analyses are representative of three independent experiments. N = 2–4. B Whole cell lysates were harvested from HNSCC PDX tumors and fractionated via SDS-PAGE, followed by an immunoblot for indicated proteins. GAPDH was used as a loading control. The densitometry values represent the mean ratios of NRG1/GAPDH from two independent experiments. Error bars: SEM (*P < 0.05, Mann–Whitney test)
Fig. 6
Fig. 6
AXL regulates NRG1 to lead to cetuximab resistance. A PCI37A-AXL cells were plated and treated with 30 nM of NRG1 siRNA or 30 nM siNT. The next day, cells were treated with 100 nM of 100 nM of IgG or 100 nM cetuximab (Ctx) for 72 h. Cell proliferation was measured after drug treatment using the CCK8 assay. Mean values, SEs, and statistical analyses are representative of seven independent experiments. N = 6–10, **P < 0.01. Whole cell lysates were collected at 24 h after treatment, fractioned by SDS-PAGE, and immunoblotted for the indicated proteins. GAPDH was used as a loading control. B Proposed model for AXL regulation of NRG1 leading to Ctx resistance

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