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ANO1 interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer.

Bill A, Gutierrez A, Kulkarni S, Kemp C, Bonenfant D, Voshol H, Duvvuri U, Gaither LA - Oncotarget (2015)

Bottom Line: However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy.By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC.Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

ABSTRACT
The epidermal growth factor receptor (EGFR) contributes to the pathogenesis of head&neck squamous cell carcinoma (HNSCC). However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy. By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC. Using structural mutants of EGFR and ANO1 we identified the trans/juxtamembrane domain of EGFR to be critical for the interaction with ANO1. Our results show that ANO1 and EGFR form a functional complex that jointly regulates HNSCC cell proliferation. Expression of ANO1 affected EGFR stability, while EGFR-signaling elevated ANO1 protein levels, establishing a functional and regulatory link between ANO1 and EGFR. Co-inhibition of EGFR and ANO1 had an additive effect on HNSCC cell proliferation, suggesting that co-targeting of ANO1 and EGFR could enhance the clinical potential of EGFR-targeted therapy in HNSCC and might circumvent the development of resistance to single agent therapy. HNSCC cell lines with amplification and high expression of ANO1 showed enhanced sensitivity to Gefitinib, suggesting ANO1 overexpression as a predictive marker for the response to EGFR-targeting agents in HNSCC therapy. Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

No MeSH data available.


Related in: MedlinePlus

Interaction between ANO1 and EGFR involves the trans/juxtamembrane domain of EGFR(A) Schematic of the EGFR-constructs tested for interaction with ANO1. (B) Immunoprecipitation of ANO1 and FLAG-tagged truncation variants of lz-EGFR in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding ANO1 and lz-EGFR-variants. EGFR/ANO1 complexes were analyzed by immunoprecipitation using an anti-ANO1 or anti-FLAG antibody coupled to magnetic beads and immunoblotting of the eluted proteins. Representative immunoblots are shown. (C) Immunoprecipitation of lz-EGFR and ANO1 truncation variants in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding lz-EGFR and ANO1-variants and ANO1/lz-EGFR complexes were analyzed as in Figure 2B. The multiple bands for ANO1 represent different glycosylation variants of ANO1 [39].
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Figure 2: Interaction between ANO1 and EGFR involves the trans/juxtamembrane domain of EGFR(A) Schematic of the EGFR-constructs tested for interaction with ANO1. (B) Immunoprecipitation of ANO1 and FLAG-tagged truncation variants of lz-EGFR in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding ANO1 and lz-EGFR-variants. EGFR/ANO1 complexes were analyzed by immunoprecipitation using an anti-ANO1 or anti-FLAG antibody coupled to magnetic beads and immunoblotting of the eluted proteins. Representative immunoblots are shown. (C) Immunoprecipitation of lz-EGFR and ANO1 truncation variants in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding lz-EGFR and ANO1-variants and ANO1/lz-EGFR complexes were analyzed as in Figure 2B. The multiple bands for ANO1 represent different glycosylation variants of ANO1 [39].

Mentions: The finding that lz-EGFR interacted with ANO1 to similar levels as wild-type EGFR (Figure 1I) demonstrates that the extracellular domain of EGFR is not required to form a complex with ANO1. To further investigate the structural requirements for the interaction between ANO1 and EGFR, we constructed deletion mutations of lz-EGFR lacking the C-terminal part of the protein or the C-terminal domain plus the kinase domain of EGFR (Figure 2A). Deletion of the complete intracellular domain of EGFR resulted in low expression levels and mislocalization of the protein to intracellular domains and was not tested for the interaction with ANO1 (data not shown). Coimmunoprecipitation using an ANO1- or FLAG-specific antibody revealed ANO1 to bind to all tested EGFR-constructs with similar affinity (Figure 2B). These results suggest that ANO1 interacts with the transmembrane domain and/or juxtamembrane domain of EGFR. To investigate the structural requirements of ANO1 for the interaction with EGFR we constructed deletion mutants of ANO1 lacking the 40–70 most C-terminal amino acids of ANO1. Expression on the plasma membrane was confirmed for all tested mutants (data not shown). Coimmunoprecipitation of ANO1 and EGFR showed that EGFR interacted with all mutants of ANO1 as determined by coimmunoprecipitation using an ANO1- or EGFR-specific antibody, suggesting that the C-terminus of ANO1 is not required for the interaction with EGFR (Figure 2C).


ANO1 interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer.

Bill A, Gutierrez A, Kulkarni S, Kemp C, Bonenfant D, Voshol H, Duvvuri U, Gaither LA - Oncotarget (2015)

Interaction between ANO1 and EGFR involves the trans/juxtamembrane domain of EGFR(A) Schematic of the EGFR-constructs tested for interaction with ANO1. (B) Immunoprecipitation of ANO1 and FLAG-tagged truncation variants of lz-EGFR in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding ANO1 and lz-EGFR-variants. EGFR/ANO1 complexes were analyzed by immunoprecipitation using an anti-ANO1 or anti-FLAG antibody coupled to magnetic beads and immunoblotting of the eluted proteins. Representative immunoblots are shown. (C) Immunoprecipitation of lz-EGFR and ANO1 truncation variants in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding lz-EGFR and ANO1-variants and ANO1/lz-EGFR complexes were analyzed as in Figure 2B. The multiple bands for ANO1 represent different glycosylation variants of ANO1 [39].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496210&req=5

Figure 2: Interaction between ANO1 and EGFR involves the trans/juxtamembrane domain of EGFR(A) Schematic of the EGFR-constructs tested for interaction with ANO1. (B) Immunoprecipitation of ANO1 and FLAG-tagged truncation variants of lz-EGFR in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding ANO1 and lz-EGFR-variants. EGFR/ANO1 complexes were analyzed by immunoprecipitation using an anti-ANO1 or anti-FLAG antibody coupled to magnetic beads and immunoblotting of the eluted proteins. Representative immunoblots are shown. (C) Immunoprecipitation of lz-EGFR and ANO1 truncation variants in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding lz-EGFR and ANO1-variants and ANO1/lz-EGFR complexes were analyzed as in Figure 2B. The multiple bands for ANO1 represent different glycosylation variants of ANO1 [39].
Mentions: The finding that lz-EGFR interacted with ANO1 to similar levels as wild-type EGFR (Figure 1I) demonstrates that the extracellular domain of EGFR is not required to form a complex with ANO1. To further investigate the structural requirements for the interaction between ANO1 and EGFR, we constructed deletion mutations of lz-EGFR lacking the C-terminal part of the protein or the C-terminal domain plus the kinase domain of EGFR (Figure 2A). Deletion of the complete intracellular domain of EGFR resulted in low expression levels and mislocalization of the protein to intracellular domains and was not tested for the interaction with ANO1 (data not shown). Coimmunoprecipitation using an ANO1- or FLAG-specific antibody revealed ANO1 to bind to all tested EGFR-constructs with similar affinity (Figure 2B). These results suggest that ANO1 interacts with the transmembrane domain and/or juxtamembrane domain of EGFR. To investigate the structural requirements of ANO1 for the interaction with EGFR we constructed deletion mutants of ANO1 lacking the 40–70 most C-terminal amino acids of ANO1. Expression on the plasma membrane was confirmed for all tested mutants (data not shown). Coimmunoprecipitation of ANO1 and EGFR showed that EGFR interacted with all mutants of ANO1 as determined by coimmunoprecipitation using an ANO1- or EGFR-specific antibody, suggesting that the C-terminus of ANO1 is not required for the interaction with EGFR (Figure 2C).

Bottom Line: However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy.By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC.Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

ABSTRACT
The epidermal growth factor receptor (EGFR) contributes to the pathogenesis of head&neck squamous cell carcinoma (HNSCC). However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy. By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC. Using structural mutants of EGFR and ANO1 we identified the trans/juxtamembrane domain of EGFR to be critical for the interaction with ANO1. Our results show that ANO1 and EGFR form a functional complex that jointly regulates HNSCC cell proliferation. Expression of ANO1 affected EGFR stability, while EGFR-signaling elevated ANO1 protein levels, establishing a functional and regulatory link between ANO1 and EGFR. Co-inhibition of EGFR and ANO1 had an additive effect on HNSCC cell proliferation, suggesting that co-targeting of ANO1 and EGFR could enhance the clinical potential of EGFR-targeted therapy in HNSCC and might circumvent the development of resistance to single agent therapy. HNSCC cell lines with amplification and high expression of ANO1 showed enhanced sensitivity to Gefitinib, suggesting ANO1 overexpression as a predictive marker for the response to EGFR-targeting agents in HNSCC therapy. Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

No MeSH data available.


Related in: MedlinePlus