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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

EGFR and ANO1 regulate each other's protein levels(A) Immunoblot of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible expression constructs for EGFR-wt, -D837A, lz-EGFR or lz-EGFR-D837A or an empty vector control, in the presence or absence of dox (48 h) and Gefitinib (1 μM, 24 h). Tubulin served as a loading control. Representative immunoblots are shown. (B) Relative mRNA levels of EGFR and ANO1 in the same samples as used in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Relative cell proliferation of Te11 cells stably expressing the indicated dox-inducible constructs analyzed by Cell Titer Glo. Signals were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments, p < 0.001*** as compared to respective no-dox condition. (D) Immunoblots of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox for 72 h. Representative immunoblots are shown. (E) Immunofluorescence of ANO1 (green) and EGFR (red) in Te11 cells treated as in A analyzed by confocal microscopy. Representative images are shown.
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Figure 3: EGFR and ANO1 regulate each other's protein levels(A) Immunoblot of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible expression constructs for EGFR-wt, -D837A, lz-EGFR or lz-EGFR-D837A or an empty vector control, in the presence or absence of dox (48 h) and Gefitinib (1 μM, 24 h). Tubulin served as a loading control. Representative immunoblots are shown. (B) Relative mRNA levels of EGFR and ANO1 in the same samples as used in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Relative cell proliferation of Te11 cells stably expressing the indicated dox-inducible constructs analyzed by Cell Titer Glo. Signals were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments, p < 0.001*** as compared to respective no-dox condition. (D) Immunoblots of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox for 72 h. Representative immunoblots are shown. (E) Immunofluorescence of ANO1 (green) and EGFR (red) in Te11 cells treated as in A analyzed by confocal microscopy. Representative images are shown.

Mentions: Next, we set out to investigate potential functional consequences of the interaction between EGFR and ANO1. EGF has been shown to increase expression of ANO1 in a human bronchial epithelial cell line, indicating a positive feedback mechanism between EGFR-signaling and ANO1-expression. To test whether EGFR-signaling regulates ANO1-expression in cancer cells, we generated Te11 cells stably expressing a dox-inducible version of EGFR or lz-EGFR (Te11-EGFR, Te11-lz-EGFR). While activation of wildtype EGFR requires EGF, lz-EGFR has been shown to be constitutively phosphorylated and signaling active because of its constitutive dimerization [46]. Dox-induced expression of lz-EGFR in Te11 cells resulted in a significant increase of ANO1 protein levels, while expression of EGFR or a kinase-inactive mutant of lz-EGFR had no effect, suggesting that EGFR-signaling regulates ANO1 protein levels in cancer cells by an EGFR-kinase-activity-dependent mechanism (Figure 3A). Consistently, treatment with Gefitinib prevented the lz-EGFR induced increase in ANO1 protein levels and reduced ANO1-protein levels in the vector-expressing cells. The lz-EGFR induced increase in ANO1 protein levels led to a significant increase in calcium-dependent chloride current in Te11 cells, indicating that ANO1 is functional and localized on the membrane (Supplementary Figure 2). Notably, unlike previously reported for a bronchial epithelial cell lines, the increase of ANO1-protein levels in Te11 cells was not caused by an increase in ANO1-mRNA levels (Figure 3B), suggesting a posttranslational effect on ANO1-protein levels. To test whether the EGFR-signaling-induced increase in ANO1 protein levels had a functional effect on the proliferation rate of Te11 cells, we measured the viability of Te11-EGFR/lz-EGFR/-wt/-D837A cells in the presence and absence of dox (Figure 3C). Induction of EGFR- and lz-EGFR-expression resulted in a profound increase in cell proliferation, whereas the expression of the kinase-dead mutants had no effect. These results are consistent with a functional link between ANO1 and EGFR and support the hypothesis that EGFR regulate proliferation of cancer cells, in part, by increasing expression of ANO1.


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)

EGFR and ANO1 regulate each other's protein levels(A) Immunoblot of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible expression constructs for EGFR-wt, -D837A, lz-EGFR or lz-EGFR-D837A or an empty vector control, in the presence or absence of dox (48 h) and Gefitinib (1 μM, 24 h). Tubulin served as a loading control. Representative immunoblots are shown. (B) Relative mRNA levels of EGFR and ANO1 in the same samples as used in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Relative cell proliferation of Te11 cells stably expressing the indicated dox-inducible constructs analyzed by Cell Titer Glo. Signals were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments, p < 0.001*** as compared to respective no-dox condition. (D) Immunoblots of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox for 72 h. Representative immunoblots are shown. (E) Immunofluorescence of ANO1 (green) and EGFR (red) in Te11 cells treated as in A analyzed by confocal microscopy. Representative images are shown.
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Related In: Results  -  Collection

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Figure 3: EGFR and ANO1 regulate each other's protein levels(A) Immunoblot of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible expression constructs for EGFR-wt, -D837A, lz-EGFR or lz-EGFR-D837A or an empty vector control, in the presence or absence of dox (48 h) and Gefitinib (1 μM, 24 h). Tubulin served as a loading control. Representative immunoblots are shown. (B) Relative mRNA levels of EGFR and ANO1 in the same samples as used in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Relative cell proliferation of Te11 cells stably expressing the indicated dox-inducible constructs analyzed by Cell Titer Glo. Signals were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments, p < 0.001*** as compared to respective no-dox condition. (D) Immunoblots of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox for 72 h. Representative immunoblots are shown. (E) Immunofluorescence of ANO1 (green) and EGFR (red) in Te11 cells treated as in A analyzed by confocal microscopy. Representative images are shown.
Mentions: Next, we set out to investigate potential functional consequences of the interaction between EGFR and ANO1. EGF has been shown to increase expression of ANO1 in a human bronchial epithelial cell line, indicating a positive feedback mechanism between EGFR-signaling and ANO1-expression. To test whether EGFR-signaling regulates ANO1-expression in cancer cells, we generated Te11 cells stably expressing a dox-inducible version of EGFR or lz-EGFR (Te11-EGFR, Te11-lz-EGFR). While activation of wildtype EGFR requires EGF, lz-EGFR has been shown to be constitutively phosphorylated and signaling active because of its constitutive dimerization [46]. Dox-induced expression of lz-EGFR in Te11 cells resulted in a significant increase of ANO1 protein levels, while expression of EGFR or a kinase-inactive mutant of lz-EGFR had no effect, suggesting that EGFR-signaling regulates ANO1 protein levels in cancer cells by an EGFR-kinase-activity-dependent mechanism (Figure 3A). Consistently, treatment with Gefitinib prevented the lz-EGFR induced increase in ANO1 protein levels and reduced ANO1-protein levels in the vector-expressing cells. The lz-EGFR induced increase in ANO1 protein levels led to a significant increase in calcium-dependent chloride current in Te11 cells, indicating that ANO1 is functional and localized on the membrane (Supplementary Figure 2). Notably, unlike previously reported for a bronchial epithelial cell lines, the increase of ANO1-protein levels in Te11 cells was not caused by an increase in ANO1-mRNA levels (Figure 3B), suggesting a posttranslational effect on ANO1-protein levels. To test whether the EGFR-signaling-induced increase in ANO1 protein levels had a functional effect on the proliferation rate of Te11 cells, we measured the viability of Te11-EGFR/lz-EGFR/-wt/-D837A cells in the presence and absence of dox (Figure 3C). Induction of EGFR- and lz-EGFR-expression resulted in a profound increase in cell proliferation, whereas the expression of the kinase-dead mutants had no effect. These results are consistent with a functional link between ANO1 and EGFR and support the hypothesis that EGFR regulate proliferation of cancer cells, in part, by increasing expression of ANO1.

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