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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 form a functional complex which regulates cancer cell proliferation(A) Immunoblots of EGFR, phospho-EGFR (Y1068) and ANO1 protein levels in Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control after treatment with dox for 72 h. Representative immunoblots are shown. (B) Relative mRNA-levels of ANO1 and EGFR in Te11 cells treated as 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) Colony formation assay of Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control. Representative images are shown. (D) Quantification of the relative colony area of Te11 cells treated as in C. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. ( p < 0.05*; p < 0.01**; p < 0.001***) (E) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox and/or Gefitinib. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments. Statistical analyses were performed using the Student's t-test or ANOVA with Tukey's post test as appropriate (*p < 0.05; **p < 0.01; ***p < 0.001); ns. not significant). (F) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against EGFR or a non-targeting control (NT) after treatment with dox and/or CaCCinh-A01. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of six independent experiments. Statistical analysis was performed as in Figure 4E.
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Figure 4: EGFR and ANO1 form a functional complex which regulates cancer cell proliferation(A) Immunoblots of EGFR, phospho-EGFR (Y1068) and ANO1 protein levels in Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control after treatment with dox for 72 h. Representative immunoblots are shown. (B) Relative mRNA-levels of ANO1 and EGFR in Te11 cells treated as 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) Colony formation assay of Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control. Representative images are shown. (D) Quantification of the relative colony area of Te11 cells treated as in C. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. ( p < 0.05*; p < 0.01**; p < 0.001***) (E) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox and/or Gefitinib. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments. Statistical analyses were performed using the Student's t-test or ANOVA with Tukey's post test as appropriate (*p < 0.05; **p < 0.01; ***p < 0.001); ns. not significant). (F) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against EGFR or a non-targeting control (NT) after treatment with dox and/or CaCCinh-A01. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of six independent experiments. Statistical analysis was performed as in Figure 4E.

Mentions: Knockdown of ANO1 inhibited cell proliferation and reduced EGFR protein levels, whereas EGFR-signaling induced ANO1 protein levels in Te11 cells. We speculated that the loss of EGFR after knockdown of ANO1 might be responsible for the inhibitory effect on cell proliferation and that overexpression of EGFR under these conditions might rescue cell viability by recovering both, EGFR-signaling and ANO1 protein levels. To test this hypothesis we infected Te11-ANO1-shRNA-#1/#2 cells with constructs coding for dox-inducible versions of EGFR or lz-EGFR or an empty vector control. Dox-treatment in the resulting cell pool is expected to induce the expression of both, the shRNAs against ANO1 and the expression constructs for EGFR/lz-EGFR or empty vector, respectively. The technical feasibility of this system was tested by immunoblotting after treatment of the cells with dox or a solvent control (Figure 4A). Addition of dox resulted in a decrease of ANO1 protein levels in empty-vector-expressing cells for both shRNAs. Furthermore, dox induced a profound expression of EGFR-wt/lz in cells infected with the constructs coding for EGFR or lz-EGFR. The induction of EGFR-/lz-EGFR-expression was accompanied by a partial rescue of ANO1-protein levels in the cells (Figure 4A). The increase in ANO1-protein levels was not caused by an increase in ANO1-mRNA-levels as measured by quantitative PCR (Figure 4B), consistent with the results obtained by overexpression of EGFR in the absence of ANO1-shRNA. To test whether the EGFR-expression induced elevation of ANO1-protein levels was sufficient to rescue ANO1-knockdown-mediated inhibition of cell viability we measured cell viability using a colony formation assay (Figure 4C/D). Knockdown of ANO1 with both shRNAs significantly reduced the number of cells in vector-expressing cells. Dox-induced expression of both EGFR and lz-EGFR was sufficient to partially rescue ANO1-knockdown induced cell killing in the presence of both shRNAs (Figure 4C/D), consistent with the partial rescue of ANO1-protein levels observed (Figure 4A). Taken together, these results demonstrate that loss of ANO1 inhibits cell proliferation by reducing EGFR-expression and that it can be partially rescued by restoring EGFR-expression in the cells which subsequently leads to a recovery of ANO1-protein and cell viability. The bidirectional interplay of EGFR and ANO1 highlights the importance of the functional complex formed between both proteins in regulating proliferation of cancer cells.


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 form a functional complex which regulates cancer cell proliferation(A) Immunoblots of EGFR, phospho-EGFR (Y1068) and ANO1 protein levels in Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control after treatment with dox for 72 h. Representative immunoblots are shown. (B) Relative mRNA-levels of ANO1 and EGFR in Te11 cells treated as 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) Colony formation assay of Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control. Representative images are shown. (D) Quantification of the relative colony area of Te11 cells treated as in C. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. ( p < 0.05*; p < 0.01**; p < 0.001***) (E) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox and/or Gefitinib. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments. Statistical analyses were performed using the Student's t-test or ANOVA with Tukey's post test as appropriate (*p < 0.05; **p < 0.01; ***p < 0.001); ns. not significant). (F) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against EGFR or a non-targeting control (NT) after treatment with dox and/or CaCCinh-A01. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of six independent experiments. Statistical analysis was performed as in Figure 4E.
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Related In: Results  -  Collection

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Figure 4: EGFR and ANO1 form a functional complex which regulates cancer cell proliferation(A) Immunoblots of EGFR, phospho-EGFR (Y1068) and ANO1 protein levels in Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control after treatment with dox for 72 h. Representative immunoblots are shown. (B) Relative mRNA-levels of ANO1 and EGFR in Te11 cells treated as 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) Colony formation assay of Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control. Representative images are shown. (D) Quantification of the relative colony area of Te11 cells treated as in C. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. ( p < 0.05*; p < 0.01**; p < 0.001***) (E) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against ANO1 or a non-targeting control (NT) after treatment with dox and/or Gefitinib. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments. Statistical analyses were performed using the Student's t-test or ANOVA with Tukey's post test as appropriate (*p < 0.05; **p < 0.01; ***p < 0.001); ns. not significant). (F) Relative colony area of Te11 cells stably expressing dox-inducible shRNAs against EGFR or a non-targeting control (NT) after treatment with dox and/or CaCCinh-A01. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of six independent experiments. Statistical analysis was performed as in Figure 4E.
Mentions: Knockdown of ANO1 inhibited cell proliferation and reduced EGFR protein levels, whereas EGFR-signaling induced ANO1 protein levels in Te11 cells. We speculated that the loss of EGFR after knockdown of ANO1 might be responsible for the inhibitory effect on cell proliferation and that overexpression of EGFR under these conditions might rescue cell viability by recovering both, EGFR-signaling and ANO1 protein levels. To test this hypothesis we infected Te11-ANO1-shRNA-#1/#2 cells with constructs coding for dox-inducible versions of EGFR or lz-EGFR or an empty vector control. Dox-treatment in the resulting cell pool is expected to induce the expression of both, the shRNAs against ANO1 and the expression constructs for EGFR/lz-EGFR or empty vector, respectively. The technical feasibility of this system was tested by immunoblotting after treatment of the cells with dox or a solvent control (Figure 4A). Addition of dox resulted in a decrease of ANO1 protein levels in empty-vector-expressing cells for both shRNAs. Furthermore, dox induced a profound expression of EGFR-wt/lz in cells infected with the constructs coding for EGFR or lz-EGFR. The induction of EGFR-/lz-EGFR-expression was accompanied by a partial rescue of ANO1-protein levels in the cells (Figure 4A). The increase in ANO1-protein levels was not caused by an increase in ANO1-mRNA-levels as measured by quantitative PCR (Figure 4B), consistent with the results obtained by overexpression of EGFR in the absence of ANO1-shRNA. To test whether the EGFR-expression induced elevation of ANO1-protein levels was sufficient to rescue ANO1-knockdown-mediated inhibition of cell viability we measured cell viability using a colony formation assay (Figure 4C/D). Knockdown of ANO1 with both shRNAs significantly reduced the number of cells in vector-expressing cells. Dox-induced expression of both EGFR and lz-EGFR was sufficient to partially rescue ANO1-knockdown induced cell killing in the presence of both shRNAs (Figure 4C/D), consistent with the partial rescue of ANO1-protein levels observed (Figure 4A). Taken together, these results demonstrate that loss of ANO1 inhibits cell proliferation by reducing EGFR-expression and that it can be partially rescued by restoring EGFR-expression in the cells which subsequently leads to a recovery of ANO1-protein and cell viability. The bidirectional interplay of EGFR and ANO1 highlights the importance of the functional complex formed between both proteins in regulating proliferation of cancer cells.

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