Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun;56(6):460-471.
doi: 10.1002/gcc.22450. Epub 2017 Apr 3.

TMEM16A/ANO1 Suppression Improves Response to Antibody-Mediated Targeted Therapy of EGFR and HER2/ERBB2

Affiliations
Free PMC article

TMEM16A/ANO1 Suppression Improves Response to Antibody-Mediated Targeted Therapy of EGFR and HER2/ERBB2

Sucheta Kulkarni et al. Genes Chromosomes Cancer. .
Free PMC article

Abstract

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

Figures

Figure 1
Figure 1. TMEM16A influences expression and activation of EGFR/HER family members and a pathway of survival in ErbB2/HER2 amplified breast cancer cells
a: SKBR3 cells were plated on 60 mm plates and allowed to attach for 2 h. Cells were left untreated as a control or treated with T16A-inh (40 μM) for 1.5 h at 37° C. Cells were lysed to obtain solubilized protein and equal amounts of protein were analyzed for pY1068 EGFR, EGFR, β-tubulin, pY1248 HER2, HER2, pY705 STAT3, STAT3, TMEM16A and Plexin B1 in western blots. b: SKBR3 cells (3 × 103/well) were plated in 96-well plates in quadruplicate and left untreated as controls or treated with human IgG or trastuzumab (20 μg/ml each) or T16A-inh (5 μM). All treatments were carried out for 3 days at 37° C followed by measurements of viability with the CellTiter-Glo reagent. Readings from the treated group were normalized to the readings from control. Representative data from triplicate results (n = 3) are presented as mean +/− SEM. The significance (***p < 0.001) was determined from one-way ANOVA analysis. c: SKBR3 cells (3 × 103/well) were plated in 96-well plates in quadruplicate and left untreated as controls or treated with trastuzumab (20 μg/ml) or trastuzumab plus varying concentrations of Cacc-inh (0.5–30 μM). All treatments were carried out for 3 days at 370 C followed by measurements of viability with the CellTiter-Glo reagent. Readings from the treated group were normalized to the readings from controls. Representative data from triplicate results (n = 3) are presented as mean +/− SEM. The significance (***p < 0.001) was determined from one-way ANOVA analysis.
Figure 2
Figure 2. Trastuzumab-resistant breast cancer cells are increasingly sensitive to TMEM16A inhibition
a: SKBR3 cells, parent or trastuzumab-resistant, (3 × 103/well) were plated in quadruplicate in 96-well plates and left untreated as controls or treated with T16A-inh (5 or 20 μM) for 3 days at 37° C. Viability was measured with the CellTiter-Glo reagent and readings from the treated group were normalized to readings from the controls. Representative data from triplicate results (n = 3) are presented as mean +/− SEM. The significance (***p < 0.001) was determined from one-way ANOVA analysis. b: SKBR3 cells, parent or trastuzumab-resistant, (1 × 103/well) were plated in 6-well plates. Cells were allowed to attach overnight after which cells were left untreated as controls or treated with trastuzumab alone (10 μg/ml) or trastuzumab (10 μg/ml) combined with T16A-inh (1 or 5 μM). Treatments were repeated every 2–3 days for a period of 2–3 weeks. Plates were fixed and stained with crystal violet. A representative plate from triplicate results (n = 3) is shown. c: The number of colonies for each condition from independent experiments (n = 3) was counted. The numbers obtained for the treated groups were normalized to the control group. Data are presented as mean +/− SEM of triplicate results (n = 3).
Figure 3
Figure 3. TMEM16A-induced HNSCC tumor growth shows EGFR activation
a, b: Tumor xenograft pairs of OSC-19 cells (control and overexpressing TMEM16A) was processed to obtain solubilized protein. Equal amounts of protein were analyzed for pY1068 EGFR, EGFR, TMEM16A and β-tubulin in western blots as shown. In b, mean tumor weight +/− SEM; significance was determined from a Student t-test (n = 4, *p < 0.05). c: FaDu cells engineered to contain either doxycycline-inducible non-targeting (NT) control shRNA or shRNA against TMEM16A were treated with doxycycline and EGF, as indicated. Cells were harvested and processed to obtain solubilized protein. Equal amounts of protein were analyzed for pY1068 EGFR, EGFR, TMEM16A, and β-tubulin as shown. Densitometric quantification of the pY1068 EGFR signal is shown; significance was determined by a Student t-test (*p < 0.05).
Figure 4
Figure 4. TMEM16A inhibition improves response to biologic therapy of EGFR in HNSCC
a: OSC-19 cells (3 × 103/well) were plated in 96-well plates in quadruplicate and left untreated as controls or treated with human IgG or cetuximab (20 μg/ml each) or T16A-inh (20 μM). Cells were treated also with cetuximab (20 μg/ml) and T16A-inh (20 μM) together. All treatments were carried out for 4 days at 370 C. Viability was measured with the CellTiter-Glo reagent. Readings from the treated groups were normalized to the readings from the control. Representative data from triplicate results (n = 3) are presented as mean +/− SEM. The significance (***p < 0.001) was determined from one-way ANOVA analysis. b: OSC-19 cells (3 × 103/well) were plated in 96-well plates in quadruplicate and left untreated as controls or treated with cetuximab (20 μg/ml) or cetuximab along with varying concentrations of T16A-inh (1–20 μM). All treatments were carried out for 3 days at 37° C followed by measurements of viability with the CellTiter-Glo reagent. Readings from the treated groups were normalized to the readings from the controls. Representative data from triplicate results (n = 3) are presented as mean +/− SEM. The significance (***p < 0.001) was determined from one-way ANOVA analysis. c: OSC-19 cells (3 × 103/well) were plated in 96-well plate in quadruplicate and left untreated as controls or treated with cetuximab (20 μg/ml) or cetuximab along with varying concentrations of Cacc-inh (0.5–30 μM). All treatments were carried out for 3 days at 37° C followed by measurements of viability with the CellTiter-Glo reagent. Readings from the treated group were normalized to readings from the controsl. Representative data from triplicate results (n = 3) are presented as mean +/− SEM. The significance (***p < 0.001) was determined from one-way ANOVA analysis.
Figure 5
Figure 5. Cl channel function of TMEM16A regulates activity of EGFR pathway
a: SKBR3 cells were plated in 60 mm plates and allowed to attach for 2 h. Cells were subjected to medium containing a physiologically normal level of Cl as a control (N) or medium in which Cl was depleted (Low) for 3 h at 37° C. Cells were lysed to obtain solubilized protein and equal amounts of protein were analyzed for pY1068 EGFR, EGFR, pY705 STAT3 and β-tubulin in western blots. b: OSC-19 cells were plated in 60 mm plates and allowed to attach for 2 h. Cells were subjected to medium containing physiologically normal level of Cl as a control (N) or T16A-inh (40 μM) and also to medium in which Cl was depleted (Low). All treatments were carried out for 2.5 h at 37° C at which time cells were lysed to obtain solubilized protein. Equal amounts of protein were analyzed for pY1068 EGFR, EGFR, pY705 STAT3 and β-tubulin in western blots. c: OSC-19 cells were plated in 100 mm plates in triplicate. One plate was mock-transfected as a control and the other two were transfected with plasmids encoding TMEM16A mutants (E727K or L759Q) known to be deficient in Cl channel activity. Cells were lysed to obtain solubilized protein that was analyzed for TMEM16A, EGFR, pY705 STAT3 and β-tubulin in western blots.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

MeSH terms

Feedback