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PP2A inhibition overcomes acquired resistance to HER2 targeted therapy.

McDermott MS, Browne BC, Conlon NT, O'Brien NA, Slamon DJ, Henry M, Meleady P, Clynes M, Dowling P, Crown J, O'Donovan N - Mol. Cancer (2014)

Bottom Line: In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells.PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells.Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Therapeutics for Cancer Ireland, National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Norma.ODonovan@dcu.ie.

ABSTRACT

Background: HER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling.

Results: Long-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L.

Conclusions: Our data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies.

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The role of PP2A in eEF2 phosphorylation. (A) Activity of PP2A in SKBR3-par and SKBR3-L cells, untreated and treated with 5 nM OA for 24 hr. (B) Immunoblot examining the effect of OA alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (C) Effect of 5 nM okadaic acid (OA) on growth of SKBR3-par and SKBR3-L cells. (D) Effect of OA alone and in combination with lapatinib on the growth of SKBR3-par cells and (E) SKBR3-L cells. Error bars represent the mean ± SD (n = 3). *denotes p ≤ 0.05, **denotes p ≤ 0.01.
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Figure 4: The role of PP2A in eEF2 phosphorylation. (A) Activity of PP2A in SKBR3-par and SKBR3-L cells, untreated and treated with 5 nM OA for 24 hr. (B) Immunoblot examining the effect of OA alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (C) Effect of 5 nM okadaic acid (OA) on growth of SKBR3-par and SKBR3-L cells. (D) Effect of OA alone and in combination with lapatinib on the growth of SKBR3-par cells and (E) SKBR3-L cells. Error bars represent the mean ± SD (n = 3). *denotes p ≤ 0.05, **denotes p ≤ 0.01.

Mentions: Protein phosphatase 2A (PP2A) activity results in the direct dephosphorylation of multiple proteins including eEF2 and AKT. Therefore we hypothesized that increased PP2A activity in SKBR3-L cells may account for the decreased levels of p-eEF2 and p-AKT observed. PP2A activity was measured in extracts from SKBR3-par and SKBR3-L cells using a phosphatase assay under conditions selective for PP2A activity. SKBR3-L cells displayed 1.8-fold higher PP2A activity compared to SKBR3-par cells (p = 0.02) (Figure 4A). Okadaic acid (OA), a potent lab-grade PP2A inhibitor was used to confirm PP2A activity, and caused a decrease in PP2A activity in both cells lines. Treatment of SKBR3-L cells with OA resulted in a significant increase in the levels of p-eEF2 (p = 0.02) and this effect was sustained when OA was combined with lapatinib (p = 0.03) (Figure 4B). Levels of p-AKT were also increased following OA treatment, although to a lesser extent. SKBR3-L cells were significantly more sensitive to OA (5 nM) treatment than SKBR3-par cells in proliferation assays (69.9 ± 3.7% versus 26.8 ± 9.1% growth inhibition, p = 0.007) (Figure 4C). Combined treatment of SKBR3-par cells with lapatinib and OA resulted in significantly greater inhibition of growth compared to either lapatinib (p = 0.007) or OA alone (p = 0.012) (Figure 4D). Importantly, significantly enhanced growth inhibition was also observed in SKBR3-L cells following combined treatment compared to single agent lapatinib (p = 0.005) or OA (p = 0.012) (Figure 4E). In contrast, treatment of SKBR3-L cells with trastuzumab and okadaic acid did not result in enhanced growth inhibition compared to okadaic acid alone (Additional file4: Figure S3A). To determine if okadaic acid combined with lapatinib was cytostatic or cytocidal, cell cycle assays were performed. Lapatinib alone (500 nM) induced G1 arrest (51.8 ± 1.6% compared to 41.8 ± 2.9% for untreated cells), OA alone induced a small increase in the sub-G1 population (12.8 ± 6.3% versus 8.9 ± 3.4% for untreated cells) and the combination induced an increase in the sub-G1 fraction (23.1 ± 8.5%) compared to either lapatinib (9.1 ± 6.7%, p = 0.044) or OA (12.8 ± 6.3%, p = 0.083) alone (Additional file4: Figure S3B).


PP2A inhibition overcomes acquired resistance to HER2 targeted therapy.

McDermott MS, Browne BC, Conlon NT, O'Brien NA, Slamon DJ, Henry M, Meleady P, Clynes M, Dowling P, Crown J, O'Donovan N - Mol. Cancer (2014)

The role of PP2A in eEF2 phosphorylation. (A) Activity of PP2A in SKBR3-par and SKBR3-L cells, untreated and treated with 5 nM OA for 24 hr. (B) Immunoblot examining the effect of OA alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (C) Effect of 5 nM okadaic acid (OA) on growth of SKBR3-par and SKBR3-L cells. (D) Effect of OA alone and in combination with lapatinib on the growth of SKBR3-par cells and (E) SKBR3-L cells. Error bars represent the mean ± SD (n = 3). *denotes p ≤ 0.05, **denotes p ≤ 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: The role of PP2A in eEF2 phosphorylation. (A) Activity of PP2A in SKBR3-par and SKBR3-L cells, untreated and treated with 5 nM OA for 24 hr. (B) Immunoblot examining the effect of OA alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (C) Effect of 5 nM okadaic acid (OA) on growth of SKBR3-par and SKBR3-L cells. (D) Effect of OA alone and in combination with lapatinib on the growth of SKBR3-par cells and (E) SKBR3-L cells. Error bars represent the mean ± SD (n = 3). *denotes p ≤ 0.05, **denotes p ≤ 0.01.
Mentions: Protein phosphatase 2A (PP2A) activity results in the direct dephosphorylation of multiple proteins including eEF2 and AKT. Therefore we hypothesized that increased PP2A activity in SKBR3-L cells may account for the decreased levels of p-eEF2 and p-AKT observed. PP2A activity was measured in extracts from SKBR3-par and SKBR3-L cells using a phosphatase assay under conditions selective for PP2A activity. SKBR3-L cells displayed 1.8-fold higher PP2A activity compared to SKBR3-par cells (p = 0.02) (Figure 4A). Okadaic acid (OA), a potent lab-grade PP2A inhibitor was used to confirm PP2A activity, and caused a decrease in PP2A activity in both cells lines. Treatment of SKBR3-L cells with OA resulted in a significant increase in the levels of p-eEF2 (p = 0.02) and this effect was sustained when OA was combined with lapatinib (p = 0.03) (Figure 4B). Levels of p-AKT were also increased following OA treatment, although to a lesser extent. SKBR3-L cells were significantly more sensitive to OA (5 nM) treatment than SKBR3-par cells in proliferation assays (69.9 ± 3.7% versus 26.8 ± 9.1% growth inhibition, p = 0.007) (Figure 4C). Combined treatment of SKBR3-par cells with lapatinib and OA resulted in significantly greater inhibition of growth compared to either lapatinib (p = 0.007) or OA alone (p = 0.012) (Figure 4D). Importantly, significantly enhanced growth inhibition was also observed in SKBR3-L cells following combined treatment compared to single agent lapatinib (p = 0.005) or OA (p = 0.012) (Figure 4E). In contrast, treatment of SKBR3-L cells with trastuzumab and okadaic acid did not result in enhanced growth inhibition compared to okadaic acid alone (Additional file4: Figure S3A). To determine if okadaic acid combined with lapatinib was cytostatic or cytocidal, cell cycle assays were performed. Lapatinib alone (500 nM) induced G1 arrest (51.8 ± 1.6% compared to 41.8 ± 2.9% for untreated cells), OA alone induced a small increase in the sub-G1 population (12.8 ± 6.3% versus 8.9 ± 3.4% for untreated cells) and the combination induced an increase in the sub-G1 fraction (23.1 ± 8.5%) compared to either lapatinib (9.1 ± 6.7%, p = 0.044) or OA (12.8 ± 6.3%, p = 0.083) alone (Additional file4: Figure S3B).

Bottom Line: In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells.PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells.Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Therapeutics for Cancer Ireland, National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Norma.ODonovan@dcu.ie.

ABSTRACT

Background: HER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling.

Results: Long-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L.

Conclusions: Our data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies.

Show MeSH
Related in: MedlinePlus