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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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PP2A regulates eEF2 phosphorylation in an additional cell line model of acquired lapatinib resistance. (A) Effect of lapatinib on growth of HCC1954-par and HCC1954-L cells. (B) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in HCC1954-par and HCC1954-L cells following 24 hr lapatinib treatment. (C) Activity of PP2A in HCC1954-par and HCC1954-L cells, untreated and treated with 5 nM OA for 24 hr. (D) Immunoblot examining the effect of okadaic acid (OA) alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (E) Effect of 5 nM OA on growth of SKBR3-par and SKBR3-L cells. (F) Effect of OA alone and in combination with lapatinib on the growth of HCC1954-par cells and (G) HCC1954-L cells. *denotes p ≤ 0.05, **denotes p ≤ 0.01. Error bars represent the mean ± SD (n = 3).
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Figure 5: PP2A regulates eEF2 phosphorylation in an additional cell line model of acquired lapatinib resistance. (A) Effect of lapatinib on growth of HCC1954-par and HCC1954-L cells. (B) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in HCC1954-par and HCC1954-L cells following 24 hr lapatinib treatment. (C) Activity of PP2A in HCC1954-par and HCC1954-L cells, untreated and treated with 5 nM OA for 24 hr. (D) Immunoblot examining the effect of okadaic acid (OA) alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (E) Effect of 5 nM OA on growth of SKBR3-par and SKBR3-L cells. (F) Effect of OA alone and in combination with lapatinib on the growth of HCC1954-par cells and (G) HCC1954-L cells. *denotes p ≤ 0.05, **denotes p ≤ 0.01. Error bars represent the mean ± SD (n = 3).

Mentions: To validate the potential of altered PP2A activity as a mechanism of acquired lapatinib resistance, we developed a second cell line model of acquired lapatinib resistance (HCC1954-L) using a similar long-term lapatinib treatment strategy. Continuous treatment of HCC1954 cells resulted in the development of HCC1954-par cells and HCC1954-L cells with lapatinib IC50 values of 0.42 ± 0.02 μM and 2.67 ± 0.08 μM, respectively (Figure 5A). HCC1954-L cells have significantly lower levels of p-eEF2 compared to HCC1954-par cells (p = 0.002), and lapatinib treatment significantly increased p-eEF2 levels in HCC1954-par cells, with no effect on the levels of p-eEF2 in HCC1954-L cells (Figure 5B). HCC1954-L cells had 1.3-fold higher PP2A activity compared to HCC1954-par cells (p = 0.04) (Figure 5C). OA treatment of HCC1954-L cells increased p-eEF2 and p-AKT levels (Figure 5D). HCC1954-L cells exhibited significantly greater growth inhibition in response to OA treatment (78.0 ± 1.1%) compared to HCC1954-par cells (53.9 ± 3.9%) (p = 0.005) (Figure 5E). Combined treatment with lapatinib and OA was significantly more effective than either agent alone, in both HCC1954-par (Figure 5F) and HCC1954-L cells (Figure 5G).


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)

PP2A regulates eEF2 phosphorylation in an additional cell line model of acquired lapatinib resistance. (A) Effect of lapatinib on growth of HCC1954-par and HCC1954-L cells. (B) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in HCC1954-par and HCC1954-L cells following 24 hr lapatinib treatment. (C) Activity of PP2A in HCC1954-par and HCC1954-L cells, untreated and treated with 5 nM OA for 24 hr. (D) Immunoblot examining the effect of okadaic acid (OA) alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (E) Effect of 5 nM OA on growth of SKBR3-par and SKBR3-L cells. (F) Effect of OA alone and in combination with lapatinib on the growth of HCC1954-par cells and (G) HCC1954-L cells. *denotes p ≤ 0.05, **denotes p ≤ 0.01. Error bars represent the mean ± SD (n = 3).
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Figure 5: PP2A regulates eEF2 phosphorylation in an additional cell line model of acquired lapatinib resistance. (A) Effect of lapatinib on growth of HCC1954-par and HCC1954-L cells. (B) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in HCC1954-par and HCC1954-L cells following 24 hr lapatinib treatment. (C) Activity of PP2A in HCC1954-par and HCC1954-L cells, untreated and treated with 5 nM OA for 24 hr. (D) Immunoblot examining the effect of okadaic acid (OA) alone and in combination with lapatinib on levels of total and phosphorylated eEF2(Thr56) and AKT(Ser473). (E) Effect of 5 nM OA on growth of SKBR3-par and SKBR3-L cells. (F) Effect of OA alone and in combination with lapatinib on the growth of HCC1954-par cells and (G) HCC1954-L cells. *denotes p ≤ 0.05, **denotes p ≤ 0.01. Error bars represent the mean ± SD (n = 3).
Mentions: To validate the potential of altered PP2A activity as a mechanism of acquired lapatinib resistance, we developed a second cell line model of acquired lapatinib resistance (HCC1954-L) using a similar long-term lapatinib treatment strategy. Continuous treatment of HCC1954 cells resulted in the development of HCC1954-par cells and HCC1954-L cells with lapatinib IC50 values of 0.42 ± 0.02 μM and 2.67 ± 0.08 μM, respectively (Figure 5A). HCC1954-L cells have significantly lower levels of p-eEF2 compared to HCC1954-par cells (p = 0.002), and lapatinib treatment significantly increased p-eEF2 levels in HCC1954-par cells, with no effect on the levels of p-eEF2 in HCC1954-L cells (Figure 5B). HCC1954-L cells had 1.3-fold higher PP2A activity compared to HCC1954-par cells (p = 0.04) (Figure 5C). OA treatment of HCC1954-L cells increased p-eEF2 and p-AKT levels (Figure 5D). HCC1954-L cells exhibited significantly greater growth inhibition in response to OA treatment (78.0 ± 1.1%) compared to HCC1954-par cells (53.9 ± 3.9%) (p = 0.005) (Figure 5E). Combined treatment with lapatinib and OA was significantly more effective than either agent alone, in both HCC1954-par (Figure 5F) and HCC1954-L cells (Figure 5G).

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