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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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Phospho-proteomic analysis reveals decreased levels of p-eEF2 in SKBR3-L cells. (A) Example of a 3D view of p-eEF2 protein abundance with graphs of protein abundance analyzed by DeCyder software in SKBR3-par and SKBR3-L cells. The solid line represents the average of three replicate measurements (dotted lines) of protein abundance. (B) Schematic depiction of mTOR-mediated activation of eEF2; active mTOR phosphorylates and activates p70S6k, which in turn phosphorylates and deactivates eEF2k thus preventing the phosphorylation of eEF2 resulting in active eEF2. (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in SKBR3-par and SKBR3-L cells following 24 hr lapatinib treatment.
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Figure 2: Phospho-proteomic analysis reveals decreased levels of p-eEF2 in SKBR3-L cells. (A) Example of a 3D view of p-eEF2 protein abundance with graphs of protein abundance analyzed by DeCyder software in SKBR3-par and SKBR3-L cells. The solid line represents the average of three replicate measurements (dotted lines) of protein abundance. (B) Schematic depiction of mTOR-mediated activation of eEF2; active mTOR phosphorylates and activates p70S6k, which in turn phosphorylates and deactivates eEF2k thus preventing the phosphorylation of eEF2 resulting in active eEF2. (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in SKBR3-par and SKBR3-L cells following 24 hr lapatinib treatment.

Mentions: The phospho-proteome of SKBR3-par and SKBR3-L cells was compared using a combination of phospho-protein enrichment, 2D-DIGE and mass spectrometry (MS). 2,500 spots were detected and analyzed using DeCyder differential in-gel analysis to produce 3-D images of protein abundance and graphs of relative protein abundance (Figure 2A). Of 2,500 spots detected on the DIGE gels, 81 spots exhibited altered abundance between SKBR3-par and SKBR3-L cells and were picked for identification by MS. When a p-value of ≤ 0.05 and a cut-off of 1.2-fold change were applied, 20 phospho-proteins were significantly higher and 21 were significantly lower in SKBR3-L compared to SKBR3-par cells (Additional file2: Table S1). The proteins identified were associated with cell growth/differentiation, metabolic processes, transcription, translation, protein folding, immune cell processes and response to stress (Additional file2: Table S1).


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)

Phospho-proteomic analysis reveals decreased levels of p-eEF2 in SKBR3-L cells. (A) Example of a 3D view of p-eEF2 protein abundance with graphs of protein abundance analyzed by DeCyder software in SKBR3-par and SKBR3-L cells. The solid line represents the average of three replicate measurements (dotted lines) of protein abundance. (B) Schematic depiction of mTOR-mediated activation of eEF2; active mTOR phosphorylates and activates p70S6k, which in turn phosphorylates and deactivates eEF2k thus preventing the phosphorylation of eEF2 resulting in active eEF2. (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in SKBR3-par and SKBR3-L cells following 24 hr lapatinib treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230643&req=5

Figure 2: Phospho-proteomic analysis reveals decreased levels of p-eEF2 in SKBR3-L cells. (A) Example of a 3D view of p-eEF2 protein abundance with graphs of protein abundance analyzed by DeCyder software in SKBR3-par and SKBR3-L cells. The solid line represents the average of three replicate measurements (dotted lines) of protein abundance. (B) Schematic depiction of mTOR-mediated activation of eEF2; active mTOR phosphorylates and activates p70S6k, which in turn phosphorylates and deactivates eEF2k thus preventing the phosphorylation of eEF2 resulting in active eEF2. (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) in SKBR3-par and SKBR3-L cells following 24 hr lapatinib treatment.
Mentions: The phospho-proteome of SKBR3-par and SKBR3-L cells was compared using a combination of phospho-protein enrichment, 2D-DIGE and mass spectrometry (MS). 2,500 spots were detected and analyzed using DeCyder differential in-gel analysis to produce 3-D images of protein abundance and graphs of relative protein abundance (Figure 2A). Of 2,500 spots detected on the DIGE gels, 81 spots exhibited altered abundance between SKBR3-par and SKBR3-L cells and were picked for identification by MS. When a p-value of ≤ 0.05 and a cut-off of 1.2-fold change were applied, 20 phospho-proteins were significantly higher and 21 were significantly lower in SKBR3-L compared to SKBR3-par cells (Additional file2: Table S1). The proteins identified were associated with cell growth/differentiation, metabolic processes, transcription, translation, protein folding, immune cell processes and response to stress (Additional file2: Table S1).

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