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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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Related in: MedlinePlus

Development and characterization of a cell-line model of acquired lapatinib resistance. (A) Schematic depiction of the continuous long-term lapatinib treatment strategy utilized to develop SKBR3-L cells. (B) Effect of lapatinib treatment on SKBR3-par and SKBR3-L cells. (C) Effect of trastuzumab treatment on SKBR3-par and SKBR3-L cells. **denotes p < 0.01. (D) Effect of gefitinib treatment on SKBR3-par and SKBR3-L cells. (E) Immunoblot analysis of total and phosphorylated HER2(Tyr1221/1222), EGFR(Tyr1173), AKT(Ser473) and ERK(Thr202/Tyr204) in cells following 24 hr lapatinib treatment. Error bars represent the mean ± SD (n = 3).
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Figure 1: Development and characterization of a cell-line model of acquired lapatinib resistance. (A) Schematic depiction of the continuous long-term lapatinib treatment strategy utilized to develop SKBR3-L cells. (B) Effect of lapatinib treatment on SKBR3-par and SKBR3-L cells. (C) Effect of trastuzumab treatment on SKBR3-par and SKBR3-L cells. **denotes p < 0.01. (D) Effect of gefitinib treatment on SKBR3-par and SKBR3-L cells. (E) Immunoblot analysis of total and phosphorylated HER2(Tyr1221/1222), EGFR(Tyr1173), AKT(Ser473) and ERK(Thr202/Tyr204) in cells following 24 hr lapatinib treatment. Error bars represent the mean ± SD (n = 3).

Mentions: We utilized a continuous long-term lapatinib treatment strategy in the HER2-positive breast cancer cell line SKBR3, to develop a cell line model of acquired resistance. The cells were treated twice weekly with 250 nM lapatinib, while parental control cells (SKBR3-par) were cultured alongside the treated cells (Figure 1A). After a period of 6 months continuous treatment, the sensitivity of the treated and un-treated cells was determined. SKBR3-par cells showed similar sensitivity to lapatinib as the SKBR3 cells prior to the 6 month growth period (IC50 = 0.1 ± 0.01 μM). In contrast, lapatinib-conditioned cells (SKBR3-L) exhibited a lapatinib IC50 of 6.5 ± 0.4 μM, confirming resistance (Figure 1B). SKBR3-L cells were tested for cross-resistance to other HER2 and EGFR targeted agents, trastuzumab and gefitinib. SKBR3-L cells were significantly less sensitive to trastuzumab treatment (p = 0.003) (Figure 1C), and to gefitinib treatment (p = 0.02) (Figure 1D), compared to SKBR3-par cells, suggesting that SKBR3-L cells are resistant to both HER2 and EGFR inhibition. We then examined the expression and phosphorylation of several key members of the HER2 and EGFR signaling pathways in both cell lines. While the expression of HER2, AKT and ERK was unaltered between the two cell lines, SKBR3-L cells exhibited increased expression of EGFR compared to SKBR3-par cells (p = 0.004) (Figure 1E). Significant alterations in each of these proteins were identified when we examined their phosphorylation status. SKBR3-L cells exhibited increased levels of p-HER2 (p = 0.02) and p-EGFR (p = 0.02), and significantly decreased levels of p-ERK (p = 0.01) and p-AKT (p = 0.001). Interestingly, when SKBR3-L cells were treated with lapatinib the levels of p-HER2 and p-EGFR decreased, in a similar manner to SKBR3-par cells (Figure 1E). SKBR3-L cells did not exhibit significant alterations in the expression of XIAP, or SRC phosphorylation. They also did not express ER nor exhibit loss of PTEN compared to SKBR3-par cells (Additional file1: Figure 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)

Development and characterization of a cell-line model of acquired lapatinib resistance. (A) Schematic depiction of the continuous long-term lapatinib treatment strategy utilized to develop SKBR3-L cells. (B) Effect of lapatinib treatment on SKBR3-par and SKBR3-L cells. (C) Effect of trastuzumab treatment on SKBR3-par and SKBR3-L cells. **denotes p < 0.01. (D) Effect of gefitinib treatment on SKBR3-par and SKBR3-L cells. (E) Immunoblot analysis of total and phosphorylated HER2(Tyr1221/1222), EGFR(Tyr1173), AKT(Ser473) and ERK(Thr202/Tyr204) in cells following 24 hr lapatinib treatment. Error bars represent the mean ± SD (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Development and characterization of a cell-line model of acquired lapatinib resistance. (A) Schematic depiction of the continuous long-term lapatinib treatment strategy utilized to develop SKBR3-L cells. (B) Effect of lapatinib treatment on SKBR3-par and SKBR3-L cells. (C) Effect of trastuzumab treatment on SKBR3-par and SKBR3-L cells. **denotes p < 0.01. (D) Effect of gefitinib treatment on SKBR3-par and SKBR3-L cells. (E) Immunoblot analysis of total and phosphorylated HER2(Tyr1221/1222), EGFR(Tyr1173), AKT(Ser473) and ERK(Thr202/Tyr204) in cells following 24 hr lapatinib treatment. Error bars represent the mean ± SD (n = 3).
Mentions: We utilized a continuous long-term lapatinib treatment strategy in the HER2-positive breast cancer cell line SKBR3, to develop a cell line model of acquired resistance. The cells were treated twice weekly with 250 nM lapatinib, while parental control cells (SKBR3-par) were cultured alongside the treated cells (Figure 1A). After a period of 6 months continuous treatment, the sensitivity of the treated and un-treated cells was determined. SKBR3-par cells showed similar sensitivity to lapatinib as the SKBR3 cells prior to the 6 month growth period (IC50 = 0.1 ± 0.01 μM). In contrast, lapatinib-conditioned cells (SKBR3-L) exhibited a lapatinib IC50 of 6.5 ± 0.4 μM, confirming resistance (Figure 1B). SKBR3-L cells were tested for cross-resistance to other HER2 and EGFR targeted agents, trastuzumab and gefitinib. SKBR3-L cells were significantly less sensitive to trastuzumab treatment (p = 0.003) (Figure 1C), and to gefitinib treatment (p = 0.02) (Figure 1D), compared to SKBR3-par cells, suggesting that SKBR3-L cells are resistant to both HER2 and EGFR inhibition. We then examined the expression and phosphorylation of several key members of the HER2 and EGFR signaling pathways in both cell lines. While the expression of HER2, AKT and ERK was unaltered between the two cell lines, SKBR3-L cells exhibited increased expression of EGFR compared to SKBR3-par cells (p = 0.004) (Figure 1E). Significant alterations in each of these proteins were identified when we examined their phosphorylation status. SKBR3-L cells exhibited increased levels of p-HER2 (p = 0.02) and p-EGFR (p = 0.02), and significantly decreased levels of p-ERK (p = 0.01) and p-AKT (p = 0.001). Interestingly, when SKBR3-L cells were treated with lapatinib the levels of p-HER2 and p-EGFR decreased, in a similar manner to SKBR3-par cells (Figure 1E). SKBR3-L cells did not exhibit significant alterations in the expression of XIAP, or SRC phosphorylation. They also did not express ER nor exhibit loss of PTEN compared to SKBR3-par cells (Additional file1: Figure 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