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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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mTOR and eEF2k mediated regulation of eEF2 phosphorylation. (A) Immunoblot analysis of total and phosphorylated mTOR(Ser2448) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (B) Effect of rapamycin on growth of SKBR3-par and SKBR3-L cells. Error bars represent the mean ± SD (n = 3). (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) following 24 hr. treatment with lapatinib and/or rapamycin. (D) Immunoblot analysis of total and phosphorylated eEF2k(Ser366, 359) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (E) Immunoblot examining the effect of NH125 alone and in combination with lapatinib on the phosphorylation of eEF2(Thr56) in SKBR3-par cells. *denotes p ≤ 0.05.
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Figure 3: mTOR and eEF2k mediated regulation of eEF2 phosphorylation. (A) Immunoblot analysis of total and phosphorylated mTOR(Ser2448) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (B) Effect of rapamycin on growth of SKBR3-par and SKBR3-L cells. Error bars represent the mean ± SD (n = 3). (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) following 24 hr. treatment with lapatinib and/or rapamycin. (D) Immunoblot analysis of total and phosphorylated eEF2k(Ser366, 359) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (E) Immunoblot examining the effect of NH125 alone and in combination with lapatinib on the phosphorylation of eEF2(Thr56) in SKBR3-par cells. *denotes p ≤ 0.05.

Mentions: To determine if decreased p-eEF2 levels in SKBR3-L cells are mediated by an alteration in mTOR signaling, we examined the levels of p-mTORser2448 and total mTOR. While the total levels of mTOR were unchanged between the two cell lines, SKBR3-L cells had significantly lower levels of p-mTOR compared to SKBR3-par cells (p = 0.01) (Figure 3A). However, lapatinib treatment significantly decreased the levels of p-mTOR in both SKBR3-par (p = 0.005) and SKBR3-L (p = 0.03) cells. Together, these results suggest that the downregulation of p-eEF2 observed in SKBR3-L cells is not due to increased or constitutive activation of mTOR. In fact, SKBR3-L cells were significantly less sensitive to the mTOR inhibitor, rapamycin, than SKBR3-par cells (p = 0.01) (Figure 3B), suggesting that SKBR3-L cells have reduced dependence on mTOR signaling for growth. Furthermore, SKBR3-par cells treated with rapamycin alone or in combination with lapatinib exhibited a significant increase in p-eEF2 following treatment (Figure 3C). However, treatment of SKBR3-L cells under the same conditions had no effect on p-eEF2. Taken together, these results suggest that the decreased phosphorylation of eEF2 is mediated via a non-mTOR dependent mechanism in SKBR3-L cells.


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)

mTOR and eEF2k mediated regulation of eEF2 phosphorylation. (A) Immunoblot analysis of total and phosphorylated mTOR(Ser2448) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (B) Effect of rapamycin on growth of SKBR3-par and SKBR3-L cells. Error bars represent the mean ± SD (n = 3). (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) following 24 hr. treatment with lapatinib and/or rapamycin. (D) Immunoblot analysis of total and phosphorylated eEF2k(Ser366, 359) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (E) Immunoblot examining the effect of NH125 alone and in combination with lapatinib on the phosphorylation of eEF2(Thr56) in SKBR3-par cells. *denotes p ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230643&req=5

Figure 3: mTOR and eEF2k mediated regulation of eEF2 phosphorylation. (A) Immunoblot analysis of total and phosphorylated mTOR(Ser2448) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (B) Effect of rapamycin on growth of SKBR3-par and SKBR3-L cells. Error bars represent the mean ± SD (n = 3). (C) Immunoblot analysis of total and phosphorylated eEF2(Thr56) following 24 hr. treatment with lapatinib and/or rapamycin. (D) Immunoblot analysis of total and phosphorylated eEF2k(Ser366, 359) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (E) Immunoblot examining the effect of NH125 alone and in combination with lapatinib on the phosphorylation of eEF2(Thr56) in SKBR3-par cells. *denotes p ≤ 0.05.
Mentions: To determine if decreased p-eEF2 levels in SKBR3-L cells are mediated by an alteration in mTOR signaling, we examined the levels of p-mTORser2448 and total mTOR. While the total levels of mTOR were unchanged between the two cell lines, SKBR3-L cells had significantly lower levels of p-mTOR compared to SKBR3-par cells (p = 0.01) (Figure 3A). However, lapatinib treatment significantly decreased the levels of p-mTOR in both SKBR3-par (p = 0.005) and SKBR3-L (p = 0.03) cells. Together, these results suggest that the downregulation of p-eEF2 observed in SKBR3-L cells is not due to increased or constitutive activation of mTOR. In fact, SKBR3-L cells were significantly less sensitive to the mTOR inhibitor, rapamycin, than SKBR3-par cells (p = 0.01) (Figure 3B), suggesting that SKBR3-L cells have reduced dependence on mTOR signaling for growth. Furthermore, SKBR3-par cells treated with rapamycin alone or in combination with lapatinib exhibited a significant increase in p-eEF2 following treatment (Figure 3C). However, treatment of SKBR3-L cells under the same conditions had no effect on p-eEF2. Taken together, these results suggest that the decreased phosphorylation of eEF2 is mediated via a non-mTOR dependent mechanism in SKBR3-L cells.

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