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PP2A inhibition determines poor outcome and doxorubicin resistance in early breast cancer and its activation shows promising therapeutic effects.

Rincón R, Cristóbal I, Zazo S, Arpí O, Menéndez S, Manso R, Lluch A, Eroles P, Rovira A, Albanell J, García-Foncillas J, Madoz-Gúrpide J, Rojo F - Oncotarget (2015)

Bottom Line: Interestingly, restoration of PP2A activity after FTY720 treatment reduced cell growth, induced apoptosis and decreased AKT and ERK activation.Moreover, FTY720 led to PP2A activation then enhancing doxorubicin-induced antitumor effects both in vitro and in vivo.Altogether, our results indicate that PP2A is frequently inactivated in breast cancer and determines worse outcome, and its restoration using PP2A activators represents an alternative therapeutic strategy in this disease.

View Article: PubMed Central - PubMed

Affiliation: Translational Oncology Division, Oncohealth Institute, Health Research Institute FJD-UAM, University Hospital "Fundación Jiménez Diaz", Madrid, Spain.

ABSTRACT
The protein phosphatase 2A (PP2A) is a key tumor suppressor which has emerged as a novel molecular target in some human cancers. Here, we show that PP2A inhibition is a common event in breast cancer and identified PP2A phosphorylation and deregulation SET and CIP2A as molecular contributing mechanisms to inactivate PP2A. Interestingly, restoration of PP2A activity after FTY720 treatment reduced cell growth, induced apoptosis and decreased AKT and ERK activation. Moreover, FTY720 led to PP2A activation then enhancing doxorubicin-induced antitumor effects both in vitro and in vivo. PP2A inhibition (CPscore: PP2A phosphorylation and/or CIP2A overexpression) was detected in 27% of cases (62/230), and associated with grade (p = 0.017), relapse (p < 0.001), negative estrogen (p < 0.001) and progesterone receptor expression (p < 0.001), HER2-positive tumors (p = 0.049), Ki-67 expression (p < 0.001), and higher AKT (p < 0.001) and ERK (p < 0.001) phosphorylation. Moreover, PP2A inhibition determined shorter overall (p = 0.006) and event-free survival (p = 0.003), and multivariate analysis confirmed its independent prognostic impact. Altogether, our results indicate that PP2A is frequently inactivated in breast cancer and determines worse outcome, and its restoration using PP2A activators represents an alternative therapeutic strategy in this disease.

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FTY720-induced PP2A activation potentiates antitumor effects of doxorubicin in breast cancer cells(A) MTS assays showing cell growth after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells. Cells treated with vehicle (DMSO) were used as controls. (B) MTS analysis showing that FTY720 resensitizes to doxorubicin MDA-MB-231 cells with doxorubicin acquired resistance. (C) Evaluation of mammosphere formation capability in BT-474 cells after doxorubicin and FTY720 treatments. (D) Western blot analysis showing the molecular effects induced after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells; *p < 0.05; **p < 0.01.
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Figure 3: FTY720-induced PP2A activation potentiates antitumor effects of doxorubicin in breast cancer cells(A) MTS assays showing cell growth after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells. Cells treated with vehicle (DMSO) were used as controls. (B) MTS analysis showing that FTY720 resensitizes to doxorubicin MDA-MB-231 cells with doxorubicin acquired resistance. (C) Evaluation of mammosphere formation capability in BT-474 cells after doxorubicin and FTY720 treatments. (D) Western blot analysis showing the molecular effects induced after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells; *p < 0.05; **p < 0.01.

Mentions: Anthracyclines like doxorubicin are among the chemotherapy drugs used in breast cancer standard systemic therapy [6]. Interestingly, we found that doxorubicin-induced antitumor effects in the MDA-MB-231 and BT-474 cell lines were markedly enhanced when cells were treated simultaneously with the PP2A activator FTY720 (Figure 3A). The microscope images obtained were in concordance with the results showed by MTS assays in both cell lines (Figure S3B). Chou-Talalay analyses showed that the FTY720/doxorubicin combination has additive effects in MDA-MB-231 cells (Combination index [CI] = 0.99), and synergistic effects in BT-474 cells (CI = 0.87). Altogether, these results show that FTY720 treatment potentiates doxorubicin-induced antitumor effects in breast cancer cells. To further confirm our hypothesis that PP2A activation sensitizes to doxorubicin treatment, we performed a genetic PP2A activation by overexpressing PP2A in MDA-MB-231 and BT-474 cells, observing that doxorubicin showed significantly enhanced antitumor effects in those cells ectopically expressing PP2A (Figure S4). Finally, we analyzed the effect of FTY720 treatment in a MDA-MB-231-derived clone with a doxorubicin resistance (in fold change compared to parental cells) of 1,92. Of importance, we observed that FTY720 was able to resensitize to doxorubicin MDA-MB-231 clones with an acquired resistance to this drug (Figure 3B).


PP2A inhibition determines poor outcome and doxorubicin resistance in early breast cancer and its activation shows promising therapeutic effects.

Rincón R, Cristóbal I, Zazo S, Arpí O, Menéndez S, Manso R, Lluch A, Eroles P, Rovira A, Albanell J, García-Foncillas J, Madoz-Gúrpide J, Rojo F - Oncotarget (2015)

FTY720-induced PP2A activation potentiates antitumor effects of doxorubicin in breast cancer cells(A) MTS assays showing cell growth after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells. Cells treated with vehicle (DMSO) were used as controls. (B) MTS analysis showing that FTY720 resensitizes to doxorubicin MDA-MB-231 cells with doxorubicin acquired resistance. (C) Evaluation of mammosphere formation capability in BT-474 cells after doxorubicin and FTY720 treatments. (D) Western blot analysis showing the molecular effects induced after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells; *p < 0.05; **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414191&req=5

Figure 3: FTY720-induced PP2A activation potentiates antitumor effects of doxorubicin in breast cancer cells(A) MTS assays showing cell growth after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells. Cells treated with vehicle (DMSO) were used as controls. (B) MTS analysis showing that FTY720 resensitizes to doxorubicin MDA-MB-231 cells with doxorubicin acquired resistance. (C) Evaluation of mammosphere formation capability in BT-474 cells after doxorubicin and FTY720 treatments. (D) Western blot analysis showing the molecular effects induced after FTY720 treatment in combination with doxorubicin in MDA-MB-231 and BT-474 cells; *p < 0.05; **p < 0.01.
Mentions: Anthracyclines like doxorubicin are among the chemotherapy drugs used in breast cancer standard systemic therapy [6]. Interestingly, we found that doxorubicin-induced antitumor effects in the MDA-MB-231 and BT-474 cell lines were markedly enhanced when cells were treated simultaneously with the PP2A activator FTY720 (Figure 3A). The microscope images obtained were in concordance with the results showed by MTS assays in both cell lines (Figure S3B). Chou-Talalay analyses showed that the FTY720/doxorubicin combination has additive effects in MDA-MB-231 cells (Combination index [CI] = 0.99), and synergistic effects in BT-474 cells (CI = 0.87). Altogether, these results show that FTY720 treatment potentiates doxorubicin-induced antitumor effects in breast cancer cells. To further confirm our hypothesis that PP2A activation sensitizes to doxorubicin treatment, we performed a genetic PP2A activation by overexpressing PP2A in MDA-MB-231 and BT-474 cells, observing that doxorubicin showed significantly enhanced antitumor effects in those cells ectopically expressing PP2A (Figure S4). Finally, we analyzed the effect of FTY720 treatment in a MDA-MB-231-derived clone with a doxorubicin resistance (in fold change compared to parental cells) of 1,92. Of importance, we observed that FTY720 was able to resensitize to doxorubicin MDA-MB-231 clones with an acquired resistance to this drug (Figure 3B).

Bottom Line: Interestingly, restoration of PP2A activity after FTY720 treatment reduced cell growth, induced apoptosis and decreased AKT and ERK activation.Moreover, FTY720 led to PP2A activation then enhancing doxorubicin-induced antitumor effects both in vitro and in vivo.Altogether, our results indicate that PP2A is frequently inactivated in breast cancer and determines worse outcome, and its restoration using PP2A activators represents an alternative therapeutic strategy in this disease.

View Article: PubMed Central - PubMed

Affiliation: Translational Oncology Division, Oncohealth Institute, Health Research Institute FJD-UAM, University Hospital "Fundación Jiménez Diaz", Madrid, Spain.

ABSTRACT
The protein phosphatase 2A (PP2A) is a key tumor suppressor which has emerged as a novel molecular target in some human cancers. Here, we show that PP2A inhibition is a common event in breast cancer and identified PP2A phosphorylation and deregulation SET and CIP2A as molecular contributing mechanisms to inactivate PP2A. Interestingly, restoration of PP2A activity after FTY720 treatment reduced cell growth, induced apoptosis and decreased AKT and ERK activation. Moreover, FTY720 led to PP2A activation then enhancing doxorubicin-induced antitumor effects both in vitro and in vivo. PP2A inhibition (CPscore: PP2A phosphorylation and/or CIP2A overexpression) was detected in 27% of cases (62/230), and associated with grade (p = 0.017), relapse (p < 0.001), negative estrogen (p < 0.001) and progesterone receptor expression (p < 0.001), HER2-positive tumors (p = 0.049), Ki-67 expression (p < 0.001), and higher AKT (p < 0.001) and ERK (p < 0.001) phosphorylation. Moreover, PP2A inhibition determined shorter overall (p = 0.006) and event-free survival (p = 0.003), and multivariate analysis confirmed its independent prognostic impact. Altogether, our results indicate that PP2A is frequently inactivated in breast cancer and determines worse outcome, and its restoration using PP2A activators represents an alternative therapeutic strategy in this disease.

Show MeSH
Related in: MedlinePlus