Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235

Abstract

Small molecule inhibitors of HER2 are clinically active in women with advanced HER2-positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach, we performed a genome wide loss-of-function short hairpin RNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumor suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we show that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that phosphatidylinositol 3-kinase (PI3K)-induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance, which can be effectively reversed by NVP-BEZ235.

Fig.1

PTEN silencing decreases sensitivity to trastuzumab and lapatinib. When required cells were either treated with trastuzumab (5 ug/ml), or lapatinib (27 nM) unless otherwise stated. A) Colony formation assay in BT474 cells infected with either NKI library vector PTEN^kdA, a second independent shRNA vector PTEN^kdB, or relevant controls. Infected cells were left untreated or treated with trastuzumab, or lapatinib, or both. After 4 weeks cells were photographed and stained with crystal violet. B) Western Blot analysis monitoring PTEN expression in stably infected PTEN knockdown cells. C). Growth curves of stably infected PTEN knockdown BT474 cells treated for 3 weeks with trastuzumab, or lapatinib, or both. Cell numbers were quantified as described in Experimental Procedures. Growth curves were performed in triplicate with the error bars depicting the mean ± s.d. D) Western blot analysis of stably infected BT474 cells with shRNA PTEN^kdB vector treated overnight with trastuzumab, or lapatinib, or both in 10% serum. Whole cell extracts were analyzed with indicated antibodies.

Fig. 2

PTEN silencing decreases sensitivity to lapatinib at clinically relevant concentrations A) Colony formation assay in BT474 cells infected with vector PTEN^kdA. Infected cells were left untreated or treated with lapatinib (500 nM). After 4 weeks (controls) or after 12 weeks (lapatinib treated) cells were photographed and stained with crystal violet. B) pAKT in relation to the total level of unphosphorylated protein in lapatinib treated samples (0, 25, 50, 100, 500 nM) in the presence or absence of PTEN^kdA vector. C) Mouse xenograft assay with BT474 VH2 cells stably infected with vector PTEN^kdB, or relevant controls. Mice were treated daily with lapatinib (50 mg/kg) or vehicle. The results shown are the mean tumour volume ± SE. A two-tailed student T test compares the two treated populations *p< 0.02 D) Western blot analysis of mouse xenograft BT474 VH2 tumours stably infected with vector PTEN^kdB or relevant controls. Whole cell extracts were analyzed with indicated antibodies.

Fig 3

Constitutive activation of the PI3K pathway decreases sensitivity to trastuzumab and lapatinib. When required cells were either treated with trastuzumab (5 ug/ml), or lapatinib (27 nM) unless otherwise stated. A) Colony formation assay in BT474 cells infected with either WT PIK3CAα or PIK3CAα mutants E545K and H1047R, or relevant controls. Infected cells were treated with trastuzumab, or lapatinib, or both. After 4 weeks cells were photographed and stained with crystal violet. B) Growth curves of stably infected PI3K wildtype or mutant PIK3CAα BT474 cells treated for 3 weeks with trastuzumab, or lapatinib, or both. Cell numbers were quantified as described in Experimental Procedures. Growth curves were performed in triplicate with the error bars depicting the mean ± s.d. C) Western blot analysis of BT474 cells infected with HA-tagged PI3K PIK3CAα (upper left panel), PI3K(E545K) PIK3CAα (lower left panel), PI3K(H1047R) PIK3CAα (right panel), or controls were treated overnight with trastuzumab, or lapatinib, or both. Whole cell extracts were analyzed with indicated antibodies.

Fig.4

Constitutive activation of the PI3K pathway decreases sensitivity to lapatinib at clinically relevant concentrations A) Colony formation assay in BT474 cells infected with either WT PIK3CAα or PIK3CAα mutants E545K and H1047R, or relevant controls. Infected cells were left untreated or treated with lapatinib (500 nM). After 5 weeks cells were photographed and stained with crystal violet. B) Western blot analysis showing pAKT in relation to the total level of unphosphorylated protein in lapatinib treated samples (0, 25, 50, 100, 500 nM) in the presence or absence of WT PIK3CAα vector. C) Western blot analysis showing pAKT in relation to the total level of unphosphorylated protein in lapatinib treated samples (0, 25, 50, 100, 500 nM) in the presence or absence of PIK3CAα mutant E545K. D) Western blot analysis showing pAKT in relation to the total level of unphosphorylated protein in lapatinib treated samples (0, 25, 50, 100, 500 nM) in the presence or absence of PIK3CAα mutant H10147R.

Fig. 5

Lapatinib and NVP-BEZ235 suppress the PI3K-AKT-mTOR axis driven by loss-of-function PTEN mutations. When required cells were either treated with trastuzumab (5 ug/ml), or lapatinib (27 nM), or NVP-BEZ235 (15nM) unless otherwise stated. A) Colony formation assay in BT474 cells infected with either PTEN^kdA or relevant controls. Infected cells were treated with trastuzumab, or lapatinib, or NVP-BEZ235, or in combination. After 4 weeks cells were photographed and stained with crystal violet. B).Growth curves of stably infected PTEN knockdown BT474 cells (PTEN^kdA or PTEN^kdB) treated for 3 weeks with lapatinib or NVP-BEZ235, or both. Cell numbers were quantified as described in Experimental Procedures. Growth curves were performed in triplicate with the error bars depicting the mean ± s.d. C) Western blot analysis of stably infected BT474 cells with shRNA PTEN^kdA vector treated overnight with lapatinib (27 nM), or NVP-BEZ235 (100nM) or both. Whole cell extracts were analyzed with the indicated antibodies. D) Western blot analysis of stably infected BT474 cells with shRNA PTEN^kdA vector treated overnight with lapatinib (27 nM), or NVP-BEZ235 (500nM) or both. Whole cell extracts were analyzed with the indicated antibodies.

Fig. 6

Lapatinib and NVP-BEZ235 suppress the PI3K-AKT-mTOR axis driven by gain of function PIK3CA mutations. When required cells were either treated with trastuzumab (5 ug/ml), or lapatinib (27 nM), or NVP-BEZ235 (15nM) unless otherwise stated. A) Colony formation assay in BT474 cells infected with either WT PIK3CAα or PIK3CAα mutants E545K and H1047R, or relevant controls. Infected cells were treated with trastuzumab, or lapatinib, or NVP-BEZ235, or in combination. After 4 weeks cells were photographed and stained with crystal violet. B) Growth curves of stably infected PI3K wildtype or mutant PIK3CAα BT474 cells treated for 3 weeks with lapatinib, or NVP-BEZ235 or both. Cell numbers were quantified as described in Experimental Procedures. Growth curves were performed in triplicate with the error bars depicting the mean ± s.d. C) Western blot analysis of BT474 cells infected with HA-tagged PI3K PIK3CAα (left panel), PI3K(E545K) PIK3CAα (middle panel), PI3K(H1047R) PIK3CAα (right panel), or controls were treated overnight with lapatinib or NVP-BEZ235, or both. Whole cell extracts were analyzed with indicated antibodies.