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. 2014 Sep 11;16(5):430.
doi: 10.1186/s13058-014-0430-x.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-_targeting mammalian _target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase

Xiaoyong Fu, Chad J Creighton, Nrusingh C Biswal, Vijetha Kumar, Martin Shea, Sabrina Herrera, Alejandro Contreras, Carolina Gutierrez, Tao Wang, Sarmistha Nanda, Mario Giuliano, Gladys Morrison, Agostina Nardone, Kristen L Karlin, Thomas F Westbrook, Laura M Heiser, Pavana Anur, Paul Spellman, Sylvie M Guichard, Paul D Smith, Barry R Davies, Teresa Klinowska, Adrian V Lee, Gordon B Mills, Mothaffar F Rimawi, Susan G Hilsenbeck, Joe W Gray, Amit Joshi, C Kent Osborne, Rachel Schiff

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-_targeting mammalian _target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase

Xiaoyong Fu et al. Breast Cancer Res. .

Abstract

Introduction: Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.

Methods: Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.

Results: Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian _target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.

Conclusions: Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

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Figures

Figure 1
Figure 1
PTEN-KD enhances PI3K signaling, reduces expression of ER and its regulated genes, and promotes expression profile of genes associated with luminal B subtype. (A) Diagram of the lentiviral pINDUCER vector. (B) Fluorescence microscopy (20×) shows the eGFP and tRFP expression in MCF7L-shPTEN cells after Dox induction for 48 h. (C) The quantification of the positive population of eGFP and tRFP cells was analyzed by flow cytometry. (D) After three days of Dox induction, the cell lysates of shPTEN cell models were subjected to Western blotting. Numbers under each blot indicate protein densitometry normalized to β-actin (values in -Dox cells were set as 1). (E) Cell lysates from MCF7L-shPTEN and T47D-shPTEN cells after five days induction with a range of Dox doses were blotted by the indicated antibodies (densitometry shown under the PTEN blotting). (F) PTEN-low gene signature derived from PTEN-KD cells representing the differentially expressed genes compared to -WT cells was correlated with known gene signatures related to growth factor signaling [28]-[30]. Heat map of t statistic indicates the global similarity between signatures based on the Pearson’s correlations. (G) Up- or downregulated (DN) genes in PTEN-KD cells were analyzed for gene enrichment (one-sided Fisher’s exact test) in the gene sets of endocrine resistant xenograft tumors (Group 1 to 5) [31] or E2-induced MCF7 cells (Cluster B) [30]. (H) Box plot shows the PTEN-low gene signature scores of each ER+ luminal tumor from datasets of TCGA [11] and Compendium [18]. The mean value ± standard deviation of all samples in each subtype is marked within the box plot in red (****P <0.0001, Student’s t test). Dox, doxycycline; E2, β-estradiol; ED, estrogen deprivation; eGFP, enhanced GFP; ER, estrogen receptor α; KD, knockdown; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; TCGA, The Cancer Genome Atlas; tRFP, turbo-RFP.
Figure 2
Figure 2
Moderate reduction in PTEN decreases endocrine sensitivity in ER+/HER2- breast cancer cell models. MCF7L-shPTEN cells with two different shRNA sequences (#1 and #2) (A and B), T47D-shPTEN (#1) cells (C), and BT483-shPTEN (#1) cells (D) were grown in PRF medium with 5% CS-FBS under -/+Dox for three days, then treated with E2 (1 nM) as control, continuing the same medium (ED), Tam (100 nM), or Ful (100 nM) for five days in 96-well plates. Cell growth (%) at day 5 in all anti-estrogen groups (ED, Tam, and Ful) was normalized to E2 -/+Dox control. There is no noticeable change in cell growth between E2-Dox and E2 + Dox groups. Cell growth was monitored daily by in situ cell cytometry (Celigo). (E and F) MCF7L-shPTEN and T47D-shPTEN cells were prepared as before with an additional range of Dox induction and subjected to endocrine treatment. Cell growth (%) at day 5 in all anti-estrogen groups (ED, Tam, and Ful) was normalized to E2 -/+Dox control. The Bonferroni post hoc test was used for all pairwise comparisons (*P <0.05, **P <0.01, ***P <0.001). CS-FBS, charcoal stripped-fetal bovine serum; Dox, doxycycline; E2, β-estradiol; ED, estrogen deprivation; ER, estrogen receptor α; Ful, fulvestrant; HER2, human epidermal growth factor receptor 2; PRF, phenol-red free; PTEN, phosphatase and tensin homolog; shRNA, short hairpin RNA; Tam, tamoxifen.
Figure 3
Figure 3
PTEN-KD confers endocrine resistance in the xenograft mouse model. (A) Fluorescent live images showed MCF7L-shPTEN xenograft tumors at week 6 after randomization. Fluorescent red suggests the expression of tRFP with the color bar indicated at bottom. The tumors without a red signal (arrow indicated) show the fluorescent green (eGFP expression) in the insets. (B-E) Growth curves of xenograft tumors under E2, ED, Tam, or Ful, all -/+Dox (n ≥10 in each arm). The IHC staining of PTEN (F), pAKT-S473 (H), and Ki67 (J) in tissue microarrays of xenograft tumors after two weeks of treatment was quantified as the Allred scores of cytoplasmic PTEN (G) and cytoplasmic pAKT-S473 (I), or as the proportion of Ki67 positive cells (K). n = 6 for each arm; scale bar, 50 μm (F and H), 100 μm (J). The Bonferroni post hoc test was applied for paired comparisons between -/+Dox in each endocrine group, or between the anti-estrogen and E2 groups (*P <0.05, **P <0.01, ***P <0.001). Dox, doxycycline; E2, β-estradiol; ED, estrogen deprivation; eGFP, enhanced GFP; Ful, fulvestrant; IHC, immunohistochemical; KD, knockdown; PTEN, phosphatase and tensin homolog; Tam, tamoxifen; tRFP, turbo-RFP.
Figure 4
Figure 4
Efficacy of combination therapy varies by PTEN levels, type of endocrine therapy, and combined kinase inhibitors. MCF7-shPTEN cells were pre-treated in PRF medium with 5% CS-FBS and -/+Dox for three days. Bar charts presented the % of cell growth of MCF7L-shPTEN cells treated for five days with single or two-agent combination kinase inhibitors (mTORi, 0.2 μm; AKTi, 1 μm; MEKi, 1 μm), under E2 (A), ED (B), or Tam (C) condition, all -/+Dox (gray/red color). Cell growth in five days of the E2 (-/+Dox) groups was used as the normalization control. The Bonferroni post hoc test was performed for paired comparisons between single-kinase inhibitors and DMSO (drug carrier control) (#P <0.05), or between a two-agent kinase inhibitor combination and either agent alone (*P <0.05, **P <0.01, ***P <0.001). Heat maps were used for all calculated % of growth inhibition (scaled so as to not exceed 100% by dividing by the maximum growth inhibition within each matrix) in two-agent combinations of kinase inhibitors: mTORi plus AKTi (D and G), mTORi plus MEKi (E and H), or AKTi plus MEKi (F and I), all under ED or Tam. Cell growth under ED or Tam without kinase inhibitor was set as 100% (0% of growth inhibition) for the normalization control. Percentages of combinations among each matrix with enhanced (P ≤0.05) or attenuated (P ≥0.95) effect compared to single drug alone (tested by the Min test) were summarized in (J) (under ED) and (K) (under Tam). AKT, protein kinase B; CS-FBS, charcoal-stripped-FBS; Dox, doxycycline; E2, β-estradiol; ED, estrogen deprivation; Ful, fulvestrant; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian _target of rapamycin; PRF, phenol-red free; PTEN, phosphatase and tensin homolog; Tam, tamoxifen.
Figure 5
Figure 5
Fulvestrant combined with the AKT inhibitor potently suppresses GFRs downstream signaling, induces apoptosis, and accelerates tumor regression. (A and B) MCF7L-shPTEN cells were prepared as before and treated with Ful combined with single or two-agent kinase inhibitors, under -/+Dox (gray/red color) conditions. Cell growth was analyzed the same way as in Figure 4. (C) Cells were treated with Ful combined with single-kinase inhibitors (-/+Dox) for 48 hours and stained with Annexin-V-APC. Flow cytometer analysis was performed to quantify the apoptotic cells with positive staining. (D) MCF7L-shPTEN cells were prepared as before and then treated with Ful alone, or E2 or Ful in combination with mTORi (0.2 μm), AKTi (1 μm), or MEKi (1 μm). Cell lysates were harvested after 48 hours of treatment and immunoblotted with the indicated antibodies. Cells of MCF7L (E), T47D (F), and BT483 (G) -shPTEN models were prepared as before (all + Dox) and then treated with E2, Ful, or each in combination with AKTi (1 μm). Cell growth within five days was normalized to the E2 groups. (H) Kaplan-Meier plots of proportion of tumor burden without response (defined `response' as tumor size halving since randomization) within 60 days of treatment of AKTi alone in the presence of supplemented E2 pellets, or Ful combined with drug carrier (Veh) or AKTi without E2 pellets (n ≥10 in each arm). The AKTi (120 mg/kg) or drug carrier (Veh) was administered twice daily by gavage for both E2 and Ful arms. The Wilcoxon method with adjusted pairwise comparison was applied. All the pairwise comparisons of cell growth were performed by the Bonferroni post hoc test (*P <0.05, **P <0.01, ***P <0.001). AKT, protein kinase B; Dox, doxycycline; E2, β-estradiol; Ful, fulvestrant; GFRs, growth factor receptors; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian _target of rapamycin; PTEN, phosphatase and tensin homolog.
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