Anti-tumor efficacy of BEZ235 is complemented by its anti-angiogenic effects via downregulation of PI3K-mTOR-HIF1alpha signaling in HER2-defined breast cancers

Abstract

Activation of the PI3K-mTOR pathway via HER2: HER3-mediated signaling in HER2+ breast cancers pose one of the major threats towards the success of trastuzumab. First, trastuzumab cannot perturb survival/proliferative signals following HER2: HER3 heterodimerization in HER2+ tumor cells. Second, trastuzumab treatment has been reported to cause drug-mediated resistance in over 50% of HER2+ breast cancers. We have reported that treatment with an anti-angiogenic drug imparted a significant anti-tumor advantage when combined with trastuzumab plus pertuzumab in the trastuzumab-resistant model of HER2+ breast cancers (PMID: 23959459). The very fact as revealed by our study that an inclusion of anti-angiogenic drug conferred a significant anti-tumor advantage when combined with dual anti-HER2 therapy clearly indicated a critical and indispensable role of angiogenesis in these tumors. Hence, we hypothesized that BEZ235 a dual PI3K/mTOR inhibitor will have an effect on the tumor as well as the angiogenic stromal compartments. In vitro and in vivo efficacy of BEZ235 was determined in HER2+ trastuzumab-sensitive, trastuzumab-resistant and HER2 amplified/PIK3CA mutated cell lines. BEZ235 alone and in combination with trastuzumab was tested on the tumor as well as stromal compartments. AKT-mTOR signal was suppressed following BEZ235 treatment in a concentration and time-dependent manner. AnnexinV, cl-CASPASE3, SURVIVIN and p-FOXO1 indicated that BEZ235-induced cell death occurred predominantly via an apoptotic pathway. Heregulin-induced HIF1α synthesis was also significantly decreased. Oncoprint data (cBioPortal) representing PAM50 Her2 enriched tumors (TCGA, Nature 2012) and Her2-positive breast tumors (TCGA, cell 2015) showed 91.4% genetic alterations and 79.2% genetic alterations in a set of four genes comprised of PIK3CA, ERBB2, VEGFA and HIF1alpha. The co-occurrence of HIF1alpha with VEGFA in PAM50 Her2 enriched tumors (TCGA, Nature 2012) and the co-occurrence of HIF1alpha with VEGFA pair as well as HIF1alpha with PIK3CA pair in Her2-positive breast tumors (TCGA, cell 2015) were found statistically significant. In xenograft models, BEZ235 blocked tumor growth and decreased Ki67, CD31, p-AKT, p-S6RP, p-4EBP1 IHC-expressions. These decreases were more pronounced when BEZ235 was combined with trastuzumab in HER2+/trastuzumab-sensitive, trastuzumab-resistant and HER2+/PIK3CA mutated models. We demonstrated that combined targeting of HER2 and the PI3K-AKT-mTOR pathway is superior to HER2-directed therapy alone. Mechanistically the inhibition of tumor-induced angiogenesis by BEZ235 caused by the down-regulation of PI3K-mTOR-HIF1alpha signaling irrespective of the trastuzumab-sensitivity status of HER2+ breast cancers proving evidence for the first time that the inhibition of angiogenesis is an important component of the anti-tumor efficacy of BEZ235 in HER2 defined breast cancers.

Keywords: Breast cancer; HER2+; PIK3CA mutation; angiogenesis; apoptosis; trastuzumab-sensitive and trastuzumab-resistant.

Figure 6

Involvement of PI3K, AKT, and mTOR in signaling from HER2 to HIF1α: (A) Hypoxia-induced HIF1α stabilization is not blocked by PI3K/mTOR dual inhibitor, BEZ235, Nor: normoxia. (B) Effects of kinase inhibitors (BEZ235 or LY294002) or trastuzumab on hypoxia-induced HIF1α stabilization. (C, D) Effects of heregulin-induced HIF1α expression in trastuzumab-sensitive (BT474) (C) and trastuzumab-resistant (BT474HerR) (D) breast tumor cells were abrogated by BEZ235. Lanes 1 & 5 are heregulin stimulation for 6 and 12 hours respectively in (C). Similarly lane 2 is heregulin stimulation for 6 hours in (D). Lanes 2, 3, 6, 7 in (C) and lanes 3, 4 in (D) are preincubated with BEZ235 (indicated concentrations of drug). Lane 4 in (C) and lane 1 in (D) are no stimulation condition. Data suggest that heregulin stimulates HIF1α expression and heregulin-induced HIF1α expression is more in resistance cells. BEZ235 dose-dependently attenuates heregulin-induced HIF1α expression in both these cell lines. (E) Serum-starved cells (BT474 & BT474HerR), was pretreated for 1 hour with vehicle or inhibitor (dual PI3K/mTOR inhibitor, BEZ235 or mTOR1 inhibitor, RAD001) then exposed to no treatment or 10 ng/ml of heregulin for 6 hours prior to HIF1α immunoblot assay of whole cell lysates. (F) Similar experiments were performed with HER2+/PIK3CA mutated cells (HCC1954 & UACC893). Data show that treatment with BEZ235 or RAD001 was associated with decreased HIF1α expression following heregulin stimulation. Furthermore, compare to RAD001, dual PI3K/mTOR inhibitor, BEZ235 was more effective for the inhibition of HIF1α expression/synthesis. (G) Integrin-directed endothelial cell migration is one of the critical steps for tumor-induced angiogenesis. Our data show that BEZ235 inhibits HUVEC cells migration on vitronectin (αvβ3/αvβ5) (lower panel). Bevacizumab was used as a positive control. BEZ235 at 100 nM concentration significantly abrogates endothelial cells tube formation on Matrigel (upper panel). These data indicate that unlike LY294002 (in B lane 3), BEZ235 cannot control hypoxia-mediated HIF1α stabilization; rather a BEZ235 dose-dependently regulates HIF1α expression level following heregulin stimulation. Taken together (expression of HIF1α, endothelial cell migration, and tube formation on Matrigel) our data clearly indicate that BEZ235 effectively controls tumor-induced angiogenesis. (H) Oncoprint data represents samples from two published sets (cBioPortal). The queried samples represent PAM50 HER2 enriched tumors (58 samples; Breast Invasive Carcinoma; TCGA, Nature 2012). A set of four genes comprised of PIK3CA, ERBB2, VEGFA and HIF1alpha was found to be altered in 53 (91.4%) of queried samples. The type of genetic alterations included in both sets is amplification, gain, deep deletion, shallow deletion and a missense mutation. The table of co-occurrent alterations is presented below. Out of four gene pairs with co-occurrent alterations in PAM50 Her2 enriched tumors (58 samples), TCGA, Nature 2012, the co-occurrence between HIF1alpha and VEGF was found statistically significant. p value was derived from Fisher Exact Test. Log Odds Ratio quantifies how strongly the presence or absence of alterations in gene A are associated with the presence or absence of alterations in gene B in the selected tumors, PAM50 Her2 enriched tumors (58 samples; Breast Invasive Carcinoma; TCGA, Nature 2012). Log Odds Ratio>0: Association towards co-occurrence p-Value<0.05: Significant association. The table was generated using cBioPortal. cBioPortal data is subjected to scheduled updates. (I) The second queried samples represent HER2-positive breast tumors (120 samples; Breast Invasive Carcinoma; TCGA, cell 2015). A set of four genes comprised of PIK3CA, ERBB2, VEGFA and HIF1alpha was found to be altered in 95 (79.2%) of queried samples. The type of genetic alterations included in both sets is amplification, gain, deep deletion, shallow deletion and a missense mutation. Oncoprint data represents samples from two published sets (cBioPortal). The table of co-occurrent alterations is presented below. Out of six gene pairs with co-occurrent alterations, of the HER2-positive breast tumors (120 samples) TCGA, Cell 2015 the co-occurrence between HIF1alpha and VEGF pair and HIF1alpha and PIK3CA pair were found statistically significant. p value was derived from Fisher Exact Test. Log Odds Ratio quantifies how strongly the presence or absence of alterations in gene A are associated with the presence or absence of alterations in gene B in the selected tumors, Her2-positive breast tumors (120 samples; Breast Invasive Carcinoma; TCGA, cell 2015). Log Odds Ratio>0: Association towards co-occurrence p-Value<0.05: Significant association. The table was generated using cBioPortal. cBioPortal data is subjected to scheduled updates.

Figure 1

Effect of BEZ235 and BEZ235 plus trastuzumab on proliferation assays: BT474, BT474HR, UACC893 and HCC1954 cells exhibit differential susceptibility to trastuzumab but are nearly equally sensitive to BEZ235. HCC1954 (HER2+, PIK3CAmutant [H1047R]) cell was particularly sensitive to this effect. Dose-response curves were obtained by MTT assay (A) after 48 hrs exposures of these four cell lines to BEZ235 (left panel) and the combination of Trastuzumab (10 µg/ml fixed dose) plus different doses of BEZ235 (right panel). Data clearly show that BEZ235 is highly effective in all four cell lines irrespective of PIK3CA mutation or not. (B) Representative clonogenic survival of HER2+/trastuzumab-sensitive (BT474) and HER2+/trastuzumab-resistant (BT474HerR) breast tumor cells were grown in the presence of trastuzumab (10 µg/ml) or BEZ235 (50 nM) or trastuzumab plus BEZ235. BT474 and BT474HerR cell lines were incubated with BEZ235, trastuzumab or a combination of trastuzumab plus BEZ235 for 48 hours and subsequently allowed to grow into colonies for both 96 hours and 7 days. Data clearly show as a single agent BEZ235 significantly inhibits cells growth as indicated by 2D and 3D assays at both 96 hours and 7 days. a: BT474, 2D assay at 96 hours, b: BT474, 2D assay at 7 days, c: BT474, 3D-ON-TOP clonogenic assay at 96 hours, d: BT474, 3D-ON-TOP clonogenic assay at 7 days, e: BT474HerR, 2D assay at 96 hours, f: BT474HerR, 2D assay at 7 days, g: BT474HerR, 3D-ON-TOP clonogenic assay at 96 hours, h: BT474HerR, 3D-ON-TOP clonogenic assay at 7 days. More importantly, the additive effect of the BEZ235 plus trastuzumab combination achieves a more powerful inhibition of clonogenic growth (both 2D and 3D-ON-TOP assays) than either agent in isolation.

Figure 2

Effect of BEZ235 and comparison on the effects of BEZ235 and RAD001 in HER2+ breast cancer cells: Western blot showing BEZ235 time/dose-dependently blocked activation of AKT and its downstream effectors in total lysates from HER2+ [trastuzumab-sensitive BT474 (A) & SKBR3 (B)], trastuzumab-resistant [BT474HerR (C)] breast cancer cells. Moreover, time course experiments revealed that long-term exposure to low concentration of BEZ235 resulted in an increase in p-AKT levels. Such an effect on p-AKT was completely abolished in cells incubated at higher concentrations (100 and 200 nM) of the inhibitor (see lane 8, 9 and 11, 12 in all three figures (A-C), BEZ235. Differential effects of BEZ235 versus RAD001 on intracellular signaling: BT474 (D), BT474HerRR (E), HER2+/PIK3CA mutated HCC1954 (F) and UACC893 (G) cells were treated with BEZ235 (50 nM) or RAD001 (1 and 2 nM) for different time periods (1 hr, 6 hrs, 24 hrs, and 48 hrs) and then lysed and analyzed by Western blots. Data show that both drugs completely blocked p-P70S6K, p-S6RP but only BEZ235 consistently blocked phosphorylation of AKT (Ser 474 and Thr 308) and 4EBP1. As expected RAD001-induced AKT activation was observed. Data also showed that a combination of BEZ235 plus RAD001 at sub-optimal concentration might have an additive effect on the PI3K-AKT-mTOR pathway molecules (lane 5, 9, 13 & 17 in D, E). The effect of BEZ235 was independent of the mutation profile of the cell cultures used. (H) PI3K pathway inhibition and ERK phosphorylation in HER2+ or HER2+/PIK3CA mutated breast cancer cells: Immunoblots of BT474, BT474HR, HCC1954 and UACC893 for indicated time points with different PI3K pathway inhibitors (dual PI3K/mTOR inhibitor, BEZ235 or mTOR1 inhibitor, RAD001) at the indicated concentration (nM). Our data show that mTOR1 inhibition induces ERK phosphorylation in all 4 cell lines. Importantly, BEZ235 at 50 nM concentration blocked phosphorylation ERK except UACC 893 cell lines where phosphorylation of ERK is upregulated following the treatment of BEZ235 (for the indicated time points). It has been known that level of HER3 is high in UACC cell line and it is resistant against trastuzumab and lapatinib (Slamon DJ 2010 Mol Can Ther 9: 1489).

Figure 3

(A) BEZ235 interrupts heregulin-induced PI3K-AKT signaling: Under the basal condition in serum free medium (overnight serum starved, NS), the PI3K signaling was assessed by the p-AKT level. On ligand (heregulin) stimulation p-AKT was upregulated in both sensitive (BT474) and trastuzumab-resistant (BT474HerR) cell lines (lane 2 and 9). The activity of AKT in both the cell lines was significantly inhibited by BEZ235 at 25 and 50 nM concentration (lane 5, 6 for sensitive cell line and lane 12, 13 in resistant cell line), suggesting its effectiveness on blocking the PI3K-AKT signal pathway in HER2+ breast cancer cells. Moreover, this inhibition is more when trastuzumab (10 µg/ml) was treated along with BEZ235 (25 nM). BEZ235 has no effect on AKT activation at 10 nM concentration (lane 4 and 11). Expression of the corresponding total target protein (AKT) and β-ACTIN was used as loading control. (B) Combination of BEZ235 along with trastuzumab more efficiently blocked the PI3K-AKT-mTOR pathway: Western blot showing combination of BEZ235 as low as 25 nM concentration plus trastuzumab at 10 µg/ml is significantly blocked AKT (both Ser473 and Thr308), and its downstream mTOR and P70S6 Kinase activations compare to either BEZ235 or trastuzumab alone (lane 5 in BT474, BT474HerR (a), SKBR3 (b) and lane 8 & 9 in HCC1954 cells, see Figure 2F). Additionally, this combination is more effective than the combination of RAD001 and trastuzumab in terms of pathway inactivation (compare lane 8 & 9 with lane 10 & 11, Figure 2F). Data show that combination is more effective compared to either drug alone and, more importantly, the combination is also effective against trastuzumab-resistant and PIK3CA mutated cell line.

Figure 4

Effect of BEZ235 on cell cycle progression, apoptosis in HER2 amplified trastuzumab-sensitive, trastuzumab-resistant and PIK3CA mutated breast cancer cells: Effect of three doses of BEZ 235 (100, 200 and 500 nM) on cell cycle distribution (A) in BT474, BT474HerR and HCC1954. Cells were treated as indicated for 24 hours. Cells were released and ethanol fixed before staining with propidium iodide for analysis by flow cytometry. (B) Effect of BEZ235 (100, 200 and 500 nM) on apoptotic (early) response analyzed by annexin V/7AAD staining in BT474, BT474HerR and HCC1954. Cells were treated for 48 hours, released, rinsed, and placed in the annexin V binding buffer. Cells were labeled with annexin V-PE and 7AAD for analysis. Error bars represent SEM from triplicates. Interestingly, RAD001 has a very insignificant response to the early phase of apoptosis in HER2+ cells when compared with BEZ235. (C) BEZ235 induces apoptosis through induction of cleaved CASPASE3 and inhibition of the expression of SURVIVIN: BT474 (a), BT474HerR (b) and HCC1954 (c) cell lines were incubated for different time periods (6, 24, 48 and 72 hours) with the indicated amount of BEZ235. Data showed that cleaved CASPASE3 expression was more at higher concentration of BEZ235. Furthermore, the expression was more in trastuzumab-resistant cells (lane 6 and 8 in Cb) might be due to addiction of the PI3K-AKT-mTOR pathway in resistant cells. Data also showed that SURVIVIN expression was more in resistant cells and SURVIVIN expression was significantly inhibited following the treatment of BEZ235 in trastuzumab-sensitive (a), trastuzumab-resistant (b) and PIK3CA mutant (c) cells. (Cd) Levels of p-AKT(Ser473) expression following the treatment of BEZ235 was reciprocally correlated (both time and dose) with the expression levels of cleaved CASPASE3. Data suggest that BEZ235 increases apoptosis through the AKT-SURVIVIN-CASPASE3 pathway. (Da) Effects of BEZ235 on the expression of p-FOXO1 and its target gene: BT474 and BT474HerR cells were treated with BEZ235 (100 nM) for 24, 48 and 72 hrs. Cells were harvested to measure the expression of phosphorylation (Ser256) of FOXO1 and one of its transcriptional target genes, p27. Data suggest BEZ235 induced dephosphorylation of FOXO1, leads to upregulation of p27 which is the transcriptional target gene of FOXO1 and upregulated p27 may be (one of the key factors) responsible for induction of cell cycle arrest. (Db) FOXO1 plays a pivotal role in tumor growth suppression by inducing growth arrest and apoptosis: Loss of function of FOXO1 protein due to phosphorylation and proteasomal degradation has been implicated in cell transformation and malignancy. It has been known that SKP2 interacts with ubiquitinates, and promotes the degradation of FOXO1. This effect of SKP2 requires AKT-specific phosphorylation of FOXO1 and translocation of phosphorylated FOXO1 (inactive form) from nucleus to cytosol. Activation of FOXO1 by upstream inhibition of the PI3K-AKT pathway, results in upregulation of p27kip, cyclin-dependent kinase inhibitor and downregulation of cyclin D, thereby increasing apoptosis (please see the cartoon).

Figure 5

Blocking of the PI3K-AKT pathway inhibited HER2+ cell migration and decreased RAC1 activation: A. Wound healing assay. A confluent monolayer of cells (HER2+/PIK3CA mutated, HCC1954 and HER2+/PTEN inactivation mutation, HCC1569) was scratched using a sterile pipette tip. At 24 hours after treatment, migrated cells into the scratched area were photomicrographed and measured the scratch area covered by the migrated cells. Transwell assay was carried out with HCC1954 cells following the treatment of BEZ235 (100 nM) or RAD001 (5 nM) (bottom panel). Photomicrograph and counting of cells that migrate through the filter to the fibronectin coated surface after crystal violet staining were showed in the bar diagram (*<0.001, n=3). BEZ235-mediated abrogation of integrin (α4β1/α5β1) -induced HER2+ cell migration was significantly well-defined than an mTOR1 inhibitor, RAD001 in both cells and in both assay types. B. Integrin (α4β1/α5β1)-induced RAC1 activation was significantly attenuated following the treatment of dual PI3K/mTOR inhibitor, BEZ235 but not by RAD001. The block of RAC1 activation has been correlated with an observed reduction in cell migration.

Figure 7

Efficacy of BEZ235 alone and in combination with trastuzumab in HER2+/trastuzumab-sensitive, BT474 (A), HER2+/trastuzumab-resistant, BT474HerR (B) and HER2+/PIK3CA mutated, HCC1954 (C) human tumor xenograft models: A pilot study was conducted with HCC1954 cells to determine 1) the number of cells required to inject for the establishment of tumors and their maintenance in animals throughout the period of drug administration and 2) the maximum tolerable dose of drugs in animals with tumor burden. The number of cells injected was adjusted on the basis of the tolerable tumor burden in untreated animals (following IACUC guidelines). On the basis of the results of the pilot study, cells were injected in matrigel subcutaneously into the flank of immunocompromised female nude (nu/nu) mice. Established xenograft tumors were treated with BEZ235 (45 mg/kg, oral, every other day) alone and in combination with trastuzumab (10 mg/kg i.p., twice weekly for 3 weeks). The table [(lower panel of (Aa), (Ba) & (Ca)] shows the change in volumes of xenograft tumors and body weights of the mice in response to drug combinations. PD data of BT474 tumor (Ab), BT474HerR (Bb) and HCC1954 (Cb) for cell proliferation marker (Ki67), tumor-induced angiogenic markers (CD31 & pVEGFR and cell signaling markers (pAKT & pS6RP) were presented. (D) A dual PI3K/mTOR inhibitor (BEZ235) plus trastuzumab showed in vivo efficacy in mouse xenograft models. Quantification of the mean changes (relative values) in tumor volume of xenografts after 3 weeks treatment, negative values indicate tumor regression.

Figure 8

Schematic representation of the central theme of our study: PI3K-mTOR dual inhibitor BEZ235 blocks the transcriptional activity of HIF1alpha by decreasing its synthesis which in turn downregulates tumor-associated stromal endothelial cells in a paracrine manner and thereby inhibits tumor-angiogenesis.