Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation.

INTRODUCTION: Estrogen receptor α-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of insulin-like growth factor (IGF)-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation. METHODS: Inhibition of AKT using the catalytic inhibitor AZD5363 was examined in four ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the fibroblast growth factor receptor (FGFR) inhibitor AZD4547. RESULTS: Treatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3α/ß and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of three of four ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1 and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERα mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting that receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation. CONCLUSIONS: These data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer.

Novel actions of estrogen to promote proliferation: integration of cytoplasmic and nuclear pathways.

Both steroids and growth factors stimulate proliferation of steroid-dependent tumor cells, and interaction between these signaling pathways occurs at several levels. Steroid receptors are classified as ligand-activated transcription factors, and steps by which they activate target gene transcription are well understood. Several steroid responses have now been functionally linked to other intracellular signaling pathways, including c-Src or tyrosine kinase receptors. Steroids such as 17beta-estradiol (E2), via binding to cytoplasmic or membrane-associated receptors, were also shown to rapidly activate intracellular signaling cascades such as ERK, PI3K and STATs. These E2-stimulated phosphorylations can then contribute to altered tumor cell function. ER-positive breast cancer cells, in which proliferation is stimulated by E2 and suppressed by antiestrogens, have been of particular interest in dissecting nuclear and cytoplasmic roles of estrogen receptors (ER). In some cell contexts, ER interacts directly with the intracellular tyrosine kinase c-Src and other cytoplasmic signaling and adaptor molecules, such as Shc, PI3K, MNAR, and p130 Cas. Although the hierarchy among these associations is not known, it is clear that c-Src plays a fundamental role in both growth factor and E2-stimulated cell growth, and this may also require other growth factor receptors such as those for EGF or IGF-1. STAT transcription factors represent one pathway to integrate E2 cytoplasmic and nuclear signaling. STAT5 is phosphorylated in the cytoplasm at an activating tyrosine in response to E2 or EGF, and then is translocated to the nucleus to stimulate target gene transcription. E2 stimulates recruitment of STAT5 and ER to the promoter of several proliferative genes, and STAT5 knockdown prevents recruitment of either protein to these promoters. STAT5 activation by E2 in breast cancer cells requires c-Src and EGF receptor, and inhibition of c-Src or EGFR, or knockdown of STAT5, prevents E2 stimulation of several genes and breast cancer cell proliferation. Hyperactivation of the growth factor receptor-c-Src pathway can in some contexts decrease growth responses to E2, or render cells and tumors resistant to suppressive actions of endocrine therapies. Crosstalk between growth factors and steroids in both the cytoplasm and nucleus may thus have a profound impact on complex biological processes such as cell growth, and may play a significant role in the treatment of steroid-dependent breast cancers.

Signal transducer and activator of transcription 5b, c-Src, and epidermal growth factor receptor signaling play integral roles in estrogen-stimulated proliferation of estrogen receptor-positive breast cancer cells.

17beta-Estradiol (E2) acts through the estrogen receptor alpha (ERalpha) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERalpha and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.

ERbeta in breast cancer--onlooker, passive player, or active protector?

The role of estrogen exposure in breast cancer risk is well-documented, and both estrogen synthesis and actions through the estrogen receptor (ER) have been targeted by therapies to control hormone-dependent breast cancer. The discovery of a second ER form and its therapeutic implications sparked great interest. Both the original ERalpha and the more recently identified ERbeta subtypes bind and respond similarly to many physiological and pharmacological ligands. However, differences in phytoestrogen binding have been noted, and subtype-specific ligands have been developed. Cell-based assays show that ERbeta and its variants are generally less active on gene transcription than ERalpha, and may influence ERalpha activity; however, both gene- and cell-specific responses occur, and nongenomic activities are less well explored. Specific ligands, and methods to disrupt or eliminate receptor subtype expression in animal and cell models, demonstrate that the ERs have both overlapping and distinct biological functions. Overall, in cell-based studies, ERalpha appears to play a predominant role in cell proliferation, and ERbeta is suggested to be antiproliferative. The potential for distinct populations of breast tumors to be identified based on ER subtype expression, and to exhibit distinct clinical behaviors, is of greatest interest. Several studies suggest that the majority of ER-positive tumors contain both subtypes, but that some tumors contain only ERbeta and may have distinct clinical behaviors and responses. Expression of ERbeta together with ERalpha favors positive responses to endocrine therapy in most studies, and additional studies to determine if the addition of ERbeta to ERalpha as a tumor marker is of clinical benefit are warranted. In contrast, the positive association between ERbeta and HER2 expression in high-grade ERalpha-negative breast cancer does not favor positive responses to endocrine therapy. Expression of ERbeta in specific clinical subpopulations, and the potential for therapies targeting ERbeta specifically, is discussed.

Estrogen negatively regulates epidermal growth factor (EGF)-mediated signal transducer and activator of transcription 5 signaling in human EGF family receptor-overexpressing breast cancer cells.

Breast cancer cell growth may be stimulated by 17beta-estradiol (E2) or growth factors like epidermal growth factor (EGF). However, tumors typically depend on only one of these pathways and may overexpress either estrogen receptor (ER) or EGF receptor (EGFR) and related family members. Tumors overexpressing EGFR are more aggressive than those expressing ER. Intracellular mediators of these growth-stimulatory pathways are not completely defined, but one potential common mediator of EGF and E2 signaling is the transcription factor signal transducer and activator of transcription 5 (STAT5). To investigate the role of STAT5 in potential crosstalk between E2 and EGF, MDA-MB231 and SKBr3 breast cancer cells, which are ER-negative and overexpress human EGF family receptors, were used. Introduction of ERalpha and treatment with E2 decreased EGF-induced tyrosine phosphorylation of STAT5b, basal and EGF-induced STAT5-mediated transcription, and EGF-stimulated DNA synthesis in these cells. Suppressive effects of E2-EpsilonRalpha were specific for STAT5, as EGF stimulation of MAPK was unaffected. Deletion/mutation analysis of ERalpha demonstrated that the DNA-binding domain was insufficient, and that the ligand-binding domain was required for these responses. ERalpha transcriptional activity was not necessary for suppression of STAT5 activity. Overexpression of c-Src did not prevent suppression of STAT5 activity by E2 and ERalpha. However, ERalpha did prevent basal increases in STAT5 activity with overexpressed c-Src. In the context of human EGF receptor family overexpression, E2-ER opposes EGF signaling by regulating STAT5 activity. STAT5 may be a crucial point of signaling for both E2 and growth factors in breast cancer cells, allowing targeted therapy for many types of breast tumors.

LYN-activating mutations mediate antiestrogen resistance in estrogen receptor-positive breast cancer.

Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.

Abrogating endocrine resistance by targeting ERα and PI3K in breast cancer.

Antiestrogen therapies targeting estrogen receptor α (ER) signaling are a mainstay for patients with ER+ breast cancer. While many cancers exhibit resistance to antiestrogen therapies, a large body of clinical and experimental evidence indicates that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway promotes antiestrogen resistance. In addition, continued ligand-independent ER signaling in the setting of estrogen deprivation may contribute to resistance to endocrine therapy. PI3K activates several proteins which promote cell cycle progression and survival. In ER+ breast cancer cells, PI3K promotes ligand-dependent and -independent ER transcriptional activity. Models of antiestrogen-resistant breast cancer often remain sensitive to estrogen stimulation and PI3K inhibition, suggesting that clinical trials with combinations of drugs targeting both the PI3K and ER pathways are warranted. Herein, we review recent findings on the roles of PI3K and ER in antiestrogen resistance, and clinical trials testing drug combinations which target both pathways. We also discuss the need for clinical investigation of ER downregulators in combination with PI3K inhibitors.

A kinome-wide screen identifies the insulin/IGF-I receptor pathway as a mechanism of escape from hormone dependence in breast cancer.

Estrogen receptor α (ER)-positive breast cancers adapt to hormone deprivation and become resistant to antiestrogens. In this study, we sought to identify kinases essential for growth of ER(+) breast cancer cells resistant to long-term estrogen deprivation (LTED). A kinome-wide siRNA screen showed that the insulin receptor (InsR) is required for growth of MCF-7/LTED cells. Knockdown of InsR and/or insulin-like growth factor-I receptor (IGF-IR) inhibited growth of 3 of 4 LTED cell lines. Inhibition of InsR and IGF-IR with the dual tyrosine kinase inhibitor OSI-906 prevented the emergence of hormone-independent cells and tumors in vivo, inhibited parental and LTED cell growth and PI3K/AKT signaling, and suppressed growth of established MCF-7 xenografts in ovariectomized mice, whereas treatment with the neutralizing IGF-IR monoclonal antibody MAB391 was ineffective. Combined treatment with OSI-906 and the ER downregulator fulvestrant more effectively suppressed hormone-independent tumor growth than either drug alone. Finally, an insulin/IGF-I gene expression signature predicted recurrence-free survival in patients with ER(+) breast cancer treated with the antiestrogen tamoxifen. We conclude that therapeutic targeting of both InsR and IGF-IR should be more effective than targeting IGF-IR alone in abrogating resistance to endocrine therapy in breast cancer.

ERα-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer.

Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER(+) xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.

Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer.

Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor-positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17beta-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways.

Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin.

A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.