Reversal of endocrine resistance in breast cancer: interrelationships among 14-3-3?, FOXM1, and a gene signature associated with mitosis.
ABSTRACT: Despite the benefits of estrogen receptor (ER)-targeted endocrine therapies in breast cancer, many tumors develop resistance. 14-3-3 ?/YWHAZ, a member of the 14-3-3 family of conserved proteins, is over-expressed in several types of cancer, and our previous work showed that high expression of 14-3-3? in ER-positive breast cancers was associated with a poor clinical outcome for women on tamoxifen. Therefore, we now probe the role of 14-3-3? in endocrine resistance, and we examine the functional dimensions and molecular basis that underlie 14-3-3? activities.From analyses of four independent breast cancer microarray datasets from nearly 400 women, we characterized a gene signature that correlated strongly with high expression of 14-3-3? in breast tumors and examined its association with breast cancer molecular subtypes and clinical-pathological features. We investigated the effects of altering 14-3-3? levels in ER-positive, endocrine sensitive and resistant breast cancer cells on the regulation of 14-3-3? signature genes, and on cellular signaling pathways and cell phenotypic properties.The gene signature associated with high 14-3-3? levels in breast tumors encompassed many with functions in mitosis and cytokinesis, including aurora kinase-B, polo-like kinase-1, CDC25B, and BIRC5/survivin. The gene signature correlated with early recurrence and risk of metastasis, and was found predominantly in luminal B breast cancers, the more aggressive ER-positive molecular subtype. The expression of the signature genes was significantly decreased or increased upon reduction or overexpression of 14-3-3? in ER-positive breast cancer cells, indicating their coregulation. 14-3-3? also played a critical role in the regulation of FOXM1, with 14-3-3? acting upstream of FOXM1 to regulate cell division-signature genes. Depletion of 14-3-3? markedly increased apoptosis, reduced proliferation and receptor tyrosine kinase (HER2 and EGFR) signaling, and, importantly, reversed endocrine resistance.This study reveals that 14-3-3? is a key predictive marker for risk of failure on endocrine therapy and serves a pivotal role impacting growth factor signaling, and promoting cell survival and resistance to endocrine therapies. Targeting 14-3-3? and its coregulated proteins, such as FOXM1, should prove valuable in restoring endocrine sensitivity and reducing risk of breast cancer recurrence.
Project description:The forkhead transcription factor FOXM1 coordinates expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We previously showed that FOXM1 acts downstream of 14-3-3? signaling, the elevation of which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high has not been clearly defined yet.We analyzed FOXM1 protein expression by immunohistochemistry in 501 ER-positive breast cancers. We also mapped genome-wide FOXM1, extracellular signal-regulated kinase 2 and ER? binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. These binding profiles were integrated with gene expression data derived from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. We also modulated the levels of FOXM1 and newly discovered FOXM1-regulated genes and examined their impact on the cancer stem-like cell population and on cell invasiveness and resistance to endocrine treatments.FOXM1 protein expression was high in 20% of the tumors, which correlated with significantly reduced survival in these patients (P = 0.003 by logrank Mantel-Cox test). ChIP-seq analyses revealed that FOXM1 binding sites were enriched at the transcription start site of genes involved in cell-cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are associated with a poor prognosis in ER?-positive patients treated with tamoxifen. Integration of binding profiles with gene expression highlighted FOXM1 transcriptional networks controlling cell proliferation, stem cell properties, invasion and metastasis. Increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness.Collectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance. They highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments.
Project description:BACKGROUND:The forkhead transcription factor FOXM1 is a key regulator of the cell cycle. It is frequently over-expressed in cancer and is emerging as an important therapeutic target. In breast cancer FOXM1 expression is linked with estrogen receptor (ER?) activity and resistance to endocrine therapies, with high levels correlated with poor prognosis. However, the precise role of FOXM1 in ER positive breast cancer is not yet fully understood. RESULTS:The study utilizes chromatin immunoprecipitation followed by high-throughput sequencing to map FOXM1 binding in both ER?-positive and -negative breast cancer cell lines. The comparison between binding site distributions in the two cell lines uncovered a previously undescribed relationship between binding of FOXM1 and ER?. Further molecular analyses demonstrated that these two factors can bind simultaneously at genomic sites and furthermore that FOXM1 regulates the transcriptional activity of ER? via interaction with the coactivator CARM1. Inhibition of FOXM1 activity using the natural product thiostrepton revealed down-regulation of a set of FOXM1-regulated genes that are correlated with patient outcome in clinical breast cancer samples. CONCLUSIONS:These findings reveal a novel role for FOXM1 in ER? transcriptional activity in breast cancer and uncover a FOXM1-regulated gene signature associated with ER-positive breast cancer patient prognosis.
Project description:It is unclear if earlier onset (<40 years) and greater proliferative capacity confer an equally poor prognosis to endocrine-dependent and endocrine-independent breast cancers. Available outcome (distant metastasis-free survival, DMFS) and expression microarray data from 621 adjuvant treatment-naïve, node-negative primary breast cancers were pooled for prognostic evaluation of age-at-diagnosis (< 40 years vs. ? 40 years) and tumor proliferative capacity relative to estrogen receptor status (n = 400 ER-positive, n = 221 ER-negative). Transcriptome measures of proliferative capacity included a proliferation score (PS) based on a 61-gene proliferation signature and the single gene surrogate, FOXM1. Kaplan-Meier analyses revealed no significant difference in DMFS between ER-positive and ER-negative cases >5 years after diagnosis. In contrast, younger age and higher proliferative capacity resulted in significantly more metastatic events cumulated over 15 years, but only in ER-positive breast cancers where positive correlations between age and proliferation were observed. While strongly correlated, FOXM1 and PS did not appear equivalent in relation to age and prognosis. The poor prognosis associated with breast cancer arising before age 40 or with higher proliferative capacity pertains only to endocrine-dependent (ER-positive) breast cancer, indicating that different biological processes drive the metastatic potential of ER-negative breast cancer.
Project description:The transcription factor FOXM1 coordinates the expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We have previously shown that FOXM1 acts downstream of 14-3-3ζ signaling, which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering the resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high, has not been clearly defined. To understand the role of FOXM1 in endocrine resistance and cancer aggressiveness, we analyzed FOXM1 protein expression by IHC in 501 ER-positive breast cancers; this revealed high FOXM1 expression in 20% of the tumors and a significantly reduced survival in these patients (p=0.003, log rank Mantel-Cox). We also mapped genome-wide FOXM1 binding events by chromatin immunoprecipitation, followed by high-throughput sequencing (ChIP-seq), in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. FOXM1 binding sites were enriched at the transcription start site of genes involved in cell cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are correlated with the worst prognosis in ERα-positive patients treated with tamoxifen. Binding profiles were also integrated with gene expression data from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. By modulating the levels of FOXM1 and newly discovered FOXM1-regulated genes [in breast cancer cells], we demonstrate that increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness. Collectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance, and highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments. MCF-7 human breast adenocarcinoma cells were tranfected with control, and FOXM1 siRNA for 72 hours and treated with 0.1% EtOH (Vehicle) or 1 uM TOT for 24 hours, and cDNA microarray analyses were carried out using Affymetrix [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array.
Project description:ER? positive breast cancer accounts for 70% of breast malignancies. Compared with ER? negative types, ER? positive breast cancer could be effective controlled by endocrine therapy. However, more than half of the patients will develop endocrine resistance, making it an important clinical issue for breast cancer therapy. Endocrine resistance might be caused by multiple alternations, including the components of ER? signaling, during tumor progression. Thus, it is urgent and necessary to uncover the molecular mechanisms that controls ER? expression and stability to improve breast cancer therapeutics. In our current study, we identifies that the ubiquitin ligase RNF181 stabilizes ER? and facilitates breast cancer progression. The expression of RNF181 is correlated with ER? level in human breast tumors and relates to poor survival in endocrine-treated patients. RNF181 depletion inhibits breast cancer progression in vivo and in vitro, reduces ER? protein level and its target gene expression, such as PS2 and GREB1. Unbiased RNA sequencing analysis indicates RNF181 is necessary for ER? signature gene expression in whole genomic level. Immuno-precipitation assays indicate that RNF181 associates with ER? and promotes its stability possibly via inducing ER? K63-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by RNF181 via stabilizing ER? protein controls ER? target gene expression linked to breast cancer progression.
Project description: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.
Project description:Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer must overcome the loss of this estrogen-induced negative feedback loop that normally constrains the growth of ER+ tumors.
Project description:Quite a few estrogen receptor (ER)-positive breast cancer patients receiving endocrine therapy are at risk of disease recurrence and death. ER-related genes are involved in the progression and chemoresistance of breast cancer. In this study, we identified an ER-related gene signature that can predict the prognosis of ER-positive breast cancer patient receiving endocrine therapy. We collected RNA expression profiling from Gene Expression Omnibus database. An ER-related signature was developed to separate patients into high-risk and low-risk groups. Patients in the low-risk group had significantly better survival than those in the high-risk group. ROC analysis indicated that this signature exhibited good diagnostic efficiency for the 1-, 3- and 5-year disease-relapse events. Moreover, multivariate Cox regression analysis demonstrated that the ER-related signature was an independent risk factor when adjusting for several clinical signatures. The prognostic value of this signature was validated in the validation sets. In addition, a nomogram was built and the calibration plots analysis indicated the good performance of this nomogram. In conclusion, combining with ER status, our results demonstrated that the ER-related prognostic signature is a promising method for predicting the prognosis of ER-positive breast cancer patients receiving endocrine therapy.
Project description:<h4>Background</h4>One of the major tenets in breast cancer research is that early detection is vital for patient survival by increasing treatment options. To that end, we have previously used a novel unsupervised approach to identify a set of genes whose expression predicts prognosis of breast cancer patients. The predictive genes were selected in a well-defined three dimensional (3D) cell culture model of non-malignant human mammary epithelial cell morphogenesis as down-regulated during breast epithelial cell acinar formation and cell cycle arrest. Here we examine the ability of this gene signature (3D-signature) to predict prognosis in three independent breast cancer microarray datasets having 295, 286, and 118 samples, respectively.<h4>Methods and findings</h4>Our results show that the 3D-signature accurately predicts prognosis in three unrelated patient datasets. At 10 years, the probability of positive outcome was 52, 51, and 47 percent in the group with a poor-prognosis signature and 91, 75, and 71 percent in the group with a good-prognosis signature for the three datasets, respectively (Kaplan-Meier survival analysis, p<0.05). Hazard ratios for poor outcome were 5.5 (95% CI 3.0 to 12.2, p<0.0001), 2.4 (95% CI 1.6 to 3.6, p<0.0001) and 1.9 (95% CI 1.1 to 3.2, p = 0.016) and remained significant for the two larger datasets when corrected for estrogen receptor (ER) status. Hence the 3D-signature accurately predicts breast cancer outcome in both ER-positive and ER-negative tumors, though individual genes differed in their prognostic ability in the two subtypes. Genes that were prognostic in ER+ patients are AURKA, CEP55, RRM2, EPHA2, FGFBP1, and VRK1, while genes prognostic in ER- patients include ACTB, FOXM1 and SERPINE2 (Kaplan-Meier p<0.05). Multivariable Cox regression analysis in the largest dataset showed that the 3D-signature was a strong independent factor in predicting breast cancer outcome.<h4>Conclusions</h4>The 3D-signature accurately predicts breast cancer outcome across multiple datasets and holds prognostic value for both ER-positive and ER-negative breast cancer. The signature was selected using a novel biological approach and hence holds promise to represent the key biological processes of breast cancer.
Project description:The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.