ESR1 F404 Mutations and Acquired Resistance to Fulvestrant in ESR1-Mutant Breast Cancer.

Fulvestrant is used to treat patients with hormone receptor-positive advanced breast cancer, but acquired resistance is poorly understood. PlasmaMATCH Cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Baseline ESR1 mutations Y537S are associated with poor outcomes and Y537C with good outcomes. Sequencing of baseline and EOT ctDNA samples (n = 69) revealed 3/69 (4%) patients acquired novel ESR1 F404 mutations (F404L, F404I, and F404V), in cis with activating mutations. In silico modeling revealed that ESR1 F404 contributes to fulvestrant binding to estrogen receptor-alpha (ERα) through a pi-stacking bond, with mutations disrupting this bond. In vitro analysis demonstrated that single F404L, E380Q, and D538G models were less sensitive to fulvestrant, whereas compound mutations D538G + F404L and E380Q + F404L were resistant. Several oral ERα degraders were active against compound mutant models. We have identified a resistance mechanism specific to fulvestrant that can be targeted by treatments in clinical development. SIGNIFICANCE: Novel F404 ESR1 mutations may be acquired to cause overt resistance to fulvestrant when combined with preexisting activating ESR1 mutations. Novel combinations of mutations in the ER ligand binding domain may cause drug-specific resistance, emphasizing the potential of similar drug-specific mutations to impact the efficacy of oral ER degraders in development. This article is featured in Selected Articles from This Issue, p. 201.

Olaparib and Ceralasertib (AZD6738) in Patients with Triple-Negative Advanced Breast Cancer: Results from Cohort E of the plasmaMATCH Trial (CRUK/15/010).

PURPOSE: Approximately 10% to 15% of triple-negative breast cancers (TNBC) have deleterious mutations in BRCA1 and BRCA2 and may benefit from PARP inhibitor treatment. PARP inhibitors may also increase exogenous replication stress and thereby increase sensitivity to inhibitors of ataxia telangiectasia and Rad3-related (ATR) protein. This phase II study examined the activity of the combination of PARP inhibitor, olaparib, and ATR inhibitor, ceralasertib (AZD6738), in patients with advanced TNBC. PATIENTS AND METHODS: Patients with TNBC on most recent biopsy who had received 1 or 2 lines of chemotherapy for advanced disease or had relapsed within 12 months of (neo)adjuvant chemotherapy were eligible. Treatment was olaparib 300 mg twice a day continuously and celarasertib 160 mg on days 1-7 on a 28-day cycle until disease progression. The primary endpoint was confirmed objective response rate (ORR). Tissue and plasma biomarker analyses were preplanned to identify predictors of response. RESULTS: 70 evaluable patients were enrolled. Germline BRCA1/2 mutations were present in 10 (14%) patients and 3 (4%) patients had somatic BRCA mutations. The confirmed ORR was 12/70; 17.1% (95% confidence interval, 10.4-25.5). Responses were observed in patients without germline or somatic BRCA1/2 mutations, including patients with mutations in other homologous recombination repair genes and tumors with functional homologous recombination deficiency by RAD51 foci. CONCLUSIONS: The response rate to olaparib and ceralasertib did not meet prespecified criteria for activity in the overall evaluable population, but responses were observed in patients who would not be expected to respond to olaparib monotherapy.

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant in PIK3CA-Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.

Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer.

Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors.

Lucitanib for the Treatment of HR+/HER2- Metastatic Breast Cancer: Results from the Multicohort Phase II FINESSE Study.

PURPOSE: The FGFR1 gene is amplified in 14% of patients with HR + /HER2 - breast cancer. Efficacy and safety of lucitanib, an inhibitor of VEGFR1-3, FGFR1-3, and PDGFRα/ß, were assessed. PATIENTS AND METHODS: Patients with HR + /HER2 - metastatic breast cancer (MBC) received oral lucitanib in three centrally confirmed cohorts: (i) FGFR1 amplified, (ii) FGFR1 nonamplified, 11q13 amplified, and (iii) FGFR1 and 11q13 nonamplified. Key inclusion criteria included Eastern Cooperative Oncology Group Performance Status ≤2, ≥1 line of anticancer therapy, but ≤2 lines of chemotherapy. Primary endpoint was overall response rates (ORR) by RECIST1.1. Simon's two-stage design was used: If ≥2 patients responded among 21 patients, 20 additional patients could be enrolled in each cohort. FGFR1 copy-number variation (CNV) was determined by FISH and droplet digital PCR, whereas FGFR1 expression was determined by IHC. RESULTS: Seventy-six patients (32/18/26 in cohorts 1/2/3) from nine countries were enrolled. The prespecified primary endpoint was met in cohort 1 with ORR of 19% [95% confidence interval (CI), 9%-35%], but not in cohorts 2 and 3 with ORR of 0% (95% CI, 0%-18%) and 15% (95% CI, 6%-34%), respectively. Frequent adverse events included hypertension (87%), hypothyroidism (45%), nausea (33%), and proteinuria (32%). Exploratory biomarker analyses suggested higher ORR in patients with high FGFR1 amplification (≥4 CNV) than those without high amplification (22% vs. 9%). ORR in patients with FGFR1-high tumors (IHC, H-score ≥50) was 25% versus 8% in FGFR1-low cancers. CONCLUSIONS: Lucitanib had modest antitumor activity and significant hypertension-related toxicity in patients with HR + /HER2 - MBC. Although based on small sample sizes, exploratory biomarker analyses suggested that patients with high FGFR1 amplification or expression might derive greater benefit.

Inactivating NF1 Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

ATR Inhibition Potentiates the Radiation-induced Inflammatory Tumor Microenvironment.

PURPOSE: ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment.Experimental Design: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy. RESULTS: Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT. CONCLUSIONS: We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid-sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity.

Molecular characterisation of aromatase inhibitor-resistant advanced breast cancer: the phenotypic effect of ESR1 mutations.

BACKGROUND: Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. MATERIALS AND METHODS: The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. RESULTS: Eighty-nine mutations were identified in 41 matched pairs (PIK3CA in 27%; CDH1 in 20%). ESR1 (n = 5), ERBB2 (n = 1) and MAP2K4 (n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1-mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1-mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. CONCLUSIONS: Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours.

FFPE breast tumour blocks provide reliable sources of both germline and malignant DNA for investigation of genetic determinants of individual tumour responses to treatment.

BACKGROUND: Bio-banked formalin-fixed paraffin-embedded (FFPE) tissues provide an excellent opportunity for translational genomic research. Historically matched blood has not always been collected as a source of germline DNA. This project aimed to establish if normal FFPE breast tissue could be used as an alternative to blood. METHODS: Exome sequencing was carried out on matched tumour tissue, normal breast tissue and blood on five patients in the START trial. Retrieved samples had been archived at different centres for at least 13 years. Following tissue macro-dissection and DNA extraction, targeted exome capture was performed using SureSelect Human All Exome v5 reagents (Agilent). Illumina paired-end libraries were prepared from the captured target regions and sequenced on a HiSeq2500 (Illumina) acquiring 2 × 75 bp reads. Somatic variants were called using the MuTect software analysis tool and copy number abnormalities (CNA) were identified using CNVkit. Targeted sequencing and droplet digital PCR were used to validate somatic variants and CNA, respectively. RESULTS: Overlap of somatic variants and CNA called on tumour versus blood and tumour versus normal breast tissue was good. Agreement in somatic variant calling ranged from 76.9 to 93.6%. Variants with an allele frequency lower than 10% were more difficult to validate irrespective of the type of germline DNA used. Pearson's correlation coefficients for paired comparisons of CNA using blood or normal tissue as reference ranged from 0.70 to 0.94. CONCLUSIONS: There is good correlation between the somatic mutations and CNA called using archived blood or normal breast tissue as germline reference material.

Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer.

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity.Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging.Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes.Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561-72. ©2017 AACR.

EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells.

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH+ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH+ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH+ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH+ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.

High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial.

UNLABELLED: FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. SIGNIFICANCE: Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838-51. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer.

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high-sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor-positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.

Parallel RNA interference screens identify EGFR activation as an escape mechanism in FGFR3-mutant cancer.

UNLABELLED: Activation of fibroblast growth factor receptors (FGFR) is a common oncogenic event. Little is known about the determinants of sensitivity to FGFR inhibition and how these may vary between different oncogenic FGFRs. Using parallel RNA interference (RNAi) genetic screens, we show that the EGF receptor (EGFR) limits sensitivity to FGFR inhibition in FGFR3-mutant and -translocated cell lines, but not in other FGFR-driven cell lines. We also identify two distinct mechanisms through which EGFR limits sensitivity. In partially FGFR3-dependent lines, inhibition of FGFR3 results in transient downregulation of mitogen-activated protein kinase signaling that is rescued by rapid upregulation of EGFR signaling. In cell lines that are intrinsically resistant to FGFR inhibition, EGFR dominates signaling via repression of FGFR3, with EGFR inhibition rescued by delayed upregulation of FGFR3 expression. Importantly, combinations of FGFR and EGFR inhibitors overcome these resistance mechanisms in vitro and in vivo. Our results illustrate the power of parallel RNAi screens in identifying common resistance mechanisms to targeted therapies. SIGNIFICANCE: Our data identify a novel therapeutic approach to the treatment of FGFR3-mutant cancer, emphasizing the potential of combination approaches targeting both FGFR3 and EGFR. Our data extend the role of EGFR in mediating resistance to inhibitors targeting a mutant oncogene, showing that EGFR signaling can repress mutant FGFR3 to induce intrinsic resistance to FGFR targeting.

FGFR signaling promotes the growth of triple-negative and basal-like breast cancer cell lines both in vitro and in vivo.

PURPOSE: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer. EXPERIMENTAL DESIGN: We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity. RESULTS: TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K-AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo. CONCLUSIONS: Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers.

FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer.

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in approximately 10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase-AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B-type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance.

Characterization of cis-regulatory elements conferring mercury-induced interleukin-4 gene expression in rat mast cells: a role for signal transducer and activator of transcription 6 and TATA box binding sites.

We have previously shown that oxidative stress is involved in the pathogenesis of a mercuric chloride (HgCl(2))-induced, T helper type 2 (Th2)-driven autoimmune syndrome in Brown Norway (BN) rats. In the context of the syndrome, the oxidative stress-induced mast cell response seems to determine the development of the early phase of vasculitis, while oxidative stress-mediated interleukin (IL)-4 production may contribute to the subsequent Th2-driven autoimmune response. However, the molecular basis of IL-4 gene transcription induced by HgCl(2) in mast cells remains unknown. In the present study, we dissect the critical regulatory mechanisms in the IL-4 gene promoter in the rat mast cell line RBL-2H3. Immunoprecipitation provided evidence that treatment with HgCl(2) increased phosphorylation of signal transducer and activator of transcription 6 (STAT6). Transient transfection reporter analyses with a series of 5' end deletions of the IL-4 promoter produced evidence that STAT6 and TATA box binding sites are important in HgCl(2)-induced IL-4 gene expression. Subsequent elimination of one or both sites by site-directed mutagenesis significantly inhibited IL-4 promoter activity. Our results provide evidence that STAT6 and TATA box regulatory elements play an important role in HgCl(2)-induced IL-4 transcription in rat mast cells.

Albumin induces interleukin-6 release from primary human proximal tubule epithelial cells.

BACKGROUND: Proximal tubule epithelial cells (PTECs) release proinflammatory and profibrogenic mediators when exposed to serum albumin that may contribute to progression of kidney disease. Interleukin 6 (IL-6) may influence renal fibrosis by modulating transforming growth factor beta1 (TGFbeta1) signalling. PTECs have been demonstrated to produce IL-6 in response to albumin treatment, but the mechanism has not been investigated. We hypothesized that albumin would induce release of IL-6 from PTECs, which would be sensitive to inhibition of PI3K, ERK1,2, p38 MAPK and NFkB. METHODS: Primary human PTECs were exposed to albumin (0.75-150 micronM) for 8 and 24 hours. IL-6 release was determined using enzyme-linked immunosorbent assay (ELISA). The effects of LY294002 (10 micronM), NH4Cl (10 mM), pyrrolidine dithiocarbamate (PDTC) (20 micronM), CAPE (17.5 micronM), PD098059 (20 micronM), SB202190 (5 micronM) and MG132 (10 micronM) on albumin-mediated IL-6 release were studied. RESULTS: Albumin caused a significant time- and concentration-dependent increase in IL-6 release by PTECs. LY294002, NH4Cl, CAPE, PD098059 and SB202190 all reduced albumin-mediated IL-6 release, but neither PDTC nor MG132 had any effect. CONCLUSIONS: These data demonstrate that albumin induces IL-6 release by primary human PTECs, and support a role for endocytosis, p38 MAPK, ERK1,2 and in this process.