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Purpose: The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2 + breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER + /HER2 + tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib. Experimental Design: Mice with established ER + /HER2 + MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses., Results: Mice receiving "extended adjuvant" therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER + /HER2 + cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER + /HER2 - MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER + /HER2 + breast cancer cells., Conclusions: These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER + /HER2 + breast cancer., (©2018 American Association for Cancer Research.)

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor-positive (ER + ) breast cancers treated with prolonged neoadjuvant letrozole. Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER + breast cancers from patients treated with preoperative letrozole (median, 7 months). Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes ( P = 2.56E-15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER + breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER + tumors in METABRIC. Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER + breast cancer cells and in patients' ER + tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER + breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517-29. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: FGFR1 amplification occurs in approximately 15% of estrogen receptor-positive (ER + ) human breast cancers. We investigated mechanisms by which FGFR1 amplification confers antiestrogen resistance to ER + breast cancer. Experimental Design: ER + tumors from patients treated with letrozole before surgery were subjected to Ki67 IHC, FGFR1 FISH, and RNA sequencing (RNA-seq). ER + / FGFR1 -amplified breast cancer cells, and patient-derived xenografts (PDX) were treated with FGFR1 siRNA or the FGFR tyrosine kinase inhibitor lucitanib. Endpoints were cell/xenograft growth, FGFR1/ERα association by coimmunoprecipitation and proximity ligation, ER genomic activity by ChIP sequencing, and gene expression by RT-PCR. Results: ER + / FGFR1 -amplified tumors in patients treated with letrozole maintained cell proliferation (Ki67). Estrogen deprivation increased total and nuclear FGFR1 and FGF ligands expression in ER + / FGFR1- amplified primary tumors and breast cancer cells. In estrogen-free conditions, FGFR1 associated with ERα in tumor cell nuclei and regulated the transcription of ER-dependent genes. This association was inhibited by a kinase-dead FGFR1 mutant and by treatment with lucitanib. ChIP-seq analysis of estrogen-deprived ER + / FGFR1 -amplified cells showed binding of FGFR1 and ERα to DNA. Treatment with fulvestrant and/or lucitanib reduced FGFR1 and ERα binding to DNA. RNA-seq data from FGFR1 -amplified patients' tumors treated with letrozole showed enrichment of estrogen response and E2F target genes. Finally, growth of ER + / FGFR1- amplified cells and PDXs was more potently inhibited by fulvestrant and lucitanib combined than each drug alone. Conclusion s : These data suggest the ERα pathway remains active in estrogen-deprived ER + / FGFR1 -amplified breast cancers. Therefore, these tumors are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Clin Cancer Res; 23(20); 6138-50. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Unlabelled: Neoadjuvant chemotherapy (NAC) induces a pathologic complete response (pCR) in approximately 30% of patients with triple-negative breast cancers (TNBC). In patients lacking a pCR, NAC selects a subpopulation of chemotherapy-resistant tumor cells. To understand the molecular underpinnings driving treatment-resistant TNBCs, we performed comprehensive molecular analyses on the residual disease of 74 clinically defined TNBCs after NAC, including next-generation sequencing (NGS) on 20 matched pretreatment biopsies. Combined NGS and digital RNA expression analysis identified diverse molecular lesions and pathway activation in drug-resistant tumor cells. Ninety percent of the tumors contained a genetic alteration potentially treatable with a currently available targeted therapy. Thus, profiling residual TNBCs after NAC identifies targetable molecular lesions in the chemotherapy-resistant component of the tumor, which may mirror micrometastases destined to recur clinically. These data can guide biomarker-driven adjuvant studies targeting these micrometastases to improve the outcome of patients with TNBC who do not respond completely to NAC., Significance: This study demonstrates the spectrum of genomic alterations present in residual TNBC after NAC. Because TNBCs that do not achieve a CR after NAC are likely to recur as metastatic disease at variable times after surgery, these alterations may guide the selection of targeted therapies immediately after mastectomy before these metastases become evident., (2013 AACR)

Basal-like breast cancer (BLBC) is an aggressive disease that lacks a clinically approved targeted therapy. Traditional chemotherapy is effective in BLBC, but it spares the cancer stem cell (CSC)-like population, which is likely to contribute to cancer recurrence after the initial treatment. Dual specificity phosphatase-4 (DUSP4) is a negative regulator of the mitogen-activated protein kinase (MAPK) pathway that is deficient in highly aggressive BLBCs treated with chemotherapy, leading to aberrant MAPK activation and resistance to taxane-induced apoptosis. Herein, we investigated how DUSP4 regulates the MAP-ERK kinase (MEK) and c-jun-NH2-kinase (JNK) pathways in modifying CSC-like behavior. DUSP4 loss increased mammosphere formation and the expression of the CSC-promoting cytokines interleukin (IL)-6 and IL-8. These effects were caused in part by loss of control of the MEK and JNK pathways and involved downstream activation of the ETS-1 and c-JUN transcription factors. Enforced expression of DUSP4 reduced the CD44(+)/CD24(-) population in multiple BLBC cell lines in a MEK-dependent manner, limiting tumor formation of claudin-low SUM159PT cells in mice. Our findings support the evaluation of MEK and JNK pathway inhibitors as therapeutic agents in BLBC to eliminate the CSC population., (©2013 AACR.)