Rinath Jeselsohn

Emergence of Constitutively Active Estrogen Receptor-α Mutations in Pretreated Advanced Estrogen Receptor–Positive Breast Cancer

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER+/HER2−) and, as controls, 115 ER-negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%–41%). These mutations were not detected in primary or treatment-naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER+ breast cancer. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

D538G Mutation in Estrogen Receptor-α: A Novel Mechanism for Acquired Endocrine Resistance in Breast Cancer

Resistance to endocrine therapy occurs in virtually all patients with estrogen receptor α (ERα)-positive metastatic breast cancer, and is attributed to various mechanisms including loss of ERα expression, altered activity of coregulators, and cross-talk between the ERα and growth factor signaling pathways. To our knowledge, acquired mutations of the ERα have not been described as mediating endocrine resistance. Samples of 13 patients with metastatic breast cancer were analyzed for mutations in cancer-related genes. In five patients who developed resistance to hormonal therapy, a mutation of A to G at position 1,613 of ERα, resulting in a substitution of aspartic acid at position 538 to glycine (D538G), was identified in liver metastases. Importantly, the mutation was not detected in the primary tumors obtained prior to endocrine treatment. Structural modeling indicated that D538G substitution leads to a conformational change in the ligand-binding domain, which mimics the conformation of activated ligand-bound receptor and alters binding of tamoxifen. Indeed, experiments in breast cancer cells indicated constitutive, ligand-independent transcriptional activity of the D538G receptor, and overexpression of it enhanced proliferation and conferred resistance to tamoxifen. These data indicate a novel mechanism of acquired endocrine resistance in breast cancer. Further studies are needed to assess the frequency of D538G-ERα among patients with breast cancer and explore ways to inhibit its activity and restore endocrine sensitivity.

Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.

Transplantation studies have demonstrated the existence of mammary progenitor cells with the ability to self-renew and regenerate a functional mammary gland. Although these progenitors are the likely targets for oncogenic transformation, correlating progenitor populations with certain oncogenic stimuli has been difficult. Cyclin D1 is required for lobuloalveolar development during pregnancy and lactation as well as MMTV-ErbB2- but not MMTV-Wnt1-mediated tumorigenesis. Using a kinase-deficient cyclin D1 mouse, we identified two functional mammary progenitor cell populations, one of which is the target of MMTV-ErbB2. Moreover, cyclin D1 activity is required for the self-renewal and differentiation of mammary progenitors because its abrogation leads to a failure to maintain the mammary epithelial regenerative potential and also results in defects in luminal lineage differentiation.

PDEF Promotes Luminal Differentiation and Acts as a Survival Factor for ER-Positive Breast Cancer Cells

Breast cancer is a heterogeneous disease and can be classified based on gene expression profiles that reflect distinct epithelial subtypes. We identify prostate-derived ETS factor (PDEF) as a mediator of mammary luminal epithelial lineage-specific gene expression and as a factor required for tumorigenesis in a subset of breast cancers. PDEF levels strongly correlate with estrogen receptor (ER)-positive luminal breast cancer, and PDEF transcription is inversely regulated by ER and GATA3. Furthermore, PDEF is essential for luminal breast cancer cell survival and is required in models of endocrine resistance. These results offer insights into the function of this ETS factor that are clinically relevant and may be of therapeutic value for patients with breast cancer treated with endocrine therapy.

Cyclin D1 activity regulates autophagy and senescence in the mammary epithelium

Overexpression of cyclin D1 is believed to endow mammary epithelial cells (MEC) with a proliferative advantage by virtue of its contribution to pRB inactivation. Accordingly, abrogation of the kinase-dependent function of cyclin D1 is sufficient to render mice resistant to breast cancer initiated by ErbB2. Here, we report that mouse cyclin D1(KE/KE) MECs (deficient in cyclin D1 activity) upregulate an autophagy-like process but fail to implement ErbB2-induced senescence in vivo. In addition, immortalized cyclin D1(KE/KE) MECs retain high rates of autophagy and reduced ErbB2-mediated transformation in vitro. However, highlighting its dual role during tumorigenesis, downregulation of autophagy led to an increase in senescence in cyclin D1(KE/KE) MECs. Autophagy upregulation was also confirmed in human mammary epithelial cells (HMEC) subjected to genetic and pharmacologic inhibition of cyclin D1 activity and, similar to our murine system, simultaneous inhibition of Cdk4/6 and autophagy in HMECs enhanced the senescence response. Collectively, our findings suggest a previously unrecognized function of cyclin D1 in suppressing autophagy in the mammary epithelium.

Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis.

IntroductionReal-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.MethodsCTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.ResultsIn total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.ConclusionThis study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer

Significance One of the mechanisms of endocrine resistance in estrogen receptor α (ER)-positive (+) breast cancer is the cross-talk between the ER and growth factor receptor pathways leading to altered ER activity and a reprogrammed ER-dependent transcriptome. However, key mediators of this ER-dependent transcriptional reprogramming remain elusive. Here we demonstrate that forkhead box protein A1 (FOXA1) up-regulation via gene amplification or overexpression contributes to endocrine resistance and increased invasiveness phenotypes by altering the ER-dependent transcriptome. We further show that IL-8, one of the top altered FOXA1/ER effectors, plays a key role in mediating these phenotypes and is a potential target to treat ER+/FOXA1-high breast cancer. Our findings provoke a new interplay of FOXA1 in the ER transcriptional program in endocrine-resistant breast cancer. Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance

Significance Resistance to endocrine treatment remains a significant clinical obstacle. ESR1 mutations were found to be the mechanism of endocrine resistance in a substantial number of patients with metastatic ER-positive breast. However, these mutations are primarily linked to aromatase inhibitor resistance and are not strongly associated with tamoxifen resistance. Herein, we show that tamoxifen treatment promotes a RUNX2–ER complex, which mediates an altered ER cistrome that facilitates the up-regulation of SOX9. We show that up-regulation of SOX9, an embryonic transcription factor with key roles in metastases, is a driver of endocrine resistance in the setting of tamoxifen treatment. Our data provide putative targets for the development of new strategies to treat tamoxifen-resistant breast cancer. The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2–ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

The Evolving Role of the Estrogen Receptor Mutations in Endocrine Therapy-Resistant Breast Cancer

Recurrent ligand-binding domain ESR1 mutations have recently been detected in a substantial number of patients with metastatic ER+ breast cancer and evolve under the selective pressure of endocrine treatments. In this review, we evaluate the current understanding of the biological and clinical significance of these mutations. The preclinical studies revealed that these mutations lead to constitutive ligand-independent activity, indicating resistance to aromatase inhibitors and decreased sensitivity to tamoxifen and fulvestrant. Retrospective analyses of ESR1 mutations in baseline plasma circulating tumor DNA from completed clinical trials suggest that these mutations are prognostic and predictive of resistance to aromatase inhibitors in metastatic disease. Currently, we are lacking prospective studies to confirm these results and to determine the optimal treatment combinations for patients with the ESR1 mutations. In addition, the clinical development of novel agents to overcome resistance engendered by these mutations is also needed.

Blockade of AP-1 Potentiates Endocrine Therapy and Overcomes Resistance

The transcription factor AP-1 is downstream of growth factor (GF) receptors (GFRs) and stress-related kinases, both of which are implicated in breast cancer endocrine resistance. Previously, we have suggested that acquired endocrine resistance is associated with increased activity of AP-1 in an in vivo model. In this report, we provide direct evidence for the role of AP-1 in endocrine resistance. First, significant overlap was found between genes modulated in tamoxifen resistance and a gene signature associated with GF-induced estrogen receptor (ER) cistrome. Interestingly, these overlapping genes were enriched for key signaling components of GFRs and stress-related kinases and had AP-1 motifs in their promoters/enhancers. Second, to determine a more definitive role of AP-1 in endocrine resistance, AP-1 was inhibited using an inducible dominant-negative (DN) cJun expressed in MCF7 breast cancer cells in vitro and in vivo. AP-1 blockade enhanced the antiproliferative effect of endocrine treatments in vitro, accelerated xenograft tumor response to tamoxifen and estrogen deprivation in vivo, promoted complete regression of tumors, and delayed the onset of tamoxifen resistance. Induction of DN-cJun after the development of tamoxifen resistance resulted in dramatic tumor shrinkage, accompanied by reduced proliferation and increased apoptosis. These data suggest that AP-1 is a key determinant of endocrine resistance by mediating a global shift in the ER transcriptional program. Implications: AP-1 represents a viable therapeutic target to overcome endocrine resistance. Mol Cancer Res; 14(5); 470–81. ©2016 AACR.