Amy M. Fowler

STAT1-deficient mice spontaneously develop estrogen receptor α-positive luminal mammary carcinomas

IntroductionAlthough breast cancers expressing estrogen receptor-α (ERα) and progesterone receptors (PR) are the most common form of mammary malignancy in humans, it has been difficult to develop a suitable mouse model showing similar steroid hormone responsiveness. STAT transcription factors play critical roles in mammary gland tumorigenesis, but the precise role of STAT1 remains unclear. Herein, we show that a subset of human breast cancers display reduced STAT1 expression and that mice lacking STAT1 surprisingly develop ERα+/PR+ mammary tumors.MethodsWe used a combination of approaches, including histological examination, gene targeted mice, gene expression analysis, tumor transplantaion, and immunophenotyping, to pursue this study.ResultsForty-five percent (37/83) of human ERα+ and 22% (17/78) of ERα- breast cancers display undetectable or low levels of STAT1 expression in neoplastic cells. In contrast, STAT1 expression is elevated in epithelial cells of normal breast tissues adjacent to the malignant lesions, suggesting that STAT1 is selectively downregulated in the tumor cells during tumor progression. Interestingly, the expression levels of STAT1 in the tumor-infiltrating stromal cells remain elevated, indicating that single-cell resolution analysis of STAT1 level in primary breast cancer biopsies is necessary for accurate assessment. Female mice lacking functional STAT1 spontaneously develop mammary adenocarcinomas that comprise > 90% ERα+/PR+ tumor cells, and depend on estrogen for tumor engraftment and progression. Phenotypic marker analyses demonstrate that STAT1-/- mammary tumors arise from luminal epithelial cells, but not myoepithelial cells. In addition, the molecular signature of the STAT1-/- mammary tumors overlaps closely to that of human luminal breast cancers. Finally, introduction of wildtype STAT1, but not a STAT1 mutant lacking the critical Tyr701 residue, into STAT1-/- mammary tumor cells results in apoptosis, demonstrating that the tumor suppressor function of STAT1 is cell-autonomous and requires its transcriptional activity.ConclusionsOur findings demonstrate that STAT1 suppresses mammary tumor formation and its expression is frequently lost during breast cancer progression. Spontaneous mammary tumors that develop in STAT1-/- mice closely recapitulate the progression, ovarian hormone responsiveness, and molecular characteristics of human luminal breast cancer, the most common subtype of human breast neoplasms, and thus represent a valuable platform for testing novel treatments and detection modalities.

Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

ABSTRACT The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription.

Increases in estrogen receptor-α concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen

A common phenotype in breast cancer is the expansion of the estrogen receptor‐α (ER+) cell population and an inappropriate elevation of ERα protein, the latter predisposing patients for a poorer prognosis than those with lower levels of the receptor. A tetracycline‐inducible ERα overexpression model was developed in the MCF‐7 cell line to assess induction of endogenous gene activation and growth in response to elevations in ERα protein. Heightened levels of ERα resulted in aberrant promoter occupancy and gene activation in the absence of hormone, which was independent of ligand and AF‐2 function. This increased receptor activity required the amino‐terminal A/B domain and was not inhibited by tamoxifen, which supports an enhancement of AF‐1 function, yet was independent of serine‐104, 106, and 118 phosphorylation. Ligand‐independent transcription was accompanied by an increase in growth in the absence of hormonal stimulation. The results suggest that elevated levels of ERα in breast cancer cells can result in activation of receptor transcriptional function in a manner distinct from classical mechanisms that involve ligand binding or growth factor‐induced phosphorylation. Further, they describe a potential mechanism whereby increases in ERα concentration may provide a proliferative advantage by augmenting ERα function regardless of ligand status.—Fowler, A. M., Solodin, N., Preisler‐Mashek, M. T., Zhang, P., Lee, A. V., Alarid, E. T. Increases in estrogen receptor‐α concentration in breast cancer cells promote serine 118/104/106‐independent AF‐1 transactivation and growth in the absence of estrogen. FASEB J. 18, 81–93 (2004)

Small-Animal PET of Steroid Hormone Receptors Predicts Tumor Response to Endocrine Therapy Using a Preclinical Model of Breast Cancer

Estrogen receptor-α (ERα) and progesterone receptor (PR) are expressed in most human breast cancers and are important predictive factors for directing therapy. Because of de novo and acquired resistance to endocrine therapy, there remains a need to identify which ERα-positive (ERα+)/PR-positive (PR+) tumors are most likely to respond. The purpose of this study was to use estrogen- and progestin-based radiopharmaceuticals to image ERα and PR in mouse mammary tumors at baseline and after hormonal therapy and to determine whether changes in these imaging biomarkers can serve as an early predictive indicator of therapeutic response. Methods: Mammary adenocarcinomas that spontaneously develop in aged female mice deficient in signal transducer and activator of transcription-1 (STAT1) were used. Imaging of ERα and PR in primary tumor–bearing mice and mice implanted with mammary cell lines (SSM1, SSM2, and SSM3) derived from primary STAT1-deficient (STAT1−/−) tumors was performed. Hormonal treatments consisted of estradiol, an ER agonist; letrozole, an aromatase inhibitor; and fulvestrant, a pure ER antagonist. Small-animal PET/CT was performed using 18F-fluoroestradiol (18F-FES) for ER, 18F-fluoro furanyl norprogesterone (18F-FFNP) for PR, and 18F-FDG for glucose uptake. Tracer uptake in the tumor was quantified and compared with receptor concentration determined by in vitro assays of resected tumors. Results: Primary STAT1−/− mammary tumors and implanted SSM2 and SSM3 tumors showed high 18F-FES and 18F-FFNP uptake and were confirmed to be ERα+/PR+. Classic estrogen-induced regulation of the progesterone receptor gene was demonstrated by increased 18F-FFNP uptake of estradiol-treated SSM3 tumors. Treatment with fulvestrant decreased 18F-FFNP, 18F-FES, and 18F-FDG uptake and inhibited growth of SSM3 tumors but decreased only 18F-FES uptake in SSM2 tumors, with no effect on growth, despite both tumors being ERα+/PR+. Decreased 18F-FFNP uptake by SSM3 tumors occurred early after initiation of treatment, before measurable tumor growth inhibition. Conclusion: Using small-animal PET, a profile was identified that distinguished fulvestrant-sensitive from fulvestrant-resistant ERα+/PR+ tumors before changes in tumor size. This work demonstrates that imaging baseline tumoral 18F-FES uptake and initial changes in 18F-FFNP uptake in a noninvasive manner is a potentially useful strategy to identify responders and nonresponders to endocrine therapy at an early stage.

A Molecular Approach to Breast Imaging

Molecular imaging is a multimodality discipline for noninvasively visualizing biologic processes at the subcellular level. Clinical applications of radionuclide-based molecular imaging for breast cancer continue to evolve. Whole-body imaging, with scintimammography and PET, and newer dedicated breast imaging systems are reviewed. The potential clinical indications and the challenges of implementing these emerging technologies are presented.

Dynamic Control of Nuclear Receptor Transcription

This Perspective highlights experiments that probe the complexity and regulation underlying cyclical association of nuclear receptor transcriptional complexes with DNA. Using advanced in vivo and in vitro techniques, these studies emphasize the importance of chromatin remodeling and histone modification in defining the timing and nature of the cycles. In addition, they reveal the multiplicity of receptor-coregulator complexes that reside on a single promoter. These conceptual and technical achievements integrate kinetic and combinatorial regulation into a new dynamic model of nuclear receptor–mediated transcription.

Utility of 6-month Follow-up Imaging after a Concordant Benign Breast Biopsy Result

PURPOSE To determine the utility of 6-month follow-up imaging after benign concordant image-guided percutaneous breast biopsy results. MATERIALS AND METHODS The institutional review board approved this retrospective, HIPAA-compliant study; informed consent was waived. Findings from consecutive stereotactic and ultrasonographically guided core breast biopsies performed from 2001 to 2005 were analyzed and included lesions with benign pathologic findings without atypia found to be concordant with imaging at a consensus conference. Rebiopsy recommendation rates and positive predictive values (PPVs) for detecting malignancy at each follow-up interval were measured and compared by using a two-tailed Fisher exact test. RESULTS In 2244 biopsies, lesions in 1465 were benign, concordant, and not excised. In 1057 of 1465 (72.2%) biopsies with imaging follow-up (average, 26.4 months; range, 4.0-49.9 months), recommended rebiopsy rates were 0.8% (four of 526), 0.5% (three of 588), and 1.0% (eight of 802) at 6-month, 12-month, and long-term follow-up intervals, respectively. When the initial follow-up did not occur until 12 months, the recommended rebiopsy rate was 0.9% (three of 322), compared with 0.8% (four of 526) at 6 months (P > .99), and no malignancies were found in either group. One malignancy was detected at the long-term follow-up interval (PPV for excision recommended, 12% [one of eight]; PPV for excision performed, 20% [one of five]). CONCLUSION Because rebiopsy recommendation rates and PPVs did not differ in the 6- and 12-month groups, a 6-month follow-up imaging examination, in the context of a formal concordancy consensus conference, may not contribute to improved breast cancer diagnosis. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.10091824/-/DC1.

Amping up estrogen receptors in breast cancer

This article highlights a recent study by Holst et al. in Nature Genetics that finds estrogen receptor-alpha (ER-α) amplification in early benign lesions and more advanced invasive carcinomas of the breast, and discusses the potential implications to our present understanding of the role of ER-α in breast tumorigenesis.

Longitudinal Noninvasive Imaging of Progesterone Receptor as a Predictive Biomarker of Tumor Responsiveness to Estrogen Deprivation Therapy

Purpose: To investigate whether longitudinal functional PET imaging of mammary tumors using the radiopharmaceuticals [18F]FDG (to measure glucose uptake), [18F]FES [to measure estrogen receptor (ER) levels], or [18F]FFNP [to measure progesterone receptor (PgR) levels] is predictive of response to estrogen-deprivation therapy. Experimental Design: [18F]FDG, [18F]FES, and [18F]FFNP uptake in endocrine-sensitive and -resistant mammary tumors was quantified serially by PET before ovariectomy or estrogen withdrawal in mice, and on days 3 and 4 after estrogen-deprivation therapy. Specificity of [18F]FFNP uptake in ERα+ mammary tumors was determined by competition assay using unlabeled ligands for PgR or glucocorticoid receptor (GR). PgR expression was also assayed by immunohistochemistry (IHC). Results: The levels of [18F]FES and [18F]FDG tumor uptake remained unchanged in endocrine-sensitive tumors after estrogen-deprivation therapy compared with those at pretreatment. In contrast, estrogen-deprivation therapy led to a reduction in PgR expression and [18F]FFNP uptake in endocrine-sensitive tumors, but not in endocrine-resistant tumors, as early as 3 days after treatment; the changes in PgR levels were confirmed by IHC. Unlabeled PgR ligand R5020 but not GR ligand dexamethasone blocked [18F]FFNP tumor uptake, indicating that [18F]FFNP bound specifically to PgR. Therefore, a reduction in FFNP tumor to muscle ratio in mammary tumors predicts sensitivity to estrogen-deprivation therapy. Conclusions: Monitoring the acute changes in ERα activity by measuring [18F]FFNP uptake in mammary tumors predicts tumor response to estrogen-deprivation therapy. Longitudinal noninvasive PET imaging using [18F]FFNP is a robust and effective approach to predict tumor responsiveness to endocrine treatment. Clin Cancer Res; 21(5); 1063–70. ©2014 AACR.

Imaging Diagnostic and Therapeutic Targets - Steroid Receptors in Breast Cancer

Estrogen receptor alpha (ERα) and progesterone receptor (PR) are important steroid hormone receptor biomarkers used to determine prognosis and to predict benefit from endocrine therapies for breast cancer patients. Receptor expression is routinely measured in biopsy specimens using immunohistochemistry, although such testing can be challenging, particularly in the setting of metastatic disease. ERα and PR can be quantitatively assayed noninvasively with PET. This approach provides the opportunity to assess receptor expression and function in real time, within the entire tumor, and across distant sites of metastatic disease. This article reviews the current evidence of ERα and PR PET imaging as predictive and early-response biomarkers for endocrine therapy.