Prognostic and Therapeutic Relevance of PELP1 in Estrogen Receptor Positive Breast Cancer: A Systematic Review

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Does the addition of chemotherapy to adjuvant endocrine treatment add any benefit in ER-positive early breast cancer: can we rely on large randomized control trials in the era of personalized medicine?

CDK4/6 inhibitors as neoadjuvant treatment in breast cancer—what can we learn?

Does adjuvant therapy reduce postmetastatic survival?

Identification of molecular signatures that allow detection of the transition from normal breast epithelial cells to malignant invasive cells is a critical component in the development of diagnostic, therapeutic, and preventative strategies for human breast cancer. Substantial efforts have been devoted to deciphering breast cancer etiology at the genome level, but only a limited number of studies have appeared at the proteome level. In this work, we compared individual in situ proteome profiles of nonpatient matched nine noncancerous, normal breast epithelial (NBE) samples with nine estrogen receptor (ER)-positive (luminal subtype), invasive malignant breast epithelial (MBE) samples by combining laser capture microdissection (LCM) and quantitative shotgun proteomics. A total of 12,970 unique peptides were identified from the 18 samples, and 1623 proteins were selected for quantitative analysis using spectral index (SpI) as a measure of protein abundance. A total of 298 proteins were differentially expressed between NBE and MBE at 95% confidence level, and this differential expression correlated well with immunohistochemistry (IHC) results reported in the Human Protein Atlas (HPA) database. To assess pathway level patterns in the observed expression changes, we developed protein set enrichment analysis (PSEA), a modification of a well-known approach in gene expression analysis, Gene Set Enrichment Analysis (GSEA). Unlike single gene-based functional term enrichment analyses that only examines pathway overrepresentation of proteins above a given significance threshold, PSEA applies a weighted running sum statistic to the entire expression data to discover significantly enriched protein groups. Application of PSEA to the expression data in this study revealed not only well-known ER-dependent and cellular morphology-dependent protein abundance changes, but also significant alterations of downstream targets for multiple transcription factors (TFs), suggesting a role for specific gene regulatory pathways in breast tumorigenesis. A parallel GOMiner analysis revealed both confirmatory and complementary data to PSEA. The combination of the two annotation approaches yielded extensive biological feature mapping for in depth analysis of the quantitative proteomic data.

Focus on breast cancer.

Targeting Androgen Receptor in Estrogen Receptor-Negative Breast Cancer

Up to one-third of women aged 30-50 years have cysts in their breasts and are presumed to be at increased risk of developing breast cancer. Here we present an extensive proteomic and immunohistochemistry (IHC) study of breast apocrine cystic lesions aimed at generating specific biomarkers and elucidating the relationship, if existent, of apocrine cysts with cancer phenotype. To this end we compared the expression profiles of apocrine macrocysts obtained from mastectomies from high risk cancer patients with those of cancerous and non-malignant mammary tissue biopsies collected from the same patients. We identified two biomarkers, 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase, that were expressed specifically by apocrine type I cysts as well as by apocrine metaplastic cells in type II microcysts, terminal ducts, and intraductal papillary lesions. No expression of these markers was observed in non-malignant terminal ductal lobular units, type II flat cysts, stroma cells, or fat tissue as judged by IHC analysis of matched non-malignant tissue samples collected from 93 high risk patients enrolled in our cancer program. IHC analysis of the corresponding 93 primary tumors indicated that most apocrine changes have little intrinsic malignant potential, although some may progress to invasive apocrine cancer. None of the apocrine lesions examined, however, seemed to be a precursor of invasive ductal carcinomas, which accounted for 81% of the tumors analyzed. Our studies also provided some insight into the origin, development, and enlargement of apocrine cysts in mammary tissue. The successful identification of differentially expressed proteins that characterize specific steps in the progression from early benign lesions to apocrine cancer opens a window of opportunity for designing and testing new approaches for pharmacological intervention, not only in a therapeutic setting but also for chemoprevention, to inhibit cyst development as both 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase are currently being targeted for chemoprevention strategies in various malignancies.

Introduction: The lack of estrogen receptor (ER) expression is the primary cause of de novo resistance of breast cancers to endocrine therapy. In contrast, in most cases of acquired endocrine resistance, ER is expressed and other mechanisms of resistance have been proposed, such as ER mutations. Pre-clinical studies demonstrated a small number of specific point mutations that can enhance ER function. However, the studies on clinical samples performed in the 1990's were limited by small sample size, lack of detailed clinical correlation and lacked the sensitivity of next-generation sequencing (NGS). Therefore, in this study we sought to comprehensively investigate the frequency and functional significance of ER mutations throughout the progression of breast cancer from primary disease to advanced metastatic disease using targeted NGS. Methods: In this retrospective study, a total of 249 tumor specimens were analyzed. The specimens include 134 ER positive and, as controls, 115 estrogen receptor negative tumors. The estrogen receptor positive samples consist of 58 primary breast cancers and 76 metastatic sample. All tumors were sequenced with high coverage using NGS targeting the coding sequence of ER and an additional 181 cancer-related genes. Results: Recurring somatic mutations at codons 537 and 538 within the ligand-binding domain of the estrogen receptor were detected in ER positive metastatic tumors. Overall, the frequency of these mutations was 12% (95% CI 6%-21%) in metastatic patients compared with none in the primary cases. In total there were 9 recurring somatic mutations; Y537C (11%), Y537N (33%), Y537S (22%) and D538G (33%). In addition in a small number of paired primary and metastatic samples from the same patient, these mutations were found only in the metastatic specimens. In a subset of heavily pre-treated patients the frequency was 20% (5/25, 95% CI 7%-41%). ER activating mutations were not detected in any stage of ER negative disease. ER alterations were not mutually exclusive with any of the other commonly altered genes and of the most frequently altered genes, all but ER alterations displayed similar frequencies across primary and metastatic specimens. Functional studies in cell line models demonstrated that these ER mutations render ER constitutively active and confer resistance to hormone deprivation, tamoxifen and fulvestrant. Conclusions: Herein, we reveal functional ER mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer. The absence of detectable mutations in the primary tumors suggests clonal evolution as the mechanism of resistance. Thus, these mutations have the potential to be an important genetic biomarker of endocrine resistance in ER positive metastatic breast cancer and could assist in clinical decision making as disease progresses. Our findings also underscore the value of repeated biopsies of metastatic lesions. Lastly, since the frequencies of these mutations are substantial when sensitive testing methods are used in the correct clinical context, pre-clinical and clinical studies to identify novel therapeutics that can overcome this resistance are warranted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-06.

MR imaging features of invasive breast cancer correlated with hormonal receptors: does progesterone receptor matter?

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERβ cells with inhibited ERβ expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERβ breast cancer cells with low levels of ERα and no expression of ERβ. In addition, data from our study indicate that ERβ-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERβ cells expressing ERβ and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERβ decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERβ cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERβ ratio) in the cells or tissue of interest.

Introduction: Current models to predict breast cancer risk do not differentiate risk for estrogen receptor (ER) positive and negative breast cancer (BC), despite growing evidence that these tumors are biologically very different. We hypothesized that women with ER+ BC cancers have different clinical risk factors and histologic findings on prior benign breast biopsies than those with ER- BC. Methods: After IRB approval, we examined associations of age at benign biopsy and histologic features of the benign biopsy with ER status of incident BCs within the Mayo Benign Breast Disease Cohort. Benign biopsy slides were reviewed for extent of lobular involution and degree of epithelial proliferation by a single breast pathologist blinded to BC events. Invasive BCs occurring within 15 years after benign biopsy were classified as ER+ if ER staining was >1%. BC case-only associations with ER status were evaluated using multivariate logistic regression. Full-cohort hazard ratios (HR) and 95% confidence intervals (CI) for risk of ER-specific subtypes were estimated using Cox proportion hazards regression. Results: Among 13,410 women undergoing a benign breast biopsy from 1967-2001, 656 invasive BCs (459 ER+, 106 ER, 106 unknown) occurred within 15 years. Women who developed ER+ and ER- BCs were similar in age at the time of their prior benign breast biopsy (p=0.34). Although benign biopsies in women who later developed ER+ BC were more likely to show complete involution (23% vs 15% for ER- BC), this was not statistically significant (p=0.06). However, the degree of epithelial proliferation was significantly associated with ER status of later BCs (p=0.001), with ER+ BCs more likely than ER- BCs to have had a prior biopsy with atypical hyperplasia (16% vs 8%), and ER+ BCs less likely than ER- BCs to have had a prior biopsy with proliferative disease without atypia (33% vs 52%); this association remained after multivariate adjustment (p=0.003). We further pursued the association of epithelial proliferation with differential risk of ER+ and ER- BC in our overall cohort of 13,410 women (Table 1). Compared to women with non-proliferative disease, women with proliferative disease +/- atypia had ∼2-fold hazard ratios for ER- BC, whereas hazard ratios for ER+ BC were higher in women with atypical hyperplasia (∼4-fold) compared to proliferative disease. Hazard Ratio of ER+ and ER- BC within 15 years of BBD based upon degree of epithelial proliferation at benign biopsyCancer TypeGroupNN EventsHazard Ratio (95% CI)p-valueInvasive ER- ≤15 yearsNP8478421.00 (ref)<0.0001 PD4229542.63 (1.76, 3.93) AH70382.60 (1.22, 5.54) Invasive ER+ ≤15 yearsNP84782321.00 (ref)<0.0001 PD42291511.33 (1.09, 1.64) AH703744.41 (3.39, 5.73) NP=nonproliferative disease, PD=proliferative disease without atypia; AH=atypical hyperplasia Conclusion: ER+ and ER- breast cancers appear to have different features on prior benign breast biopsy, with atypical hyperplasia showing increased risk for both types of breast cancer, but a greater risk for ER+ tumors. Citation Format: Amy C Degnim, Derek C Radisky, Robert A Vierkant, Ryan D Frank, Marlene H Frost, Vernon S Pankratz, Celine M Vachon, Tanya L Hoskin, Julie M Cunningham, Chen Wang, Jean-Pierre Kocher, Teresa M Allers, Joanne L Johnson, Tina J Hieken, Karthik Ghosh, Lynn C Hartmann, Daniel W Visscher. Histologic features of benign breast biopsy tissue and association with ER positive and ER negative breast cancer in the Mayo BBD cohort study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-10-06.

Breast cancer is estimated to be the leading cause of new cancer cases and second in cancer related deaths among American women (Cancer: 63, 1153-1154, 2013). Classification of breast cancers into receptor-based subtypes, including estrogen receptor (ER) positive or triple negative breast cancers, establish targets that guide individualized treatment for patients (J Clin Oncol: 27,1153-1154, 2009). Drug resistant ER positive breast cancers and triple-negative breast cancers are prolific breast cancers with poor clinical outcome. TRA-8 is a death receptor-5 (DR5) specific agonistic antibody with tumoricidal activity in vitro and in vivo without inducing normal hepatocyte apoptosis (Nature Med: 7, 954-960, 2001). ER positive and triple negative breast cancer cell lines have a range of in vitro sensitivity to TRA-8 mediated cytotoxicity (Clin Cancer Res: 9, 3731-3741, 2003, Breast Cancer Res Treat: 133, 417-426, 2012). Calmodulin (CaM) is overexpressed in breast cancer (J Clin Oncol: 4, 994-1012, 1986, Cancer Res: 42, 2571-2574, 1982). Our lab has shown CaM binds to DR5 and plays a role in DR5 mediated DISC formation in ER positive MCF-7 and triple negative MDA-MB231 breast cancer cell lines (Proc. AACR; 2014, Abstract nr 2282). The goal of this study is to characterize an interaction between CaM and DR5 in a panel of ER positive and triple negative breast cancer cell lines representing a range of sensitivity to TRA-8 mediated cytotoxicity. CaM-DR5 interaction was characterized in a panel of triple negative breast cancer cell lines: MDA-MB-231, MDA-MB-468, HCC1937, and HCC1143 and ER positive breast cancer cell lines: ZR-75-1, T47D, and MCF-7 representing the range of sensitivity to TRA-8 (Clin Cancer Res: 9, 3731-3741, 2003, Breast Cancer Res Treat: 133, 417-426 2012). DR5 was immunoprecipitated, using TRA-8 conjugated beads, from the panel of breast cancer cell lines lysates. CaM co-immunoprecipitated with DR5 from all breast cancer cell lines lysates, showing endogenous CaM and DR5 form a complex. CaM pull-down of DR5 from the breast cancer lysates in the presence of 1 mM Ca2+ or 2 mM EGTA showed CaM/DR5 interaction is calcium dependent. We present evidence for a Ca2+ dependent interaction between CaM and DR5 in a panel of ER positive and triple negative breast cancer cell lines independent of TRA-8 sensitivity. Results from this study provide the basis to further characterization of CaM-DR5 interactions and to investigate the role of CaM/DR5 binding in DR5-mediated DISC formation, which could lead to novel strategies to overcome drug resistance in breast cancers. Results suggest that direct interaction between CaM and DR5 could represent a potential site for breast cancer chemotherapeutics. Citation Format: Romone M. Fancy, Donald J. Buchsbaum, Tong Zhou, Yuhua Song. Calmodulin-DR5 binding in breast cancer: Independent of TRA-8 sensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2931. doi:10.1158/1538-7445.AM2015-2931

Established histopathological criteria divide invasive breast carcinomas into defined groups. Ductal of no specific type and lobular are the two major subtypes accounting for around 75 and 15% of all cases, respectively. The remaining 10% include rarer types such as tubular, cribriform, mucinous, papillary, medullary, metaplastic, and apocrine breast carcinomas. Molecular profiling technologies, on the other hand, subdivide breast tumors into five subtypes, basal-like, luminal A, luminal B, normal breast tissue-like, and ERBB2-positive, that have different prognostic characteristics. An additional subclass termed "molecular apocrine" has recently been described, but these lesions did not exhibit all the histopathological features of classical invasive apocrine carcinomas (IACs). IACs make up 0.5-3% of the invasive ductal carcinomas, and despite the fact that they are morphologically distinct from other breast lesions, there are presently no standard molecular criteria available for their diagnosis and as a result no precise information as to their prognosis. Toward this goal our laboratories have embarked in a systematic proteomics endeavor aimed at identifying biomarkers that may characterize and subtype these lesions as well as targets that may lead to the development of novel targeted therapies and chemoprevention strategies. By comparing the protein expression profiles of apocrine macrocysts and non-malignant breast epithelial tissue we have previously reported the identification of a few proteins that are specifically expressed by benign apocrine lesions as well as by the few IACs that were available to us at the time. Here we reiterate our strategy to reveal apocrine cell markers and present novel data, based on the analysis of a considerably larger number of samples, establishing that IACs correspond to a distinct molecular subtype of breast carcinomas characterized by the expression of 15-prostaglandin dehydrogenase alone or in combination with a novel form of acyl-CoA synthetase medium-chain family member 1 (ACSM1). Moreover we show that 15-prostaglandin dehydrogenase is not expressed by other breast cancer types as determined by gel-based proteomics and immunohistochemistry analysis and that antibodies against this protein can identify IACs in an unbiased manner in a large breast cancer tissue microarray making them potentially useful as a diagnostic aid.

Background: Hyperactivation of ERK1/2 MAPK (hMAPK) leads to loss of estrogen receptor (ER) expression and poor outcome in breast cancer. Recent evidence suggests that microRNAs (miRNAs) play important regulatory roles and serve as biomarkers of disease. We previously reported a miRNA signature indicative of hyperactivation of MAPK signaling in breast cancer and its associations with pathological features of breast cancer and breast cancer clinical outcomes. This hMAPK-miRNA signature identified ER+ breast cancers with reduced recurrence-free and overall survival. Here we report on a leave-one-out analysis of this hMAPK-miRNA signature to narrow down and identify those miRNAs that may be critically important to poor clinical outcomes associated with hyperactive MAPK signaling among patients with ER+ breast cancer. Methods: We performed a leave-one-out analysis of the full hMAPK-miRNA signature, wherein we determined the impact that removal of individual members of the signature had on the ability of the hMAPK-miRNA signature to predict poor disease survival in patients from the METABRIC breast cancer dataset with ER+ cancers. We identified a subsignature of 21 miRNAs that is very significantly associated with poor clinical outcome in patients with ER+ breast cancer from a large patient cohort, according to multivariate Cox proportional hazard analysis. Summary of Results: Of 57 miRNAs in our original hMAPK-miRNA signature, 21 were retained following leave-one-out analysis. "High-hMAPK" status as described by this signature significantly correlated with adverse pathological characteristics of breast cancer, including ER-negativity, increased tumor grade, and enrichment for Basal and HER2 subtypes. Kaplan-Meier survival analysis of primary breast cancers from the METABRIC dataset indicate that the 21 miRNA subsignature improves upon the prognostic capability of the overall signature in this large patient cohort, and particularly in providing prognostic capability in breast cancers of the luminal-A and luminal-B molecular subtypes. Furthermore, multivariate Cox proportional hazards analysis suggests that classification of breast cancers as "high-hMAPK" based on this leave-one-out miRNA signature is an independent risk factor for poor disease outcome, and is a more significant indicator of poor disease status than hormone receptor status, tumor grade, molecular subtype, and HER2 status. Patients with ER-positive breast cancer who were treated with hormone therapy and classified as "high-hMAPK" by this 21 miRNA signature displayed significantly poorer disease survival compared to patients classified as "low hMAPK". Kaplan-Meier survival analysis and multivariate analysis of independent breast cancer cohorts with miRNA expression data confirm these observations. Conclusions: We report a subset of 21 of the hMAPK-miRNAs that are important prognostic factors in ER-positive breast cancer, and that may have predictive value in estimating whether an ER-positive breast cancer may be resistant to hormone therapy. Additionally, these 21 miRNAs may be potential effectors of MAPK signaling, and could serve as novel biomarkers or therapeutic targets in breast cancer. Citation Format: Philip Miller, Jennifer Clarke, Dorraya El-Ashry. A MAPK microRNA signature significantly associated with poor outcome and predictive of response to tamoxifen therapy in ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-07.