Prognostic value of estrogen receptor α and progesterone receptor conversion in distant breast cancer metastases

BACKGROUND:

Changes in the receptor profile of primary breast cancers to their metastases (receptor conversion) have been described for the estrogen receptor α (ERα) and progesterone receptor (PR). The purpose of this study was to evaluate the impact of receptor conversion for ERα and PR on survival in a large group of distant non‐bone breast cancer metastases.

METHODS:

Receptor conversion was studied by immunohistochemistry in a group of 233 metastatic breast cancer patients. Kaplan‐Meier overall survival curves were plotted, and differences between the curves were analyzed by log‐rank analysis. The additional prognostic value of conversion to established prognosticators was studied by Cox regression.

RESULTS:

Overall survival of patients showing conversion from positive to negative ERα or PR, or from negative to positive ERα or PR, or remaining receptor negative was comparable, and significantly worse than patients remaining receptor positive. ERα or PR receptor conversion from positive in the primary breast tumor to negative in distant metastases has independent negative prognostic value.

CONCLUSIONS:

ERα or PR receptor conversion from positive in the primary breast cancer to negative in distant metastases has negative prognostic value. Cancer 2012. © 2012 American Cancer Society.     

Comparison of five different antibodies in the immunohistochemical assay of estrogen receptor α in Human breast cancer

BACKGROUND: Estrogen receptor alpha (ER) expression is the best prognostic and predictive factor of hormone dependency of human breast cancers. Unlike enzyme immunoassay (EIA), which has been widely used to evaluate ER status in breast cancer, immunohistochemical assay (IHC) can detect ER in a small amounts of tissue with detailed localization. Although there is a sufficient number of ER antibodies against various regions of the protein, the reliability of IHC staining is only well understood for a few. IHC and EIA for the evaluation of the ER status of human breast cancer, therefore, should be compared using the same breast cancer tissues. METHODS: Five different ER antibodies (1D-5, C-314, G-20, C-311 and HC-20) that identify different amino acid sequences were used. The evaluation of ER status by IHC using these antibodies was compared with EIA concomitantly in 97 primary human breast cancer tissues RESULTS: The positivity rate for EIA was 68%. That of IHC for antibodies 1D-5, C-314, G-20, C-311 and HC-20 was 50.5%, 47.4%, 46.4%, 44.3% and 57.7%, respectively. The concordance between EIA was 76.3% for 1D-5 and 77.3% for HC-20, which is statistically highly significant (p<0.0001); Other antibodies were not. CONCLUSIONS: HC-20 is most suitable in the evaluation of the ER status of human breast cancers using the IHC method. Although antibody 1D-5 is also available, C-314, G20 and C-311 are unreliable in such an evaluation.     

Clinical significance of estrogen receptor β in breast cancer

To estimate the clinical significance of estrogen receptor (ER) β in breast cancer we reviewed some reports and compared them with our preliminary results. The structure of ER β is similar to that of ER α. The DNA binding domain of ER β is 96% conserved compared with ER α, and the ligand binding domain shows 58% conserved residues, suggesting that both receptors can bind estrogen responsive elements on target genes and that they may also bind similar ligand. The target tissues of ER β such as testis, prostate, lung, brain, thymus, and ovary, are different from those of ER α, ovary, uterus, endometrium, and breast. Although the function of ER β in breast cancer progression is not well understood, 30-50% of breast cancers may express ER β mRNA signals. Additionally, ER β may be a useful prognostic factor in patients with breast cancer, because tumors that co-expressed ER α and ER β might be node positive and tend to be of higher grade. Further characterization of the function of ER β and its isoforms in breast cancer is warranted.     

Standardization of steroid receptor assays in human breast cancer—IV. Long-term within- and between-laboratory variation of estrogen and progesterone receptor assays

One batch of lyophilized calf uterine cytosol was analyzed for estrogen and progesterone receptor (ER and PgR, respectively) content by 12 members of the EORTC Receptor Group on three different occasions over a total study period of 1 yr:(1) One vial was included with each of 20 consecutive batches of routine tumor analyses between December 1983 and May 1984.(2) Two vials were simultaneously assayed between July and August 1984 (within-run variation).(3) One vial was analyzed at the end of 1984 (November-December).The overall mean ER and PgR values did not change systematically over the total study period of 1 yr. Within the various laboratories, the between-run variations of both ER and PgR assays were considerable and differed from one institution to another (7–26%). For both ER and PgR measurements the average within-run (n = 2) and between-run (n = 20) coefficients of variation were similar (8–9% and 16–17%, respectively). Comparison of the results from multiple sequential assays (c.v. = 12.9%) with those from single assays (c.v. = 21.2%) showed that about 60% of the between-laboratory variance in ER could be explained on the basis of the between-run variance. With regard to PgR analysis, however, the between-laboratory variance decreased only 25%.Standardized use of one type of protein assay (Coomassie brilliant blue) and a standard protein solution (human serum albumin) has decreased the between-laboratory variation of the protein analysis results to less than 15%.     

The expression of variant exon v7–v8 CD44 antigen in relation to lymphatic metastasis of human breast cancer

The purpose of this prospective study was to evaluate the expression of CD44 splice variant epitopes in human breast cancer and their potential as prognostic indicators.Invasive breast cancer tissues obtained from 91 patients were examined for expression of the standard CD44 antigen and variant CD44 antigens (v5, v6, v7, v7–v8, and v8–v10) by immunohistochemical staining to investigate the relations of these antigens to clinicopathological factors and prognosis.The expression of standard CD44 antigen was detected in 54.9% of 91 patients with primary human breast cancer. The variant epitopes of CD44 examined, i.e., v5, v6, v7, v7–v8, and v8–v10, were expressed in 54.9%, 54.9%, 0%, 34.1%, and 0%, respectively. There was a significant difference in tumor size, lymph nodal status, and degree of lymphatic permeation between patients who were positive for exon v7–v8 and those negative for this variant (p < 0.01). Prognosis was also significantly worse in patients positive for CD44 v7–v8 than in those negative for this variant. However, multivariate analysis with the three prognostic indicators tumor size, lymph nodal status, and the degree of lymphatic invasion, has shown that the expression of CD44 v7–v8 antigen in breast carcinoma was not a significant independent prognostic factor and was closely dependent on lymphatic invasion and nodal status. Fourteen of 31 patients who were positive for CD44 v7–v8 experienced recurrences. The mode of recurrence was lymphatic metastasis in 10 out of these 14 patients. Breast cancer cells expressing v7–v8 CD44 antigen have an extremely high affinity for lymph nodes and lymphatic vessels, and are likely to metastasize to distant lymph nodes even at a very early stage in the progression of this disease. This suggests that not only the anatomical factors but also organ affinity plays an important role in the establishment of lymph nodal metastasis of breast cancer.     

Celastrol inhibits the growth of estrogen positive human breast cancer cells through modulation of estrogen receptor α

We studied the chemopreventive efficacy of indole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables, to inhibit tobacco carcinogen-induced lung adenocarcinoma in A/J mice when given following post-initiation or progression protocol. Moreover, we assessed the potential mechanisms responsible for the anticancer effects of I3C. Post-initiation administration of I3C decreased the multiplicity of surface tumors as well as all forms of histopathological lesions, including adenocarcinoma, whereas administration of the compound during tumor progression failed to decrease the multiplicity of surface tumors and early forms of microscopic lesions but reduced the frequency of adenocarcinoma. Mechanistic studies in A549 lung adenocarcinoma cells indicated that the lung cancer preventive effects of I3C are mediated, at least in part, via modulation of the receptor tyrosine kinase/PI3K/Akt signaling pathway.     

Value of post-operative reassessment of estrogen receptor α expression following neoadjuvant chemotherapy with or without gefitinib for estrogen receptor negative breast cancer

The NICE trial was designed to evaluate the possible benefits of adding epidermal growth factor receptor targeted therapy to neoadjuvant chemotherapy in patients with estrogen receptor α (ER) negative and operable breast cancer. Preclinical data have suggested that signalling through the ErbB receptors or downstream effectors may repress ER expression. Here the authors investigated whether gefitinib, given neoadjuvant in combination with epirubicin and cyclophosphamide (EC), could restore ER expression. Eligible patients in the NICE trial were women with unilateral, primary operable, ER negative invasive breast cancer ≥2 cm. Material from patients randomized and completing treatment (four cycles of neoadjuvant EC plus 12 weeks of either gefitinib or placebo) in the NICE trial having available ER status both at baseline and after neoadjuvant treatment were eligible for this study. Tumors with indication of changed ER phenotype (based on collected pathology reports) were immunohistochemically reassessed centrally. 115 patients were eligible for this study; 59 patients in the gefitinib group and 56 patients in the placebo group. Five (4.3%) of 115 tumors changed ER phenotype from negative to positive. A change was seen in three patients in the gefitinib (5.1%) and in two patients in the placebo (3.6%) group with a difference of 1.51% (95% CI, −6.1–9.1). Results of the NICE trial have been reported previously. Post-operative reassessment of ER expression changed the assessment of ER status in a small but significant fraction of patients and should, whenever possible, be performed following neoadjuvant chemotherapy for ER negative breast cancer. Gefitinib did not affect the reversion rate of ER negative tumors.     

Stabilization of estrogen receptor α by USP37 contributes to the progression of breast cancer

Breast cancer is a major cause of cancer-related morbidity and mortality in women. Estrogen receptor-positive breast cancer accounts for roughly 70%-80% of breast tumors, and estrogen receptor alpha (ERα) has been considered as a key driver in promoting breast cancer progression. In the present study, we identified USP37 as a novel modulator in modulating ERα ubiquitination and stability. The expression of USP37 was upregulated in ERα-positive breast cancer and correlated with ERα protein level. High expression of USP37 was associated with unfavorable prognosis. USP37 depletion resulted in significantly decreased ERα protein level, ERα target genes expression as well as the estrogen response element activity in breast cancer cells. Further mechanistic study revealed the interaction between USP37 and ERα: USP37 regulated ERα signaling through modulating protein stability instead of gene expression, in which it stabilized ERα protein via inhibiting the K48-specific polyubiquitination process. Additionally, USP37 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells, which could be further rescued by ERα overexpression. Overall, our study proposed a novel post-translational mechanism of ERα in promoting breast cancer progression. Targeting USP37 may be proved to be a promising strategy for patients with ERα-positive breast cancer.     

Differential role of psoriasin (S100A7) in estrogen receptor α positive and negative breast cancer cells occur through actin remodeling

Psoriasin (S100A7) is a calcium-binding protein that has shown to be highly expressed in high-grade ductal carcinoma in situ (DCIS) and a subset of invasive breast cancers. However, its role in invasion and metastasis is not very well known. In this study, we have shown that S100A7 differentially regulates epidermal growth factor (EGF)-induced cell migration and invasion in ERα^? MDA-MB-231 cells and ERα⁺ MCF-7 and T47D breast cancer cells. Further signaling studies revealed that S100A7 enhances EGF-induced EGFR phosphorylation and actin remodeling that seems to favor lamellipodia formation in ERα^? cells. In addition, S100A7 overexpression enhanced NF-κB-mediated matrix metalloproteinase-9 (MMP-9) secretion in MDA-MB-231 cells indicating its role in enhanced invasiveness. However, S100A7 overexpression inhibited migration and invasion of MCF-7 cells by inactivating Rac-1 pathway and MMP-9 secretion. Moreover, S100A7 overexpressing MDA-MB-231 cells showed enhanced metastasis compared to vector control in in vivo nude mice as detected by bioluminescence imaging. Our tissue microarray data also revealed predominant expression of S100A7 in ERα^? metastatic carcinoma, especially in lymph node regions. Overall these studies suggest that S100A7 may enhance metastasis in ERα^? breast cancer cells by a novel mechanism through regulation of actin cytoskeleton and MMP-9 secretion.     

Increased expression of enolase α in human breast cancer confers tamoxifen resistance in human breast cancer cells

Enolase-α (ENO-1) is a key glycolytic enzyme that has been used as a diagnostic marker to identify human lung cancers. To investigate the role of ENO-1 in breast cancer diagnosis and therapy, the mRNA levels of ENO-1 in 244 tumor and normal paired tissue samples and 20 laser capture-microdissected cell clusters were examined by quantitative real-time PCR analysis. Increased ENO-1 mRNA expression was preferentially detected in estrogen receptor-positive (ER+) tumors (tumor/normal ratio >90-fold) when compared to ER-negative (tumor/normal ratio >20-fold) tumor tissues. The data presented here demonstrate that those patients whose tumors highly expressed ENO-1 had a poor prognosis with greater tumor size (>2 cm, *P = .017), poor nodal status (N > 3, *P = .018), and a shorter disease-free interval (≦1 year, *P < .009). We also found that higher-expressing ENO-1 tumors confer longer distance relapse (tumor/normal ratio = 82.8–92.4-fold) when compared to locoregional relapse (tumor/normal ratio = 43.4-fold) in postsurgical 4-hydroxy-tamoxifen (4-OHT)-treated ER+ patients (*P = .014). These data imply that changes in tumor ENO-1 levels are related to clinical 4-OHT therapeutic outcome. In vitro studies demonstrated that decreasing ENO-1 expression using small interfering RNA (siRNA) significantly augmented 4-OHT (100 nM)-induced cytotoxicity in tamoxifen-resistant (Tam-R) breast cancer cells. These results suggest that downregulation of ENO-1 could be utilized as a novel pharmacological approach for overcoming 4-OHT resistance in breast cancer therapy.     

Phosphorylation of estrogen receptor α serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer

Introduction  

Endocrine therapy is the most important treatment option for women with hormone-receptor-positive breast cancer. The potential mechanisms for endocrine resistance involve estrogen receptor (ER)-coregulatory proteins and crosstalk between ER and other growth factor signaling networks. However, the factors and pathways responsible for endocrine resistance are still poorly identified.     

Peroxiredoxin-1 protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer

Introduction

Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H₂O₂) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer.

Methods

An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H₂O₂, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor.

Results

In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ERα protein was upregulated in PRDX1 high tumors. Exogenous H₂O₂ treatment decreased ERα protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H₂O₂- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ERα levels in breast cancer cells.

Conclusions

PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ERα loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.