Local aromatase activity in human breast tissues

The presence of oestradiol in malignant breast cells is considered to be an important factor in the promotion of growth of the tumor. Therefore the regulation of the local high intra-tissue oestradiol concentrations, regardless of plasma concentrations, has been investigated. Experimental evidence suggests that in situ biosynthesis of oestrogens is at least partly responsible for the local accumulation of these steroids. In this paper we report further data on measurements in fatty and tumor tissues of local aromatase activities and of concentrations of substrates and products of this enzyme. Data are given on localization of aromatase and on steroid concentrations in tumors and in adipose tissues dissected from different quadrants of breasts with malignent tumors. In adipose tissues small variations in steroid concentrations in fatty tissues were found. No tumor-directed gradients in the adipose tissue-concentrations of the androgens dehydro-epiandrosterone, 5-androstene-3ß,17ß-diol, 4-androstene-3,17-dione and testosterone and of the oestrogens oestradiol, oestrone and their sulfates could be detected. Furthermore no consistent pattern could be recognized in the aromatase activities in the fatty tissues dissected from tumor-bearing and non-affected quadrants of the same breast. No correlations between aromatase activity measured in vitro and product concentrations in vivo were found. Therefore the mechanisms for regulation of the local oestradiol levels in breast tissues remain unknown.     

Aberrant expression of Arpin in human breast cancer and its clinical significance

Arpin (Arp2/3 complex inhibitor), a novel protein found in 2013, plays a pivotal role in cell motility and migration. However, the precise role of Arpin in cancer is unclear. This study investigated the expression of Arpin in breast cancer and evaluated its correlation with the characteristics of clinical pathology and prognosis of breast cancer patients. Immunohistochemistry (IHC) for Arpin protein was performed on formalin‐fixed, paraffin‐embedded 176 breast cancer tissues and 43 normal breast tissues while qRT‐PCR for Arpin mRNA with 104 paired tumour and paratumoural tissues from breast cancer patients respectively. The association of Arpin expression with clinical pathological features and survival was assessed in a retrospective cohort analysis of patients. The results showed that the expression of Arpin protein in cancer tissues was lower compared to that in normal breast and the expression of Arpin mRNA was also lower in cancer tissues than that in the matched paratumoural tissues. Among the 176 breast cancer patients, the lower expression of Arpin was significantly associated with advanced tumour, nodes and metastasis system stage, and the reduced Arpin expression was strongly associated with axillary lymph node metastasis using univariate and multivariate logistic regression analysis [odds ratio: 3.242; 95% confidence interval (CI): 1.526, 6.888; P < 0.05]. Furthermore, Arpin expression was an independent risk factor for recurrence‐free survival (HR: 0.373; 95% CI: 0.171, 0.813; P < 0.05). As Arpin expression was first examined in human breast cancer tissues with qRT‐PCR and IHC, our results suggest that Arpin downregulation may contribute to the initiation and development of breast cancer metastasis. Therefore, as a potential predictive marker, Arpin deserves future studies.     

Future uses for aromatase inhibitors in breast cancer

Over recent years highly potent, well-tolerated aromatase inhibitors have been developed, which essentially obliterate peripheral aromatase activity in postmenopausal women. Their role as the optimal second-line agents (post-tamoxifen) for the treatment of advanced breast cancer has recently been established in large comparative clinical trials. Their testing as adjuvant therapy is warranted, but their eventual application in this (or the prophylactic) setting will be dependent on the currently unknown effects of profound oestrogen deprivation on the physiology of postmenopausal women as well as on its efficacy. It is also possible that these new compounds could suppress oestrogen synthesis in premenopausal women, but the consequences on ovarian folliculogenesis might prevent their widespread use in this group of patients.     

Evaluation of chromogranin A expression in serum and tissues of breast cancer patients

Human chromogranin A (CgA) is a member of the granin family and is widely distributed in large dense core granules of endocrine and neuroendocrine cells. A variety of non-neuroendocrine carcinomas arising in various tissues show patterns of neuroendocrine differentiation. Expression of CgA has been documented in epithelial cells of normal mammary gland as well as in breast cancers, and elevation of serum CgA has been detected in patients with breast cancer. Our study was undertaken to evaluate the relationship between serum CgA levels and neuroendocrine features in breast cancer. In addition, we evaluated the expression of serum CgA in patients affected by breast cancer compared to controls and the relationship between serum CgA and tumor histology, extent of disease, lymph node status, tumor stage and serum CA 15.3 levels. We enrolled 266 patients with infiltrating ductal or lobular breast carcinoma and a group of 100 age-matched healthy women serving as controls. Serum CgA and CA 15.3 were assayed by specific immunoradiometric methods. The overall sensitivity of CgA and CA 15.3 was 0.06 and 0.34, respectively (chi2 19.1, p<0.0005). No relationship was found between serum levels of CgA and tumor histology, extent of disease, lymph node status or tumor stage while serum levels of CA 15.3 were strongly correlated with all these variables but tumor histology. No relationship was found between serum levels of CgA and CA 15.3. Immunostaining against CgA, CgB, NSE and synaptophysin was performed on primary tumor tissue of 14 serum CgA-positive and 24 serum CgA-negative patients and was negative in all cases. We also evaluated eight cases of pathologically-proven neuroendocrine breast cancer: only four and two of these showed positive CgA immunostaining and increased serum CgA concentration, respectively. In conclusion, serum CgA assay offers no additional information regarding the presence, the extent and the histology of breast cancer compared to the CA 15.3 assay. Moreover, serum CgA was not an accurate marker to identify or exclude the rare neuroendocrine differentiation of breast cancer. We therefore conclude that CgA is not useful as a serum marker in breast cancer.     

Aromatase, aromatase inhibitors, and breast cancer

Estrogens are known to be important in the growth of breast cancers in both pre and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also in innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs. Article from the special issue on 'Targeted Inhibitors'.     

Immunohistochemical analysis of MUC5B apomucin expression in breast cancer and non-malignant breast tissues.

A deregulation of several MUC genes (MUC1, MUC2, MUC3, MUC5AC, and MUC6) was previously demonstrated in breast carcinomas. Considering that recently we found the "non-mammary" MUC5B mRNA in primary breast tumors (Berois et al. 2003), we undertook the present study to evaluate the expression profile of MUC5B protein product in breast tissues, using LUM5B-2 antisera raised against sequences within the non-glycosylated regions of this apomucin. Expression of MUC5B by breast cancer cells was confirmed by immunocytochemistry, in situ hybridization, and Western blot on MCF-7 cancer cells. Using an immunohistochemical procedure, MUC5B apomucin was detected in 34/42 (81%) primary breast tumors, in 13/14 (92.8%) samples of non-malignant breast diseases, in 8/19 (42.1%) samples of normal-appearing breast epithelia adjacent to cancer, and in 0/5 normal control breast samples. The staining pattern of MUC5B was very different when comparing breast cancer cells (cytoplasmic) and non-malignant breast cells (predominantly apical and in the secretory material). We analyzed MUC5B mRNA expression using RT-PCR in bone marrow aspirates from 22/42 patients with breast cancer to compare with MUC5B protein expression in the primary tumors. Good correlation was observed because the six MUC5B-positive bone marrow samples also displayed MUC5B expression in the tumor. Our results show, for the first time at the protein level, that MUC5B apomucin is upregulated in breast cancer. Its characterization could provide new insights about the glycobiology of breast cancer cells.     

Aberrant AID expression and human cancer development

Cancer develops via a multistep process that occurs through the accumulation of somatic mutations of tumor-related genes that govern cell proliferation, regeneration, and apoptosis. The question how normal cells acquire the genetic changes that lead to malignant transformation is, however, unknown at present. Activation-induced cytidine deaminase (AID) produces immune-diversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes. Unfortunately, this function of AID as a genome mutator could aim at the generation of somatic mutations in various host genes of non-lymphoid tissues and contribute to tumorgenesis. Notably, aberrant AID expression can be triggered by several pathogenic factors, including Helicobacter pylori infection and proinflammatory cytokine stimulation, in human epithelial cells, whereas AID expression is absent in those cells under physiologic conditions. Thus, aberrant AID activity in epithelial tissues may provide the critical link between inflammation, somatic mutations, and cancer development.     

Overview of adjuvant trials of aromatase inhibitors in early breast cancer

The third-generation aromatase inhibitors are an important class of drugs for use in adjuvant therapy for postmenopausal women with resected estrogen receptor positive breast cancer. Multiple large prospective randomized trials have established their value in this setting and provided guidance for their use in clinical management. This review will outline the trials that have provided evidence on the value of the aromatase inhibitors in the adjuvant setting as well as the ongoing trials that will expand our knowledge of how to use them most effectively.     

Impact of aromatase inhibitors on bone health in breast cancer patients

Following the implementation of the third generation aromatase inhibitors in the treatment algorithms for early breast cancer, special attention has been given to the influence of these drugs on bone health. Due to their potent estrogen suppression, the aromatase inhibitors anastrozole and letrozole, as well as the aromatase inactivator exemestane, enhance bone loss in postmenopausal women reflected in decreasing levels of bone mineral density. Moreover, all major phase III trials involving aromatase inhibitors in the adjuvant setting have reported increased fracture rates. All in all, there is no hard evidence to suggest major differences between the individual compounds concerning their side-effects on bone. The consequences of AI therapy on bone are in addition modified by a variety of factors like the BMD level prior to therapy, time since menopause, and vitamin D status. Strategies to avoid bone loss during AI therapy have shown promising results. Thus, bisphosphonates have been shown to prohibit bone loss during AI therapy if used upfront. Novel treatment strategies, like antibodies against RANKL have been developed and promising preliminary results have been published from early trials. Standardized guidelines to avoid or minimize bone loss during AI therapy have been developed, in most countries involving calcium and vitamin D supplementation, as well as BMD measurements to identify patient subgroups demanding bisphosphonate therapy.     

The control and biological importance of intratumoural aromatase in breast cancer

The existence of aromatase activity in human breast carcinomas has been established for about 20 years but the clinical and biological importance of this remains unclear. A number of studies in clinical material suggest that aromatase activity may be a prerequisite of response to aromatase inhibitors and that aromatase activity may be enhanced in those tumours relapsing during treatment with one such inhibitor, aminoglutethimide. These results would carry more significance, however, if it was demonstrable that the growth of breast carcinomas is affected by the conversion of androgens to oestrogens by intratumoural aromatase. We have tried to address this by establishing model systems with aromatase-transfected MCF7 breast cancer cells. We have demonstrated that these cells can be stimulated mitogenically with androgen and that this proliferation is suppressible with aromatase inhibitors. Similarly the growth of aromatase transfected cells but not wild type cells as xenografts is supported by androstenedione and inhibitable by both the steroidal aromatase inhibitor, 4-hydroxyandrostenedione and the non-steroidal inhibitor, CGS 20267. Work with the former of these, which is a suicide inhibitor allowed us to demonstrate that growth can proceed with aromatase activity approximating to the highest level seen in breast carcinomas indicating that at least at this extreme level the intratumoural conversion of androgens to oestrogens may indeed be able to support tumour growth. Further work with this model system should allow us to define the minimal amount of intratumoural activity which can support tumour growth.     

Aberrant gata-3 expression in human pancreatic cancer.

Gata-3 has been shown to specifically alter its expression patterns in different types of cancers. Recent evidence suggests that an interference of Gata-3 exists in the TGF-beta signaling pathway. To determine the role of Gata-3 in pancreatic cancer, pancreatic cancer samples were analyzed in comparison to normal pancreatic tissues. Furthermore, four different pancreatic cancer cell lines with different alterations of the TGF-beta pathway were studied. To evaluate if a potential relationship with TGF-beta signaling pathway exists, we correlated mRNA expression levels with the expression of TGF-betas, TGF-beta receptors, and Smad-3. Finally, we analyzed the influence of TGF-beta on Gata-3 expression in vitro. All pancreatic cancer samples demonstrated a marked overexpression of Gata-3 mRNA and protein. Immunohistochemical staining revealed strong and persistent cytoplasmic Gata-3 immunoreactivity in cancer cells. In an electrophoretic mobility shift assay, a disturbed nuclear translocation was confirmed. The expression of Gata-3 showed a significant correlation with the expression of TGF-betas, TGF-beta receptors, and Smad-3. TGF-beta responsive cell lines showed a downregulation of Gata-3 mRNA upon TGF-beta exposure, whereas in TGF-beta-unresponsive cell lines, Gata-3 mRNA expression persisted at high levels. Furthermore, strong specific upregulation of Gata-3 impaired nuclear translocation and its cooperative action with the TGF-beta pathway, suggesting that Gata-3 plays a central role in human pancreatic cancer.     

Clinical use of aromatase inhibitors in the treatment of advanced breast cancer

The aim of endocrine therapy is to reduce the estrogenic stimulus for tumour growth. After failure of tamoxifen — the standard “first-line” hormonotherapy for advanced breast cancer (ABC) — the most frequently prescribed endocrine therapies are progestins and aromatase inhibitors (AIs) (“second-line” hormonotherapy). Estrogen deprivation through AIs provides effective treatment of hormone-dependent ABC in postmenopausal (PMP) women. Over the past few years, the goals of our research programme were to develop more potent, more selective and better tolerated AIs than our first AI, aminoglutethimide (AG). Lentaron® (4-hydroxyandrostenedione, formestane), a highly selective steroidal compound was the first of the new AIs to be used in clinical trials. It is a substrate analogue, administered as an i.m. injection every 2 weeks. It is effective in the treatment of ABC with an objective response rate (ORR) similar to tamoxifen and megestrol acetate (MA) and is generally well tolerated; rare instances of injection site reactions have been reported. Afema® (fadrozole HCl), a non-steroidal AI is active orally, and effectively suppresses estrogen levels in PMP women, but it is not completely selective for aromatase when administered at higher than therapeutic doses. At the therapeutic dose of 1 mg twice a day it has an anti-tumour efficacy similar to MA, a good tolerability and no clinically relevant effects on other hormones of the endocrine system. Letrozole is the fourth of our AIs in clinical development. It is a non-steroidal, achiral, orally active, potent and highly selective competitive AI. Clinical endocrine studies have shown that the dose of 0.5 mg a day is the lowest dose achieving maximum estrogen suppression. Over a wide dose range, a lack of clinically relevant effects on other hormones of the endocrine system has confirmed its high selectivity. In four phase Ib/IIa studies in PMP patients with ABC who failed previous therapy, letrozole produced ORRs of 9, 31, 33 and 36%. One phase IIb/III study has been completed and two others are ongoing, comparing two doses of letrozole with MA or AG to confirm the anti-tumour efficacy of letrozole in the treatment of ABC in PMP women after treatment with anti-estrogens. Preliminary results from the completed trial show that letrozole 2.5 mg is superior to 0.5 mg in terms of ORR, time to progression and time to treatment failure, and is superior to the standard dose of MA in terms of ORR and tolerability. Therefore letrozole 2.5 mg can be recommended as a first choice for the treatment of PMP patients with hormone receptor-positive or unknown ABC after anti-estrogen therapy.