Aromatization inhibition alone or in combination with GnRH agonists for the treatment of premenopausal breast cancer patients.

Aromatase inhibition in postmenopausal women causes a marked fall in the plasma levels of oestrogens and is an effective treatment for breast cancer, however, trials with aminoglutethimide found that this aromatase inhibitor was ineffective in suppressing plasma oestrogen levels in premenopausal breast cancer patients. We found that the more potent inhibitor, 4-hydroxyandrostenedione (4-OHA), which can suppress oestrogen synthesis in rodents and non-human primates with intact ovarian function, was also unsuccessful as an oestrogen suppressant in premenopausal women at its maximum tolerated dose (500 mg/week i.m.). GnRH agonists are effective suppressants of ovarian oestrogen synthesis but oestrogen production from peripheral sites is unaffected. Our studies of a combination of the GnRH agonist goserelin and 4-OHA demonstrated that the combination caused greater oestrogen suppression than goserelin alone and led to objective clinical response in 4/6 breast cancer patients after their relapse from treatment with goserelin as a single agent. The combination of a GnRH agonist and an aromatase inhibitor should be subjected to clinical trials.     

The menopause,hormone replacement therapy and breast cancer

Concern exists that the reduction in breast cancer risk associated with the onset of the menopause will be negated with exposure to hormone replacement therapy (HRT). Evidence from large-scale randomised HRT trials support observational data that have shown a modest increase in breast cancer risk with long-term use (i.e. >15 years) of combined therapy, although this falls following HRT cessation suggesting a growth-promoting effect. Randomised evidence demonstrates that the efficacy of anti-estrogens, aromatase inhibitors and raloxifene in the treatment and chemoprevention of breast cancer are restricted to women with oestrogen receptor positive (ER +ve) disease; however, HRT has not been associated conclusively with a predominance of hormone sensitive breast cancer. Despite stimulating the breast cancer cell growth, HRT has not been shown to increase breast cancer recurrence or mortality when prescribed to breast cancer survivors experiencing oestrogen deficiency symptoms and randomised trials have been recommended and commenced. In conjunction with controlled breast cancer trials demonstrating a therapeutic benefit of high dose estrogens and interest in the use of additive oestrogen therapy in patients developing resistance to oestrogen deprivation, the dogma that HRT is an absolute contra-indication following diagnosis is challenged.     

Pilot and feasibility study: comparative proteomic analysis by 2-DE MALDI TOF/TOF MS reveals 14-3-3 proteins as putative biomarkers of response to neoadjuvant chemotherapy in ER-positive breast cancer

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity? Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.     

Effect of raloxifene on vascular endothelial growth factor expression in breast carcinomas of postmenopausal women

Objective:  This study aimed to evaluate the effect of raloxifene on vascular endothelial growth factor (VEGF) expression in breast carcinomas of postmenopausal women.
Materials and methods:  Sixteen postmenopausal patients with operable stage II, oestrogen receptor-positive, infiltrating ductal breast carcinoma were treated with raloxifene at a dose of 60 mg/day, for a period of 28 days prior to definitive surgery. Tumour size varied from 3 to 5 cm (mean 3.7 cm) and mean age of patients was 61.8 years (range 49–72 years). Tumour samples were obtained by incisional biopsy at the time of diagnosis and again at the time of surgery. Immunohistochemical evaluation of VEGF expression was assessed semiquantitatively based on fraction of stained tumour cells and on intensity of staining. McNemar's test of symmetry was used to evaluate agreement between positive or negative classification of VEGF expression prior to and following raloxifene treatment ( P  < 0.05).
Results:  Fourteen of the 16 patients (88%) were classified as positive for VEGF expression prior to raloxifene treatment, while only 5 (31%) were classified as positive following treatment ( P  < 0.007).
Conclusion:  Raloxifene significantly reduced VEGF expression in these oestrogen receptor-positive breast carcinomas of postmenopausal women.     

Methylsulfonylmethane suppresses breast cancer growth by down-regulating STAT3 and STAT5b pathways

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1α, VEGF, BrK, and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90α, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis. Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.     

Oestrogen Receptor in Human Breast Cancer Tissue and Response to Endocrine Therapy

Oestrogen receptor determinations were done in metastatic breast cancer tissue of patients with advanced breast cancer. In 37 patients with progressive disease evaluation of the response to endocrine treatment was possible, following the criteria of the E.O.R.T.C. Co-operative Breast Cancer Group. In 20 patients with receptor-negative tumours two objective remissions were noted; in 17 patients with receptor-positive tumours 14 objective remissions were seen. There seems to be a striking correlation between the presence or absence of oestrogen receptor in tumour tissue and the clinical response to hormonal therapy.     

Selective cytotoxicity of ascochlorin in ER-negative human breast cancer cell lines

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.     

Endocrine approaches for the treatment of early and advanced breast cancer in postmenopausal women

Preventing clinical progression is the major treatment goal for both early and advanced breast cancer. For hormone-responsive cases (about 70% of the total), this can necessitate the use of sequential hormone therapies at various points during the patient's life. Newer hormonal therapies, such as the third-generation aromatase inhibitor anastrozole, are now competing with tamoxifen as first choice endocrine therapy in breast cancer. In addition, a further non-steroidal aromatase inhibitor letrozole has been shown to be beneficial when given at completion of 5 years adjuvant tamoxifen. In light of these new data, current treatment paradigms need to be reviewed. Already well established as second-line treatments for advanced breast cancer, the improved risk:benefit profiles of anastrozole and letrozole compared with tamoxifen mean that these agents are now also recognised alternative treatments in the first-line relapse setting. More recent studies demonstrate that anastrozole may also have an improved risk:benefit profile compared with tamoxifen when used as initial adjuvant therapy in early breast cancer. Anastrozole is also being evaluated as a preventative treatment in women at high risk of developing breast cancer. A new addition to the endocrine treatment armamentarium is the oestrogen receptor antagonist fulvestrant, which, unlike tamoxifen, has no agonist effects. Fulvestrant is at least as effective as anastrozole in the second-line treatment of advanced breast cancer, and provides similar benefits to tamoxifen when used as first-line therapy in patients with advanced, hormone receptor-positive tumours.     

The importance of local synthesis of estrogen within the breast

Tumor aromatase has been correlated with clinical response to treatment with aminoglutethimide in patients with estrogen receptor-positive advanced breast cancer. There was a significant positive relationship between aromatase status and likelihood of response to therapy, none of five patients with aromatase-negative tumors responding compared with 11 of 18 having aromatase-positive cancers. Measurement of aromatase in sequential biopsies of large primary tumors before and during treatment with aminoglutethimide-hydrocortisone showed a marked but paradoxical rise in activity following therapy. Assays of aromatase in adipose tissue from the different quadrants of mastectomy specimens from patients with breast cancer indicate that activity was always higher in quadrants associated with tumor as compared with non-involved quadrants. These results emphasize the importance of local estrogen synthesis within the breast in terms of both the natural history and behavior of breast cancers.     

Comparative trial of nafoxidine and ethinyloestradiol in advanced breast cancer: an E.O.R.T.C. study.

A randomized clinical trial of nafoxidine, a non-steroidal oestrogen antagonist, and ethinyloestradiol in postmenopausal patients with advanced breast cancer produced objective remissions in 31% of 49 women receiving nafoxidine and in 14% of 49 receiving ethinyloestradiol. The differences in remission rates was almost significant (0.05 less than P less than 0.10). Life-threatening complications were more frequent with ethinyloestradiol than with nafoxidine but the latter produced specific toxic reactions on skin and hair that may limit its practical usefulness. Synthetic oestrogen antagonists may occupy a privileged place in the treatment of breast cancer, and other representatives of this new class of compounds should be accurately assessed in randomized clinical trials.     

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.     

Effect of tamoxifen and 2-methoxyestradiol alone and in combination on human breast cancer cell proliferation

Endocrine therapy is widely accepted for the treatment of hormone receptor-positive breast cancer. However, in many cases eventually resistance will develop and tumor regrows. Combination therapy may be one way to resolve this problem. In the present study we investigated the effect of a combination of the widely used antiestrogen tamoxifen with the endogenous estradiol metabolite 2-methoxyestradiol (2-ME) on the proliferation of human estrogen receptor-positive and receptor-negative breast cancer cells.The receptor-positive cell line MCF-7 and the receptor-negative cell line BM were treated with 4-hydroxytamoxifen (4-OHTam) and 2-methoxyestradiol in the concentration range of 0.8-25 microM alone and equimolar combinations for 4 days. The proliferation of the cells was determined using the ATP-chemosensitivity test.4-Hydroxytamoxifen inhibited proliferation of MCF-7 and BM cells with IC(50) values of 31 and 10 microM, the corresponding figures for 2-methoxyestradiol were 52 and 8 microM. The combination showed IC(50) values of 6 microM and 4 microM.These results indicate that a combination of tamoxifen with 2-methoxyestradiol showed an additive inhibitory effect concerning the proliferation of estrogen receptor-positive and receptor-negative breast cancer cell lines. Thus a combination of these substances may allow ameliorating of adverse events of tamoxifen by reducing its concentrations and probably also drug resistance and should be tested in clinical trials.     

The relationship between factors affecting endogenous oestradiol levels in postmenopausal women and breast cancer

Breast cancer accounts for 1 in 4 of all female cancers worldwide; approaching 13,000 women dying per year in the UK alone. Seventy five per cent of all diagnosed breast cancers are oestrogen receptor (ER) positive. Ovarian synthesis of oestrogens ceases at menopause and as breast cancer is more prevalent in postmenopausal women the non-ovarian sources of oestrogen are important in disease progression. There is now considerable evidence that associates increased breast cancer risk with prolonged exposure to oestrogens hence greater attention is now being given to determining whether the measurement of plasma oestrogen may assist in identifying chemoprevention target groups. Studies suggest that in most postmenopausal patients the intra-tumoural concentrations of oestrogens are up to 20-fold higher than those present in the plasma however, while the extent of biosynthesis of oestrogens within breast tissue is a major determinant of local exposure, plasma levels are a useful indicator of overall metabolism in peripheral tissues. As such it is important to understand factors that influence these measurements. This review summarises the impact of lifestyle such as body mass index, together with the role of genetic polymorphisms placed within the context of designing future epidemiological studies and breast cancer risk algorithms.     

Reduction in serum IL-6 after vacination of breast cancer patients with tumour-associated antigens is related to estrogen receptor status

Elevated serum IL-6 concentrations have been associated with poor prognosis in a variety of cancers, and decreases in serum IL-6 concentrations have been reported after chemotherapy. We have demonstrated that serum IL-6 concentrations are elevated in breast cancer patients [normal women 0.7 +/- 2.5 pg/ml (n=36), breast cancer patients 38.3 +/- 138.7 pg/ml (n = 111)]. After vaccination of breast cancer patients with a combination of tumour-associated antigens and biological adjuvants (IL-2 and GM-CSF), the concentration of IL-6 decreased significantly (P<0.05) to 8.1 +/- 14.6 pg/ml (n=85). Other studies have shown that oestrogen suppresses IL-6 production in oestrogen receptor positive breast cancer cells. We have demonstrated that the decrease in IL-6 associated with vaccination is related to the oestrogen receptor status of the tumours from breast cancer patients, as a decrease in IL-6 from 124.0 +/- 267.5 pg/ml (n=26) to 6.2 +/- 11.0 pg/ml (n=34) only occurs in patients with oestrogen receptor negative tumours. The IL-6 concentration in breast cancer patients with oestrogen receptor positive tumours remained unchanged (9.5 pg/ml before vaccination, and 9.3 pg/ml after vaccination). These results suggest that postmenopausal women with oestrogen receptor negative breast cancers, who do not respond well to either hormonal therapy with tamoxifen or adjuvant chemotherapy, may have a significant response to vaccination with autologous tumour-associated antigens.     

The pineal gland and breast cancer: serum levels of melatonin in patients with mammary tumors and their relation to clinical characteristics

Several studies have suggested that the pineal gland hormone melatonin may influence the growth of breast cancer. The importance of melatonin blood concentrations in the clinical history of human breast cancer, however, has still to be defined. To further investigate this problem, we used a RIA method to assay serum levels of the pineal hormone in 74 untreated breast cancer patients, clinical stage T1-3 NO-2 MO, and in 46 age-matched healthy women as controls. Mean serum melatonin levels were significantly higher in patients than in controls. Melatonin concentrations were highest in breast cancer patients with the best prognosis (i.e. estrogen receptor-positive/node-negative cases). Mean levels of melatonin were significantly higher in estrogen receptor-positive patients than in the negative ones. They were also higher in node-negative than in node-positive cases, and in progesterone receptor-positive patients than negative ones, but none of these differences was statistically significant. No difference was observed in relation to menopausal status and to tumor histotype. These results suggest that melatonin plays a role in the hormone-dependency of human breast cancer.     

Cytotoxicity and DNA interactions of some platinum(II) complexes with substituted benzimidazole ligands

In the present study, four Pt(II) complexes with 2-ethyl (1)/or benzyl (2)/or p-chlorobenzyl (3)/or 2-phenoxymethyl (4) benzimidazole carrier ligands were evaluated for their in vitro cytotoxic activities against the human HeLa cervix, oestrogen receptor-positive MCF-7 breast, and oestrogen receptor-negative MDA-MB 231 breast cancer cell lines. The plasmid DNA interactions and inhibition of the BamHI restriction enzyme activities of the complexes were also studied. Complex 3 was found to be more active than carboplatin for all examined cell lines and comparable with cisplatin, except for the HeLa cell line.     

Clinical development of aromatase inhibitors for the treatment of breast and prostate cancer

Numerous aromatase inhibitors are under development for breast cancer treatment. The major aims are to obtain a drug which at its dose of maximum efficacy has no effect on other endocrine systems, has no clinical side-effects and its convenient to administer. During the early clinical stages of development detailed endocrine and pharmacokinetic analyses are a valuable aid in the establishment of a drug's selectivity and its optimum dose, route and frequency of administration. The optimal dose may be defined as the minimum that will achieve maximal and sustained suppression of aromatase activity. This has generally been measured indirectly by comparing the suppression of plasma oestrogen levels at a selection of dosages. This approach has major advantages in speeding dose selection for therapeutic clinical trials. However, it also has some disadvantages including the unproven assumption that clinical response has a direct relationship with the degree of oestrogen suppression. In addition there are technical difficulties of analysis, of wide variability in endocrine response between patients and of demonstrating oestrogen suppression to be equivalent between doses (necessary to show maximal suppression). The direct measurement of aromatase inhibition in vivo by isotopic infusion analysis provides support to these indirect estimates. Its value is shown by our recent results with CGS16949A. The additional value of collating pharmacokinetic and endocrine measurements is apparent from our investigations of 4-hydroxyandrostenedione (4-OHA) and pyridoglutethimide. A consideration of our experience with these inhibitors may be helpful in directing the development of future agents.

Whilst the value of aromatase inhibition in breast cancer is established its value in prostatic cancer is in doubt: we have found that 4-OHA is only poorly efficacious in advanced prostatic cancer.     

Expression of the oestrogen responsive protein pS2 in human breast cancer

The trefoil peptide pS2 was discovered in a breast cancer cell line as a result of its oestrogen responsive character. The expression of pS2 in breast tumours in vivo is also likely to be an oestrogenic effect and as such, the presence of pS2 in oestrogen receptor positive breast cancer is evidence of an intact oestrogen response pathway and an indicator of putative hormone responsiveness. Consistent with this, clinical studies of breast cancer have revealed a correlation between pS2 expression and favourable tumour characteristics as well as response to endocrine therapy.