Lev M. Berstein

Effect of metformin on life span and on the development of spontaneous mammary tumors in HER-2/neu transgenic mice.

Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. Insulin/insulin-like growth factor 1 (IGF-1) signaling molecules that have been linked to longevity include DAF-2 and InR and their homologues in mammals, and inactivation of the corresponding genes is followed by increased life span in nematodes, fruit flies and mice. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which mimetic effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. The chronic treatment of female transgenic HER-2/neu mice with metformin (100 mg/kg in drinking water) slightly decreased the food consumption but failed in reducing the body weight or temperature, slowed down the age-related rise in blood glucose and triglycerides level, as well as the age-related switch-off of estrous function, prolonged the mean life span by 8% (p < 0.05), the mean life span of last 10% survivors by 13.1%, and the maximum life span by 1 month in comparison with control mice. The demographic aging rate represented by the estimate of respective Gompertzs parameter was decreased 2.26 times. The metformin-treatment significantly decreased the incidence and size of mammary adenocarcinomas in mice and increased the mean latency of the tumors.

Phase 2 randomized trial of primary endocrine therapy versus chemotherapy in postmenopausal patients with estrogen receptor-positive breast cancer

Few studies have compared primary neoadjuvant endocrine therapy with neoadjuvant chemotherapy in breast cancer patients. The need for preoperative chemotherapy with doxorubicin or taxanes may be reduced in postmenopausal patients with estrogen receptor (ER)‐positive and/or progesterone receptor (PgR)‐positive tumors. This randomized, controlled, phase 2 study evaluated the efficacy of neoadjuvant chemotherapy compared with endocrine treatment with aromatase inhibitors in postmenopausal women with ER‐positive and/or PgR‐positive breast cancer.

Metformin slows down aging and extends life span of female SHR mice.

Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which resemble effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. Antidiabetic biguanides are most promising among them. Here we show the the chronic treatment of female outbred SHR mice with metformin (100 mg/kg in drinking water) slightly modified the food consumption but decreased the body weight after the age of 20 months, slowed down the age-related switch-off of estrous function, increased mean life span by 37.8%, mean life span of last 10% survivors by 20.8%, and maximum life span by 2.8 months (+10.3%) in comparison with control mice. On the other side, treatment with metformin failed influence blood estradiol concentration and spontaneous tumor incidence in female SHR mice. Thus, antidiabetic biguanide metformin dramatically extends life span, even without cancer prevention in this model.

Macrophages, estrogen and the microenvironment of breast cancer

Estrogen is a major mitogenic stimulus to established breast cancer. Estrogen sources include ovarian, extraglandular sites and breast tissue. Which source primarily maintains benign and breast cancer tissue estrogen concentrations remains unclear. While macrophages may comprise up to 50% of the mass of breast carcinomas, previous studies neglected to study them as possible sources of estrogen. We present evidence that breast macrophages constitute an in situ source of estradiol and that the amount produced is sufficient to mediate cellular proliferation. We utilized immunohistochemistry and RT-PCR to study cell-specific aromatase expression in (i) 29 breast biopsies, (ii) human monocytes/macrophages and (iii) a myeloid cell line (THP-1) capable of differentiating into macrophages. Use of a breast cancer cell line (MCF-7) provided biologic confirmation of the role of aromatization in cell proliferation. We demonstrated considerable amounts of immunoreactive-aromatase (irARO) in breast tissue macrophages and a positive correlation between the proportion of irARO present in macrophages and lesion severity. Using in vitro techniques, we demonstrated that monocytes and THP-1 cells require differentiation into macrophages to produce aromatase in amounts approaching placental levels. The amount of estrogen produced by THP-1 cells stimulated MCF-7 cells to proliferate, an effect blocked by aromatase inhibitors. Estrogen production by macrophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cells and to autoregulate cytokine production. These findings represent a new dimension of cellular regulation in breast tissue with major biologic implications, amenable to pharmacological manipulation.

Evidence against involvement of p53 polymorphism in breast cancer predisposition

Dear Sir p53 codon 72 Arg/Pro polymorphism appears to be one of the most promising low-penetrance candidates for breast cancer predisposition. Indeed, p53 Pro allele is among a very few genetic variants, which retain the unfavorable significance upon the systematic meta-analysis of breast cancer polymorphic genes.1 In addition, there is sound biological evidence arguing for the functional difference between Arg and Pro alleles. These variants have the distinct ability to mediate HPV E6 protein degradation, activate transcription of p53-responsive genes, induce apoptosis and suppress malignant transformation in at least some experimental systems.2–4 Furthermore, p53 codon 72 polymorphism may be a subject of natural selection by the level of sun exposure, as its frequency gradually changes with latitude.5 In recognition of the crucial impact of the p53 gene in tumor development, the epidemiology of the Arg/Pro allele variations was tested intensively for all major cancer types, including lung, cervical, colorectal, bladder and other neoplasms. Unfortunately, none of the reported gene-disease associations demonstrated consistency in the literature.2,4,6–9 Despite promising assumptions, the role of the Arg/Pro allelism in breast cancer susceptibility was examined in only a few reports and some of these had a very small number of observations.10–15 We have re-assessed the hypothesis on involvement of p53 polymorphism in the determination of breast cancer risk. In addition to a traditional comparison of breast cancer patients (n 448; mean age 55.4 years; age range: 29–83 years) and healthy female donors (n 249; mean age 38.5 years; age range: 18–54 years), we attempted to increase the demonstrability of the study by invoking the subjects with extreme degrees of either breast cancer predisposition or cancer tolerance. In particular, we also genotyped 81 bilateral breast cancer patients (mean age at onset of the first tumor: 49.5 years; age range: 30–85 years; mean age at onset of the contralateral tumor: 56.5 years; age range: 37–87 years) as well as 144 elderly tumor-free women (mean age 79.5 years; age range: 75–90 years). Histologically confirmed cases of unilateral and bilateral breast cancer were collected in N.N. Petrov Institute of Oncology (St.-Petersburg, Russia). Female donors were randomly recruited from the blood transfusion unit located in the same hospital; a medical permit to donate blood was considered to be sufficient proof of their healthy status. Elderly tumor-free women were enrolled from various hospitals of St.-Petersburg; results of the current clinical examination, previous records of health professionals and information obtained by questionnaires were taken into consideration to ensure the lack of cancer history for elderly subjects. All affected and non-affected subjects were Caucasians of Slavic origin residing in St.-Petersburg, Russia. Peripheral leukocytes were the source of normal DNA for unilateral breast cancer patients as well as for middle-aged and elderly controls. To accumulate a significant number of biBC cases, we invoked the collection of paraffin-embedded archival tissues, using specimens from nontumor parts of the affected breasts or intact lymph nodes for the DNA extraction. The details on DNA isolation have been described in our earlier report.16 p53 codon 72 polymorphism was tested by PCR-RFLP analysis using BstU1 restriction endonuclease. Because the non-coding p53 allelic variations (MspI polymorphism in intron 6 and 16 bp deletion/insertion polymorphism in intron 3) were also a subject of controversial results,1,6 we included them in our study as well. The obtained data are summarized in the Table I. Distribution of codon 72 polymorphic alleles showed striking similarity in all the groups studied. If the affected and non-affected groups were combined (i.e., bilateral and unilateral breast cancer patients vs. middle-aged and elderly tumor-free females), the power of the study was 99.8% to exclude an odds ratio (OR) of 2. The actual OR for the Pro allele carriers was equal to 0.96 (0.74–1.25), leaving the published meta-analysis1 estimate of 1.27 outside the 95% confidence interval (CI) (Table II). Contrary to earlier reports,1,6 carriers of the rare MspI or 16 bp variants did not appear to be at risk as well (OR 0.74 (0.54–1.01) and 0.86 (0.63–1.16), respectively). Furthermore, none of the studied alleles or genotypes showed a relationship with breast cancer clinical features, such as age at onset, menopausal status, TNM stage, or family history (data not shown). Finally, in contrast to earlier suggestions,6 there was no specific unfavorable significance for combined p53 genotypes, including the haplotype composed of the 3 minor allelic variants (data not shown).

Tumor estrogen content and clinico-morphological and endocrine features of endometrial cancer.

ObjectivesTo compare estrogen concentrations in endometrial cancer tissue with those in macroscopically normal endometrium and with certain morphological characteristics of the tumor and endocrine parameters in patients.MethodsThe estradiol content was evaluated by radioimmunoassay after homogenization and extraction in 78 adenocarcinomas (61 from postmenopausal patients).ResultsHigher concentrations of estradiol in tumor tissue samples than in macroscopically normal endometrium were found in patients of both reproductive and postmenopausal age. This difference was the same in patients with either endometrial carcinoma type I or type II. No association between tumor steroid receptor levels, estradiol concentrations in blood serum, and timing of menopause with intratumoral estradiol contents was discovered. Estradiol concentrations in tumor tissues correlated positively with the clinical stage of disease and rate of tumor invasion (in patients with peripheric/lower type of fat topography), and negatively with tumor differentiation stage (in patients with central/upper type of fat topography) and the percentage of intact double-stranded DNA in normal endometrium.ConclusionsTumor estrogen content in endometrial cancer has clinical significance that is modified in the presence of certain endocrine characteristics related to insulin resistance. The role of local estrogen production (aromatase activity) in this setting deserves special study.

Insulin resistance, its consequences for the clinical course of the disease, and possibilities of correction in endometrial cancer

Objectives To study the frequency of insulin resistance (IR) in endometrial cancer patients, its relation to the clinical course of the disease and DNA damage, and to evaluate possible approaches to the pharmacological correction of IR in the patients studied.Methods The signs of insulin resistance syndrome and its association with the clinical and pathological features of the disease and DNA damage in somatic cells (micronucleus frequency in peripheral blood lymphocytes) and endometrial normal and tumor tissue (alkaline unwinding) were determined in 99 endometrial cancer patients.Results The frequency of insulin resistance syndrome counted on the basis of fasting plasma glucose and insulin concentrations according to Duncan et al. is equal to 0.35 (95% CI 0.24–0.46), or 35%, in endometrial cancer patients who do not have a history of diabetes mellitus. Patients with well- or moderately differentiated endometrial adenocarcinomas (mostly type I) had statistically significantly higher basal and stimulated plasma insulin and C-peptide concentrations than patients with poorly differentiated endometrial adenocarcinomas or rarely encountered tumors of the endometrium (primarily type II). Interestingly, the level of fasting insulinemia positively correlates with disease stage and with local and regional tumor dissemination only in the group of patients with well- or moderately differentiated endometrial adenocarcinomas. On the other hand, hyperinsulinemia and other hormonal-metabolic disturbances typical of insulin resistance syndrome do not increase the probability of DNA damage of somatic cells (according to the data of micronucleus test). In addition, no association between hormonal-metabolic disturbances and the degree of DNA unwinding in tumor and visually unchanged endometrium was found.Conclusion Thus, insulin resistance/hyperinsulinemia is associated with a more aggressive course of the disease in certain groups of the patients but—in contrast to excessive estrogenic stimulation—does not result in increased genotoxic damage in tumor and normal tissues. The data obtained once more confirm the need for treatment and prevention measures aimed at correcting hormonal-metabolic disturbances in endometrial cancer patients and groups at risk of this disease. Such an approach might include use of antidiabetic biguanides, thiazolidinediones (glitazones), and statins.

CYP19 gene polymorphism in endometrial cancer patients.

Purpose: Initiation/promotion of endometrial cancer is known to be associated with estrogenic influence. Therefore, it is possible that some allelic polymorphisms of the genes involved in steroidogenesis or steroid metabolism contribute to endometrial cancer susceptibility. Methods: Here, we compared CYP19 (aromatase) gene polymorphism in 85 endometrial cancer patients and in 110 non-affected women. Results: The genotypes containing the longest alleles (A6 and A7) of CYP19 were found to be over-represented in patients as compared to controls. In addition, these genotypes demonstrated a tendency to be associated with increased concentrations of estradiol and testosterone in postmenopausal patients. Conclusions: Thus, CYP19 polymorphism might be one of the genetic risk factors for endometrial cancer development.

Neoadjuvant therapy of endometrial cancer with the aromatase inhibitor letrozole: endocrine and clinical effects

OBJECTIVE To investigate the short-term hormonal and clinical effects of the aromatase inhibitor letrozole (Femara) in patients with endometrial cancer. MATERIALS AND METHODS Ten previously untreated, post-menopausal patients (mean age 59 years) with endometrial cancer, predominantly stage I disease, received letrozole 2.5mg per day for 14 days before surgery. Clinical, sonographic, morphologic, cytologic, and hormonal-metabolic parameters (blood estradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), glucose, and cholesterol by radioimmunoassay, enzyme immune assay, or enzyme-colorimetric methods; tumor progesterone receptors by ligand-binding assay; and aromatase activity by 3H-water release assay) were evaluated before and after treatment. RESULTS Treatment was well-tolerated in all patients. In two patients, pain relief in the lower part of the belly and/or decrease in intensity of uterine discharge was reported. In the three cases, substantial decreases in endometrial M-echo (ultrasound) signal were noted; the mean value of this parameter after treatment was 31.1% lower than before treatment. Blood estradiol concentration decreased by an average of 37.8% after letrozole therapy, and tumor progesterone receptor levels and aromatase activity decreased by 34.4 and 17.5%, respectively. Treatment with letrozole did not influence surgery. CONCLUSIONS These data show that short-term treatment with letrozole in the neoadjuvant setting resulted in some positive clinical changes. Longer-term and larger-scale trials of neoadjuvant letrozole in endometrial cancer are warranted.

Long-term exposure to tamoxifen induces hypersensitivity to estradiol

In women with hormone-dependent breast cancer, tamoxifen (TAM) frequently induces tumor regression, but regrowth occurs with continuation of antiestrogen therapy. Studies of breast xenografts in nude mice suggest that this secondary resistance to TAM may reflect the development of enhanced sensitivity to the estrogenic properties of TAM. In the current study, we examined the hypothesis that TAM could also induce a state of hypersensitivity to estradiol (E2) itself. Oophorectomized nude mice with MCF-7 cell xenografts received 25-mg implants of TAM [long-term TAM treated (LTTT) mice] or cholesterol (C-MCF-7) over a 5-month period (phase 1). The LTTT group regressed to a lesser extent than did C-MCF-7 tumors. After 4 months of TAM exposure, the LTTT tumors begin to regrow, as did the C-MCF-7, as assessed by slope analysis. At 5 months, TAM or vehicle implants were removed, and the LTTT and C-MCF-7 subgroups were given vehicle or two doses of E2 to test estrogen sensitivity (phase 2). We used our “E2 clamp” technique to maintain levels of plasma E2 at either 1.25 or 20 pg/ml. Neither group responded to the very low concentrations of E2 (1.25 pg/ml) or vehicle. The LTTT tumors but not C-MCF-7 tumors exhibited a growth response on exposure to 20 pg/ml E2 during 7 weeks, as demonstrated with mixed models analysis. These studies provide evidence that long-term TAM exposure enhances sensitivity to the estrogenic effects of TAM and also to E2 itself.