Domingo Navarro

Androgens and androgen receptors in breast cancer.

Aromatase (CYP19) converts adrenal and ovarian androgens into estrogens, which supports the growth of estrogen-dependent breast cancers. Anti-aromatase agents are displacing antiestrogens as the first-line treatment for estrogen receptor positive breast cancers. Androgens can act as estrogen precursors, but besides this capability they can also directly act on breast cancer cells by binding to androgen receptors, which are present in the majority of breast cancer specimens. Epidemiological and clinical evidences suggest that higher levels of circulating androgen increase the risk of developing breast cancer. Androgen receptor gene polymorphisms which render the more transcriptionally active receptors have been related to a lower risk of breast cancer. It is currently accepted that androgens act as antiproliferative agents in the presence of estrogens in some breast cancer cell lines. However, emerging evidence suggests that direct androgenic activity might also stimulate cell growth in a subset of estrogen-resistant breast tumors. Here we discuss the supporting evidence which proposes that androgens themselves are actively involved in breast carcinogenesis and its clinical behaviour.

Transition to androgen-independence in prostate cancer

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of death as a result of cancer in men in the western countries. Withdrawal of androgens or the peripheral blockage of androgen action remain the critical therapeutic options for the treatment of advanced prostate cancer. However, after initial regression, most of the prostate cancers become androgen-independent and progress further, with eventual fatal outcome. Understanding the mechanisms of transition to androgen independence and tumor progression in prostate cancer is critical to finding new ways to treat aged patients that are ineligible for conventional chemotherapy. A large number of different molecular mechanisms might be responsible for the transition to androgen-independence. Many of these involve the androgen receptor (AR) and its signalling pathways, but they might also include genetic changes that affect several genes, which results in the activation of oncogenes or the inactivation of tumor suppressor genes. Here, we discuss the most recent and relevant findings on androgen resistance in prostate cancer in order provide a comprehensive interpretation of the clinical behaviour of tumors at molecular levels.

Endometrial stromal sarcoma expression of estrogen receptors, progesterone receptors and estrogen-induced srp27 (24K) suggests hormone responsiveness.

The endometrial stroma plays a decisive role in sustaining the gland epithelium along the menstrual cycle, and in preparing the microenvironment that allows embryo implantation. The stroma undergoes important changes during the menstrual cycle that affects both the cell number and differentiation. These changes are regulated by both estrogen and progesterone. Stromal sarcomas are extremely rare, occurring much less than any other uterine tumor. Their origin and biology are poorly understood. The purpose of this work was to try to learn more about the stromal physiology, and also to ascertain whether the stromal sarcoma has characteristics of hormone dependence. We studied the presence of estrogen receptors (ER), progesterone receptors (PR) and the stress-responsive protein of 27K (srp27, a protein first described as an estrogen-induced 24K protein in MCF-7 cells) in both normal stroma and stromal sarcoma. The ER and PR were measured by exchange assays. The srp 27 was studied both by Western-blot and by IHC by means of specific monoclonal antibodies. The stromal sarcomas studied showed a high concentration of both ER (96 to 116 fmol/mg prot.) and PR (565 to 995 fmol/mg prot.). These amounts of ER and PR were higher than the mean found in normal endometrium during the proliferative phase (43 and 637 fmol/mg prot., respectively), and much higher than that of the secretory phase (17 and 229 fmol/mg prot., respectively). The srp27 characterized by Western-blot in both the normal stroma and stromal sarcoma was found to be similar to the srp27 of breast cancer. The IHC results showed a very low expression of srp27 in the stroma during the proliferative phase that increases when the endometrium enters the secretory phase. The low-malignancy grade stromal sarcomas showed abundant expression of srp27, but the high-malignancy grade sarcomas showed no expression of srp27. The obtained results prove the stroma capability to express the srp27. A negative correlation between malignancy of stromal tumors and srp27 expression was found. The presence of ER and PR in some stromal sarcomas proves that they have characteristics of hormone responsiveness. These findings suggest that ER and PR assays should be routinely performed in stromal sarcomas as well as in endometrial adenocarcinomas, and also that antiestrogenic drugs might be considered for the treatment of ER and PR positive stromal sarcomas.

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

HER‐2/neu oncogene status and total cellular p185HER‐2 content were simultaneously analyzed in 415 invasive breast‐cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut‐off value of 1.7 for the ratio between the intensity of the HER‐2/neu gene band and the reference gene band, to consider the HER‐2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER‐2 over‐expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER‐2/neu gene amplification. Of these tumors, 87% showed over‐expression of the p185HER‐2. Of the remaining 352 specimens that did not display HER‐2/neu gene amplification, 97% showed no p185HER‐2 over‐expression (p < 0.0001). In 40 selected samples with a p185HER‐2 level lower than 260 fmol/mg protein, the degree of p185HER‐2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER‐2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER‐2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER‐2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER‐2/neu oncogene is amplified or p185HER‐2 is over‐expressed.

The two native estrogen receptor forms of 8S and 4S present in cytosol from human uterine tissues display opposite reactivities with the antiestrogen tamoxifen aziridine and the estrogen responsive element

We have investigated the capability of the different native ER forms, present in cytosols from human uterine tissues, of reacting with the antiestrogen [3H]Tamoxifen aziridine ([3H]TA) and with the Estrogen Responsive Element (ERE). Cytosols from uterine leiomyoma (myoma) prepared in buffer containing 40 mM molybdate and protease inhibitors, labelled with [3H]estradiol and analyzed in low-salt sucrose gradient showed 8S and 4S ER forms. The same cytosols labelled with [3H]TA only showed a 4S ER form, whereas the ERE only reacted with fractions from the 8S peak. The band of ERE reaction in the EMSA assay showed a lower relative mobility than the band labelled with [3H]TA, but both bands contained immunoreactive ER of 65 kDa. Electrophoretic mobility of the [3H]TA-labelled band in that system was not affected by cytosol treatment with cross-linkers or SDS, which suggests that it is a monomeric protein. The [3H]TA-binding 4S ER form was found in all studied myoma samples, as well as in human endometrium or myometrium, but not in rat tissues. These results suggest that the 8S and 4S ER form were already present before cytosol from human uterine tissues comes into contact with the molybdate buffer. They both contain the same ER molecule of 65 kDa, either in the free form or as an oligomer. Only the ER dimers, which have been described both in the cytosolic 8S form and in the nuclear 4-5S form, react with the ERE. [3H]TA only binds to the 4S ER monomer probably because its binding site is concealed in the 8S form under these experimental conditions. The opposite reactivity of the 8S and 4S ER forms with [3H]TA and the ERE support the hypothesis that they may constitute separate entities with a different physiological role.

Estrogen antagonism on T3 and growth hormone control of the liver microsomal low-affinity glucocorticoid binding site (LAGS)

Male rat liver microsomes contain a low-affinity glucocorticoid binding site (LAGS) capable of binding all natural glucocorticoids and progesterone with a Kd from 20 to 100 nM. The LAGS level is under endocrine control by T3, glucocorticoids and GH. These hormones act synergistically at physiological concentrations to increase the LAGS level. Since female rats show a LAGS level that is much lower than the males (0.15 vs 23 pmol/mg protein, respectively), here we investigated whether estradiol could decrease the LAGS in the male rat. Orchiectomized (OX) male rats showed a higher LAGS level than intact rats. This effect was reversed by implanting a Sylastic capsule containing testosterone. When the OX rats were implanted for 20 days with estrogen capsules that provided an estradiol level in serum of 40 pg/ml, their LAGS level decreased from 23 to 0.2 pmol/mg protein. This effect was not observed in intact male rats and can be partially reversed by testosterone implants into OX rats. Both hypophysectomized male rats and hypothyroid-orchiectomized male rats showed very low levels of LAGS. Administration of physiological doses of GH and/or T3 to these rats greatly increased their LAGS level (from 0.3 to 15 and 16 pmol/mg protein, respectively). Implantation of estrogen capsules to these rats two weeks prior to starting treatment completely inhibited the increase in the LAGS level in response to T3, and significantly decreased the response to hGH, and to a combination of hGH and T3. These results suggest that physiological estradiol levels can antagonize the LAGS induction by T3 and hGH in the male rat, and could be responsible for the low level of LAGS in the female rat. Moreover, estrogen capsules also inhibited the increase in the body and hepatic weights observed after hGH treatment, which suggests a powerful inhibitory effect of low estradiol levels on the male rat liver functions under regulation by T3 and/or GH.

Age-related changes in the induction of tyrosine aminotransferase by dexamethasone: correlation with the low-affinity glucocorticoid binding sites

Rat liver membranes contain Low-affinity glucocorticoid binding sites (LAGS), capable of binding with low affinity (Kd approximately 100 nM) endogenous glucocorticoids. Unlike the glucocorticoid receptor (GR), the LAGS level undergoes abrupt changes throughout life. The investigation of these changes may be useful in determining whether the LAGS are involved in the cellular response to glucocorticoids. For this purpose, we have studied glucocorticoid induction of tyrosine aminotransferase (TAT), and its relationship with the LAGS level in adrenalectomized and fasted rats of different ages. No significant differences in the GR level, or in its Kd and activation, were observed among rats of 1, 3, and 12 months of age. On the other hand, the LAGS level showed an important variation with age, from almost undetectable in 1-month-old rats, to a maximum value in 3-month-old rats. With respect to TAT activity, an increase with age in the threshold of response to dexamethasone (DEX) administration was observed. The smallest dose of DEX capable of provoking a significant TAT induction rose from 0.1 microgram/kg body wt. in 1-month-old rats to 10 micrograms/kg body wt. in 12-month-old rats. However, the smallest dose of DEX able to elicit the maximal response was 10 micrograms/kg body wt. in all the assayed ages. This dose provoked a 40% decrease in the GR level, but did not significantly modify the LAGS content. From these results, we conclude that there is an age-related change in the threshold of response to DEX that cannot be explained by the GR-glucocorticoid interaction. The possibility that the LAGS modulate the cell response to glucocorticoids arises from the coincidence of this change with that observed in the LAGS concentration throughout life.

The estradiol induction of the microsomal low-affinity glucocorticoid binding sites (LAGS) in the male rat liver is independent of the endocrine status

The low-affinity glucocorticoid binding sites (LAGS) are entities present in the microsomal fraction of the rat liver, capable of binding several glucocorticoids and progesterone with low affinity. The present work focuses on the demonstration that estradiol exerts a powerful stimulatory effect on the LAGS concentration. For this purpose, we studied the effect of this hormone in immature, hypothyroid, and hypophysectomized rats, three experimental models which present a very low level of LAGS. In all of them, estradiol showed ability to significantly increase the level of LAGS. The positive results obtained in hypophysectomized rats point to a direct action of estradiol on the liver. In immature rats, the estradiol induction of the LAGS was shown to be especially slow, 3-4 days after estradiol administration being necessary to obtain a significant rise in the level of LAGS. Moreover, the dose of estradiol necessary to obtain the LAGS induction in these rats (0.5 mg/100 g body weight) was clearly supraphysiological. From these data we concluded that: (A) estradiol is a powerful stimulator of the LAGS concentration, its effect probably being exerted directly on the liver; and (B) to elicit its effect, estradiol does not need the participation of other hormones known to be implicated in the endocrine regulation of the LAGS.

Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D

Abstract[3H]Tamoxifen Aziridine ([3H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [3H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [3H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [3H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [3H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/mg p.). The 4S peak of [3H]TAZ was partially inhibited by both estrogens and antiestrogens. When [3H]E2 was used instead of [3H]TAZ, only an 8S peak was detected. [3H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [3H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [3H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [3H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [3H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor.