Proteins induced by estrogens. Search for new markers of hormone dependence in breast cancers

The hormone dependence and the prognosis of human breast cancers are now based on the assays of the oestrogen receptor (RE) and progesterone receptor (PR) in the tumor. However, such prediction is not fully accurate since RE may be non functional in some tumors and the estrogenic regulation in PR is not directly correlated with the effects of estrogens on cell growth. Here, we review other estrogen induced proteins. A special emphasis is made on a 52,000 dalton glycoprotein released into the medium by human breast cancer cells. Studies on the regulation and biological role of this protein should improve our understanding of the mechanisms by which estrogens stimulate the growth and spreading of breast cancers. Such secreted proteins could later provide convenient circulatory markers of hormone dependency.     

Solid and mucinous varieties of so-called mammary carcinoid tumors.

Three neoplasms that had histologic features reminiscent of carcinoid tumors of other sites were encountered in a review of 3,300 examples of invasive mammary cancer in women (.09%). One of these showed cytoplasmic argyrophilia. This, as well as the two putative carcinoids, lacked argentaffinity. Attention is directed to the occurence of variable numbers of argyrophilic cells in eight of 19 so-called mucinous cancers of the breast studied. Further, neurosecretory-type granules were observed in cells of all four mucinous cancers suitably prepared for electron microscopic examination. The possible reasons for the lack of universal argyrophilic reactions in these lesions is discussed. It is concluded that there may be two types of mammary carcinoid tumors, the solid and mucinous varieties. No patient who had the latter type had experienced treatment failure after five years of observation. Various numbers of ductal epithelial cells in four of 45 examples of banal fibrocystic disease showed cytoplasmic argyrophilia, and neurosecretory-type granules were found in two of eight examples suitably prepared for electron microscopic examination. Whether this demonstration establishes the existence of precursor elements for the development of the carcinoid tumors is at present uncertain.     

乳腺交界性病变不典型囊性导管的临床病理学特征

目的 探讨乳腺的一种新的交界性病变,即不典型囊性导管(ACD)的临床病理学特征及其意义。方法 对200例术前没有做活检取材(仅经细胞学或细针穿刺活检诊断)的乳腺癌乳房切除材料作全乳腺切片检查,观察ACD的临床病理学特征和免疫组织化学特性。结果 ACD的发生率为22％(44／200),多发于绝经前女性(P=0.001),存在部位以乳腺癌巢周边多见;连续切片(间隔50张切片,150μm距离)可观察到ACD向非浸润性导管癌的移行;36％(16／44)伴有ACD的乳腺癌病例,癌组织p53染色阳性,其中12／16伴有的ACD也同时呈现阳性改变(P=0．001);α-平滑肌肌动蛋白染色显示ACD的肌上皮与非浸润性导管癌的肌上皮相似呈明显的受压变薄;伴有ACD的乳腺癌和不伴有ACD的乳腺癌两组在雌激素受体(ER),雄激素受体(PR),p53、c-erbB-2和Ki-67蛋白,肿瘤大小以及淋巴结转移方面没有明显的区别;44例ACD c-erbB-2染色呈阴性,Ki67标记指数也明显低于其所伴有的乳腺癌。结论 鉴于ACD与癌巢间的连续性及与癌组织p53蛋白的共同表达特性,将ACD作为乳腺的一种交界性病变具有一定的意义。     

Curcumin inhibits MPA-induced secretion of VEGF from T47-D human breast cancer cells

OBJECTIVE: Recent clinical trials show that women who receive combined estrogen and progestin hormone therapy (HT) have a higher risk of breast cancer than women who receive estrogen alone or placebo. We have shown that progestins stimulate expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in human breast cancer cells that express the progesterone receptors and mutant p53 protein. Because increased levels of VEGF promote tumor progression, compounds that prevent progestin-induced expression of VEGF could be clinically useful. The objective of this study was to examine whether the polyphenol compound curcumin has the capacity to block progestin-induced secretion of VEGF from T47-D human breast cancer cells. DESIGN: The estrogen and progesterone receptor containing T47-D human breast cancer cells was exposed to 10 nM progesterone or synthetic progestins and varying concentrations of curcumin to determine whether curcumin blocks progestin-dependent production of VEGF from tumor cells. RESULTS: Curcumin (0.001-10 microM for 18 h) reduced medroxyprogesterone acetate (MPA)-induced secretion of VEGF from T47-D cells in a dose-dependent manner. Secretion of VEGF from cells treated with progesterone or progestins other than MPA was unaffected by curcumin. CONCLUSIONS: MPA is the most widely used progestin in HT. Curcumin may therefore provide a clinically useful tool for the suppression of MPA-induced elaboration of VEGF by tumor cells. We propose therefore that clinical trials to assess the beneficial effects of curcumin in postmenopausal women are warranted.     

Retinoblastoma and p53 gene product expression in breast carcinoma: Immunohistochemical analysis and clinicopathologic correlation

We examined 100 breast cancers for retinoblastoma (Rb) and p53 protein expression by immunohistochemistry using the PMG3.245 and PAb 1801 antibodies. We assessed percentages of reactive cells and their intensity, as well as staining patterns. The results were correlated with neu protein reactivity and a panel of variables, including age, tumor size and type, nuclear grade, estrogen receptor/progesterone receptor content, and lymph node status. Retinoblastoma protein negativity, either partial or complete, was noted in 47% of cases. Surprisingly, a relatively stronger Rb reaction was seen in some high nuclear grade tumors. p53 positivity was found in 23% of cases and was a significant predictor of Rb loss. p53 also was correlated with poorly differentiated (nuclear grade III) neoplasms and neu expression but not with negative ER status. Tissue distribution profiles for Rb-negative and p53-positive cells were variable in this series, with both uniform and heterogeneous patterns observed. This suggests that Rb and p53 alterations may represent early or late events in transformation. Our findings further implicate Rb and p53 derangements in mammary oncogenesis.     

Immunoreactive opioid peptides in human breast cancer.

Opioid peptides have a variety of actions on inter alia pituitary hormone secretion and the immune system. Release of endogenous opioids has been found to stimulate growth of experimental breast cancers and opiate receptor blockers have reduced the growth of chemically induced rat breast tumors. Opioid peptides may therefore play a role in human breast cancer. Invasive ductal carcinomas from 61 premenopausal women were immunocytochemically analyzed for the presence of opioid peptide immunoreactivity. Positive staining was unambiguously identified in 34 of the tumors (56%). In addition, a medullary carcinoma was positive. In a smaller series of tumors, opioid peptide immunoreactive cells were detected in both primary tumors and metastases. Positive tumor cells were usually few and scattered. Therefore, underestimates of their true frequency of occurrence are likely to have occurred, making accurate correlations with clinical behavior and estrogen receptor status difficult. No correlations with estrogen receptors were established for the unambiguously opioid peptide-positive tumors. Many of the positive tumors also stained with antibodies to gamma-endorphin and alpha-melanocyte-stimulating hormone, suggesting the presence of proopiomelanocortin-derived peptides in them. However, peptides derived from other opioid precursors also may be present in breast cancer.     

Introduction of estrogen receptor-alpha into the tTA/TAg conditional mouse model precipitates the development of estrogen-responsive mammary adenocarcinoma

Conditional expression of estrogen receptor (ER)-alpha) was introduced into tetracycline-responsive MMTV-tTA/tetop-TAg mice to develop a mouse model of estrogen-responsive ER-alpha-positive mammary adenocarcinoma. Mammary adenocarcinomas developed in the mice with a mean latency of 11 months. Precursor lesions including ductal hyperplasia and hyperplastic alveolar nodules were present by the age of 4 months. The mammary adenocarcinomas exhibited histological features similar to human breast cancers. ER steroid-binding studies conducted on adenocarcinoma lysates demonstrated binding to estradiol. Tumor explant studies in the presence and absence of estradiol in ovariectomized athymic nude mice revealed that growth of mammary tumors was stimulated by estrogen. In addition, the presence of ER-alpha altered the tumor spectrum in other MMTV-targeted tissues in the tTA/TAg female mice. Lymphomas, which develop in 40% of tTA/TAg female mice, were found in only 4% of tTA/TAg/ER-alpha mice (P = 0.014, chi-square test). These experiments demonstrate that the introduction of an ER-alpha transgene targeted to mammary epithelial cells can be used to develop mouse models of ER-alpha-responsive mammary cancer.     

Incidence estimation of female breast cancer among Koreans.

The authors conducted a nationwide survey to estimate the incidence rates of female breast cancer among Korean women in 1990-1991. We identified potential breast cancer cases based on the claims sent by medical care institutions throughout Korea to the Korea Medical Insurance Corporation (KMIC) from January 1988 to December 1989, whose diagnoses in the claims included one of the following diagnostic codes; ICD-9 174-175 (malignant neoplasms of the breast), 217 (benign neoplasms of the breast), 610-611 (benign mammary dysplasia and other disorders of the breast), 233 (carcinoma in situ of the breast and genito-urinary system), or 195-199 (malignant neoplasms with uncoded sites). In order to collect the final diagnosis of the potential cases, abstracting medical records was performed through visiting or mailing an abstracting format to the corresponding medical institutions. Thereafter oncologists reviewed the abstracting formats and confirmed the incident cases of female breast cancer among the potential breast cancer cases. Using these data from the KMIC, the incidence patterns of female breast cancer among Korean women were estimated as of July 1, 1988 to June 30, 1989. The incidence rate of female breast cancer adjusted for the Korean population was estimated to be 9.9 (95% confidence interval: 9.5-10.4). The cumulative rates for the ages 0-64 and 0-74 were 0.85% and 1.0%, respectively. The standardized rate for the world population was 10.9, which was lower than those of any other Asian country including China and Japan in 1983-1987.(ABSTRACT TRUNCATED AT 250 WORDS)     

PTEN expression in breast and endometrial cancer: correlations with steroid hormone receptor status.

OBJECTIVE: The PTEN (MMAC1/TEP1) tumor suppressor gene is frequently mutated and homozygously deleted in human neoplasms, but there is only sparse information about PTEN protein expression in hormone-dependent female tumors. Therefore, we investigated PTEN expression in 68 breast and 43 endometrial carcinomas. METHODS: For PTEN protein detection, we used Western blot analysis followed by densitometry and compared these data with clinicopathologic parameters, the estrogen receptor (ER) and progesterone receptor (PR) status, HER2/neu and the proliferation marker Ki67. RESULTS: We were able to show significantly decreased PTEN protein expression in endometrial carcinomas compared with normal endometrial tissue samples, especially in the endometrioid histological subtype. In contrast, PTEN downregulation was found more rarely in breast cancer. Lower PTEN expression in breast cancer correlated significantly with high ER immunoreactivity (p = 0.008) and was weakly associated with PR expression (p = 0.055) and low histological grading (p = 0.081). No correlation with any of these parameters was observed in endometrial tumors. In both tumor types, no association of PTEN expression with any other analyzed parameter was found. CONCLUSIONS: These results suggest that PTEN expression plays different roles in the pathogenesis of endometrial carcinomas and breast cancer. In mammary carcinomas, loss of PTEN expression is mainly found in more differentiated tumors and is probably not a major event in carcinogenesis.     

Breast cancer risk with postmenopausal hormonal treatment

This review was designed to determine from the best evidence whether there is an association between postmenopausal hormonal treatment and breast cancer risk. Also, if there is an association, does it vary according to duration and cessation of use, type of regimen, type of hormonal product or route of administration; whether there is a differential effect on risk of lobular and ductal cancer; and whether hormone treatment is associated with breast cancers that have better prognostic factors? Data sources for the review included Medline, the Cochrane Database of Systematic Reviews (Cochrane Library, 2005) and reference lists in the identified citations. Eligible citations addressed invasive breast cancer risk among postmenopausal women and involved use of the estrogen products with or without progestin that are used as treatment for menopausal symptoms. Abstracted data were demographic groupings, categories of hormone use, categories of breast cancer, two-by-two tables of exposure and outcome and adjusted odds ratios, relative risks (RRs) or hazard rates. Average estimates of risk were weighted by the inverse variance method, or if heterogeneous, using a random effects model. The average risk of invasive breast cancer with estrogen use was 0.79 [95% confidence interval (95% CI) = 0.61-1.02] in four randomized trials involving 12 643 women. The average breast cancer risk with estrogen-progestin use was 1.24 (95% CI = 1.03-1.50) in four randomized trials involving 19 756 women. The average risks reported in recent epidemiological studies were higher: 1.18 (95% CI = 1.01-1.38) with current use of estrogen alone and 1.70 (95% CI = 1.36-2.17) with current use of estrogen-progestin. The association of breast cancer with current use was stronger than the association with ever use, which includes past use. For past use, the increased breast cancer risk diminished soon after discontinuing hormones and normalized within 5 years. Reasonably adequate data do not show that breast cancer risk varies significantly with different types of estrogen or progestin preparations, lower dosages or different routes of administration, although there is a small difference between sequential and continuous progestin regimens. Epidemiological studies indicate that estrogen-progestin use increases risk of lobular more than ductal breast cancer, but the number of studies and cases of lobular cancer remains limited. Among important prognostic factors, the stage and grade in breast cancers associated with hormone use [corrected] do not differ significantly from those in non-users, but breast cancers in estrogen-progestin users are significantly more likely to be estrogen receptor (ER) positive. In conclusion, valid evidence from randomized controlled trials (RCTs) indicates that breast cancer risk is increased with estrogen-progestin use more than with estrogen alone. Epidemiological evidence involving more than 1.5 million women agrees broadly with the trial findings. Although new studies are unlikely to alter the key findings about overall breast cancer risk, research is needed, however, to determine the role of progestin, evaluate the risk of lobular cancer and delineate effects of hormone use on receptor presence, prognosis and mortality in breast cancer.     

Immunohistochemical detection of Ki67 in breast cancer correlates with transcriptional regulation of genes related to apoptosis and cell death.

Ki67 is a nuclear protein that is tightly linked to the cell cycle. It is a marker of cell proliferation and has been used to stratify good and poor prognostic categories in invasive breast cancer. Its correlation with gene expression patterns has not been fully elucidated. In this study, Ki67 immunohistochemistry using the MIB-1 antibody was performed on sections cut from 21 formalin-fixed, paraffin-embedded invasive breast cancers. Scoring was determined as nil (no immunostaining), low (10% or less immunopositivity) or high (>10% immunoreactive cells) respectively. The relationship of Ki67 immunohistochemical detection with clinicopathologic parameters was evaluated. Using Affymetrix U133A GeneChips, expression profiles for these tumors were generated and correlated with Ki67 immunohistochemical findings. Analysis of variance was used to define genes that were differentially regulated between the groups. Real-time polymerase chain reaction (PCR) was used to confirm the presence of a downregulated gene. Our results showed high, low and nil Ki67 immunostaining in nine (43%), six (28.5%) and six (28.5%) invasive breast cancers respectively, with increased Ki67 protein expression correlating with high histologic grade (P=0.02), mitotic score (P=0.001) and estrogen receptor immunonegativity (P=0.002). Expression profiling trends of the Ki67 gene mirrored the observed proportions of immunostained cells when the Ki67 immunoscore was >10%. Genes related to apoptosis and cell death (bcl2, MAP2K4, TNF10) were noted to be downregulated in tumors that disclosed >40% Ki67 immunostaining (P<0.001). Downregulation of the bcl2 gene was confirmed at the RNA level by real-time RT-PCR. Differential regulation of these genes, especially bcl2, may contribute to the biological nature of clinically more aggressive and highly proliferative breast cancers.     

Breast cancer incidence in postmenopausal women using testosterone in addition to usual hormone therapy

OBJECTIVE: There is now convincing evidence that usual hormone therapy for ovarian failure increases the risk for breast cancer. We have previously shown that ovarian androgens normally protect mammary epithelial cells from excessive estrogenic stimulation, and therefore we hypothesized that the addition of testosterone to usual hormone therapy might protect women from breast cancer. DESIGN: This was a retrospective, observational study that followed 508 postmenopausal women receiving testosterone in addition to usual hormone therapy in South Australia. Breast cancer status was ascertained by mammography at the initiation of testosterone treatment and biannually thereafter. The average age at the start of follow-up was 56.4 years, and the mean duration of follow-up was 5.8 years. Breast cancer incidence in this group was compared with that of untreated women and women using usual hormone therapy reported in the medical literature and to age-specific local population rates. RESULTS: There were seven cases of invasive breast cancer in this population of testosterone users, for an incidence of 238 per 100,000 woman-years. The rate for estrogen/progestin and testosterone users was 293 per 100,000 woman-years--substantially less than women receiving estrogen/pro-gestin in the Women's Health Initiative study (380 per 100,000 woman-years) or in the "Million Women" Study (521 per 100,000 woman-years). The breast cancer rate in our testosterone users was closest to that reported for hormone therapy never-users in the latter study (283 per 100,000 woman-years), and their age-standardized rate was the same as for the general population in South Australia. CONCLUSIONS: These observations suggest that the addition of testosterone to conventional hormone therapy for postmenopausal women does not increase and may indeed reduce the hormone therapy-associated breast cancer risk-thereby returning the incidence to the normal rates observed in the general, untreated population.     

Grb2 overexpression in nuclei and cytoplasm of human breast cells: a histochemical and biochemical study of normal and neoplastic mammary tissue specimens

In 20–30 per cent of human breast cancers, the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and c-erbB2 are overexpressed. This overexpression leads to increased mitogenic signalling and is correlated with poor prognosis. Overexpression of associated adaptor proteins, like Grb2, can also induce upregulation of signalling pathways. In this study, the expression of the Grb2 adaptor protein was determined in both normal human breast tissue and mammary cancers, using immunoblotting experiments and immunostaining on paraffin-embedded tissue sections. Both biochemical and immunohistochemical techniques revealed overexpression of Grb2 in all breast cancer specimens. In addition, although Grb2 protein is described as localized in the cytoplasm, it can also be detected in the nucleus, both in normal and in tumour breast tissue. In tumour breast tissue, 58 per cent of Grb2 protein is found in the nucleus, while 37 per cent is detected in the cytoplasm. In normal breast tissue, 22 per cent of Grb2 is found in the nucleus and 70 per cent in the cytoplasm. These findings indicate that in human breast cancer, Grb2 is overexpressed and appears to be predominantly localized in the nucleus. © 1997 John Wiley & Sons, Ltd.     

Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status

A Japanese study reported that up to 16% of breast cancer samples harbor a sporadic mutation within the human Cav-1 gene, namely P132L. To date, however, no studies have examined the United States' population. Here, we developed a novel allele-specific real-time PCR assay to detect the Cav-1 P132L mutation in mammary tumor cells isolated by laser capture microdissection from formalin-fixed paraffin-embedded breast cancer samples. We report that the Cav-1 P132L mutation is present in approximately 19% of estrogen receptor alpha (ERalpha)-positive breast cancers but not in ERalpha-negative breast cancers. This is the first demonstration that the P132L mutation is exclusively associated with ERalpha-positive mammary tumors. We also identified six novel Cav-1 mutations associated with ERalpha-positive breast cancers (W128Stop, Y118H, S136R, I141T, Y148H, and Y148S). Thus, the overall incidence of Cav-1 mutations in ERalpha-positive breast cancers approaches 35% (greater than one-third). To mechanistically dissect the functional relationship between Cav-1 gene inactivation and ERalpha expression, we isolated primary mammary epithelial cells from wild-type and Cav-1-/- mice and cultured them in a three-dimensional system, allowing them to form mammary acinar-like structures. Under conditions of growth factor deprivation, Cav-1-deficient mammary acini displayed increased ERalpha levels and enhanced sensitivity toward estrogen-stimulated growth, with specific up-regulation of cyclin D1. Finally, we discuss the possibility that sporadic Cav-1 mutations may act as an initiating event in human breast cancer pathogenesis.     

Estrogen and cancers of the colorectum,breast, and lung in postmenopausal women

As estrogens play an important role in maintaining physiological function in various organs, the estrogen decrease after menopause is thought to cause various diseases frequently observed in postmenopausal or elderly women. With the aging of society and a decrease in infectious or vascular diseases, neoplasms have now become the most frequent cause of death in Japan. Cancers of the colorectum, breast, and lung have been rapidly increasing both in incidence and death, especially among postmenopausal women. Interestingly, all three of these cancers are associated with estrogens. In premenopausal women, ovarian estrogens plays major roles in the female reproductive organs through the classic estrogen receptor, ER‐α. In postmenopausal women, however, estrogens produced/activated by peripherally localized estrogen‐metabolizing enzymes such as aromatase, which converts androgen into estrogens, are thought to play physiologically and pathobiologically important roles in various organs through second ER, namely ER‐β, distributing systemically. In this article, the association of estrogens with these cancers in postmenopausal or elderly women are reviewed, especially focusing on the role of ER‐β and peripheral estrogen metabolism. The possibility of prevention or treatment of these diseases through estrogenic control is also discussed.     

Human epidermal growth factor receptor 2, Na+/H+ exchanger regulatory factor 1, and breast cancer susceptibility gene-1 as new biomarkers for familial breast cancers

The aim of this study is to evaluate the analysis of markers related with progression, to further characterize familial breast cancers. Here, we investigated the expression of breast cancer susceptibility gene-1, hypoxia-inducible factor-1α, vascular endothelial growth factor receptor 1, and Na+/H+ exchanger regulatory factor 1 in 187 microarrayed breast carcinomas from 94 familial and 93 sporadic breast cancer patients by immunohistochemical staining. Furthermore, the expression levels of these biomarkers were compared with triple-negative phenotype. Familiarity was significantly associated with younger age (P < .000), higher tumor grade (P = .038), negative estrogen receptor hormonal status (P = .036), and high proliferative activity (P = .029). The familial cancers were immunonegative for membranous Na+/H+ exchanger regulatory factor 1 expression compared with sporadic cancers (P = .001); notably, vascular endothelial growth factor receptor 1 staining correlated with cytoplasmic Na+/H+ exchanger regulatory factor 1 expression in familial tumors (P = .009). In multivariate analysis, the "new biomarkers," including negative human epidermal growth factor receptor 2 status (odds ratio, 4.538; 95% confidence interval, 1.756-11.728), negative membranous Na+/H+ exchanger regulatory factor 1 expression (odds ratio, 7.686; 95% confidence interval, 1.876-31.483) and positive nuclear breast cancer susceptibility gene-1 (odds ratio, 0.3982; 95% confidence interval, 0.169-0.936), significantly correlated with family history of breast cancer. We hypothesize that the evaluation of human epidermal growth factor receptor 2, Na+/H+ exchanger regulatory factor 1, and breast cancer susceptibility gene-1 could be clinically useful to identify familial breast tumors and to select patients candidate to breast cancer susceptibility genes 1/2 gene sequencing.     

Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in invasive breast cancer in women

Introduction

Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression are crucial in the biology of breast carcinoma. HER-2/neu gene is amplified and overexpressed in 15-30% of invasive breast cancers. HER-2-positive breast cancers have worse prognosis than HER-2 negative tumors and possess distinctive clinical features. The aim of this study was to assess the expression of HER2 in cancer tissue of patients with invasive breast cancer in correlation with tumor type, histological grade, tumor size, lymph node status, and expression of estrogen receptor and progesterone receptor.

Material and methods

Formalin-fixed, paraffin-embedded tissues from 40 patients with invasive HER-2-positive breast cancer and from 191 patients with HER-2-negative breast cancer were used in this study. HER2 expression was determined using the test HerceptTest™ DAKO.

Results

Among 231 cases of breast cancer, 18 invasive lobular carcinomas and 213 invasive ductal carcinomas were diagnosed. Sixty percent of HER-2-positive breast cancers were ER-positive compared with 77% in the HER-2-negative group (p = 0.002). The expression of PR was observed in 43% of HER-2-positive breast cancers and in 72% of HER2-negative tumors (p = 0.003). Excessive expression of HER2 protein was detected in 60% of patients positive for estrogen receptors, which may worsen prognosis in these patients.

Conclusions

Determination of HER2 overexpression in breast cancer patients, allows for a determination of a group of patients with a worse prognosis.