Estrogen Receptor-negative Breast Carcinoma Research Articles

Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival.

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3- cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3- cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3- cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3- cotransport associates with severe breast cancer: enlarged CO2/HCO3--dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3--dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3- cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

Immunohistochemical and clinicopathologic features of estrogen receptor-negative, progesterone receptor-positive, HER-2 negative breast carcinomas.

Currently, there is an ongoing debate whether progesterone receptor positive and estrogen receptor negative breast carcinomas represent a true distinct subtype of tumor or a mere immunohistochemical artifact. In this study, we conducted an immunohistochemistry panel with the antibodies TFF1, EGFR, and CK5 to reclassify this phenotype in a luminal or basal-like subtype. Tumors estrogen receptor -/progesterone receptor +, Her-2 - from a large population of breast cancer patients were selected to be studied. Immunohistochemistry with the antibodies TFF1, EGFR, and CK5 was performed. Tumors showing positivity for TFF1, regardless of EGFR and CK5 results, were classified as luminal-like carcinomas. Those lesions that were negative for TFF1, but were positive for EGFR and/or CK5, were classified as basal-like triple-negative carcinomas. When the three markers were negative, tumors were classified as undetermined. Clinical pathologic characteristics of patients and tumor recurrence were evaluated. Out of 1188 breast carcinomas investigated, 30 cases (2.5%) presented the estrogen receptor -/progesterone receptor +/HER2- phenotype. Of them, 27 tumors (90%) were classified as basal-like triple-negative carcinomas, one as luminal-like (3.3%), and two as undetermined tumors (6.7%). The mean follow-up for the study group was 27.7 (2.7 to 50) months. Out of the 26 patients, 6 had cancer recurrence: 2 local and 4 systemic recurrences. The average time for recurrence was 17 (8 to 38) months. Estrogen receptor -/progesterone receptor +/tumors exhibit aggressive behavior, similar to triple-negative tumors. An appropriate categorization of these tumors should be made to improve their therapeutic management.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. CAFs were demonstrated to increase expressions and activities of 17βHSD2, 17βHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17βHSD2, 17βHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17βHSD2 and 17βHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17βHSD2 and 17βHSD5 status in TNBC tissues, especially AR-positive cases. Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract P3-05-11: Clinical outcomes of estrogen-receptor negative breast carcinoma are associated with protein hormone-cognate receptor gene expression

Abstract Background: Since certain protein hormones appear to be associated with progression of breast cancer cells in vitro, we employed a global approach by evaluating relationships between genes for these hormones and their cognate receptors in human breast cancer biopsies as independent predictors of risk of recurrence. Our goal is to derive clinically relevant molecular signatures correlating various ER/PR subtypes. Methods: Microarray analyses of LCM-procured carcinoma cells from 247 de-identified biopsies determined expression of genes for 61 protein hormones (the ligands) and 81 cognate receptors. Total RNA was extracted, purified and amplified from cells to determine expression of 22,000 genes. Univariable and multivariable Cox regressions were determined using expression levels of each hormone/receptor gene. Kaplan-Meier plots were constructed with an adjusted p-value <0.30 (discovery cutoff) for each gene candidates with/without ER status of the lesion. Cox regression analyses with/without interaction models deciphered candidates of hormone-receptor complexes that predicted risk of recurrence as well as overall survival using clinical follow-up that extended as much as 12 years. LASSO was used to derive gene subsets to predict overall survival (OS) and progression free survival (PFS) within ER/PR subtypes. Results: Expression levels of 7 genes for protein hormones and 10 receptor genes were significant for OS at an adjusted p-value of <0.30 while expression of 15 hormone and 19 receptor genes were significant for PFS without regard to ER/PR subtypes. When expression levels were considered with ER status only, 3 genes for ER+ lesions and 6 genes for ER- lesions were identified that predicted OS/PFS. Categorization of cancers according to combined ER and PR status revealed one gene for ER+/PR+, 3 genes for ER+/PR- and 2 genes for ER-/PR- associated with PFS/OS. After stratifying gene expression into above/below median for the linear predictor, Kaplan-Meier plots revealed patient groups with each signature for ER+/PR+, ER-/PR- and ER- lesions that exhibited significantly different survival. Molecular signatures comprising 16 genes for ER+/PR+ carcinomas, 10 genes for ER-/PR- lesions and 6 genes for ER- carcinomas predicted clinical outcomes with C-indices of 0.79, 0.76 and 0.71, respectively. Conclusion: We revealed many breast carcinomas synthesize mRNA species for various protein hormones and their cognate receptors by determining gene expression directly on pure populations of these cells procured by LCM. We report a novel ten-gene ER-/PR- signature containing four genes in common with that of a six-gene ER- only signature that predicts breast cancer recurrence. Collectively, results of mRNA expression suggest that often breast carcinomas exhibit substantial elements of endocrine autonomy for regulating progression, warranting investigation of protein products of gene candidates in isolated populations of breast carcinoma cells. Citation Format: Daniels MW, Wittliff JL, Brock GN. Clinical outcomes of estrogen-receptor negative breast carcinoma are associated with protein hormone-cognate receptor gene expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-11.

Abstract A120: Dissecting the role of AGR2 in breast cancer

Abstract Breast cancer is the most common women′s malignity, with growing incidence primarily in advanced countries. Hormone sensitive tumors, characterized by expression of estrogen and progesterone receptors, represent the largest group of breast carcinomas. The presence of estrogen receptors (ER) and progesterone receptors (PgR) indicates response to endocrine therapy and improved disease-free survival (1). The most frequent therapy for estrogen receptor positive breast cancer malignancies is functional blockage of estrogen receptors by tamoxifen. Although tamoxifen is treatment of choice and estrogen receptor targeting is responsible for improvements in cure rates of breast cancers, the therapy itself is limited by frequently developed drug resistance (2). As the endocrine resistance is a significant problem in breast cancer treatment, identification and validation of novel resistance determinants is important to improve treatment efficacy and patient outcome. In our previous work we have found that AGR2 expression is elevated rather than inhibited in response to tamoxifen, thus we have evaluated the function of AGR2 during anti-hormone therapy. Our in vitro results clearly showed that AGR2 promotes viability of cells exposed to tamoxifen. Consequently AGR2 activation through the ER was confirmed by chromatin immunoprecipitation and reporter assays (3). To reveal if AGR2 expression correlates with patient outcome or just mirrors hormonal levels in particular patients, we have determined the AGR2 expression in Tru-Cut needle biopsies from tamoxifen treated postmenopausal breast cancer patients. We found that AGR2 mRNA levels are inversely associated with primary treatment response (p=0.0011) and progression free survival (p=0.0366) indicating that elevated AGR2 expression predicts decreased efficacy of tamoxifen treatment. From this perspective, AGR2 is a potential predictive biomarker enabling selection of an optimal algorithm for adjuvant hormonal therapy in postmenopausal ER-positive breast cancer patients. What is interesting, AGR2 expression could be less frequently detected also in estrogen receptor negative breast carcinomas. Analysis of consecutive group of breast tumors revealed that except estrogen receptor positive cases, AGR2 expression is associated with the HER2 molecular subtype especially, indicating that HER2 overexpression may influence AGR2 levels. These data are consistent with our recently published work showing involvement of PDPK1-AKT signaling pathway in regulation of AGR2 expression (4). Taken together it seems that apart from direct control of AGR2 expression by estrogen receptors, alternative pathway(-s) inclusive HER2 and AKT could be involved in regulation of AGR2 expression. This work was supported by GACR P301/13/00956S, IGA NT/13794-4/2012 and RECAMO CZ.1.05/2.1.00/03.0101 (from European Regional Development Fund).

Impact of anthracycline-based adjuvant chemotherapy on quality of life in women over 70 with estrogen receptor-negative breast carcinoma: Results from the unicancer gerico-06 Phase II multicentre program

Impact of anthracycline-based adjuvant chemotherapy on quality of life in women over 70 with estrogen receptor-negative breast carcinoma: Results from the unicancer gerico-06 Phase II multicentre program

Artemin and Its Emerging Role in Pathogenesis of Systemic Tumors Besides Pancreatic Cancers

TO THE EDITOR: The recent article by Demir et al. (2012) in your esteemed journal provided for highly stimulating and interesting reading. Interestingly, over the past few years artemin has been identified as a significant player in the enhancement of oncogenicity of various other tumors besides pancreatic cancers. For instance, artemin enhances transcription of bcl2 – leading to it's up regulation and thereby augments tumor growth in human non-small cell lung carcinomas (Tang et al., 2010). Similarly, in estrogen receptor negative breast carcinomas, artemin shows synergization with TWIST1 and thereby accentuates the metastatic potential of the primary breast tumor (Banerjee et al., 2011). As a result, a poor clinical outcome is associated with this combination of artemin and TWIST1. Attenuated artemin expression is seen as a result of tamoxifen administration (Kang et al., 2010). Interestingly, the sensitivity to tamoxifen of tamoxifen resistant mammary tissue is accentuated following antibody mediated inhibition of artemin. Increased expression of artemin is also seen in esophageal carcinomas. Interestingly transfection with a mir-223 vector decreases expression of artemin and thereby suppresses tumor growth in esophageal carcinomas (Li et al., 2011). Similarly, artemin augments the expression of AKT1 and thereby accentuates the invasive potential of endometrial carcinomas (Pandey et al., 2010). The invasive potential of endometrial cancer tissue is significantly abrogated following antibody mediated inhibition of artemin. The above examples clearly illustrate the significant enhancement of oncogenicity secondary to artemin in tumors ranging from lung carcinomas to endometrial carcinomas. There is a clear and urgent need to identify inhibitors of artemin function in order to improve the prognosis in these tumors.

Tamoxifen Inhibits TRPV6 Activity via Estrogen Receptor–Independent Pathways in TRPV6-Expressing MCF-7 Breast Cancer Cells

The epithelial calcium channel TRPV6 is upregulated in breast carcinoma compared with normal mammary gland tissue. The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy. Previously, we showed that tamoxifen inhibits calcium uptake in TRPV6-transfected Xenopus oocytes. In this study, we examined the effect of tamoxifen on TRPV6 function and intracellular calcium homeostasis in MCF-7 breast cancer cells transiently transfected with EYFP-C1-TRPV6. TRPV6 activity was measured with fluorescence microscopy using Fura-2. The basal calcium level was higher in transfected cells compared with nontransfected cells in calcium-containing solution but not in nominally calcium-free buffer. Basal influxes of calcium and barium were also increased. In transfected cells, 10 mumol/L tamoxifen reduced the basal intracellular calcium concentration to the basal calcium level of nontransfected cells. Tamoxifen decreased the transport rates of calcium and barium in transfected cells by 50%. This inhibitory effect was not blocked by the estrogen receptor antagonist, ICI 182,720. Similarly, a tamoxifen-induced inhibitory effect was also observed in MDA-MB-231 estrogen receptor-negative cells. The effect of tamoxifen was completely blocked by activation of protein kinase C. Inhibiting protein kinase C with calphostin C decreased TRPV6 activity but did not alter the effect of tamoxifen. These findings illustrate how tamoxifen might be effective in estrogen receptor-negative breast carcinomas and suggest that the therapeutic effect of tamoxifen and protein kinase C inhibitors used in breast cancer therapy might involve TRPV6-mediated calcium entry. This study highlights a possible role of TRPV6 as therapeutic target in breast cancer therapy.

17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis

17beta-Hydroxysteroid dehydrogenase type 12 (17beta-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17beta-HSD12 expression was also reported in breast carcinomas but its functions have remained unknown. In this study, we examined the correlation between mRNA expression profiles determined by microarray analysis and tissue E2 concentrations obtained from 16 postmenopausal breast carcinoma cases. No significant correlations were detected between 17beta-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17beta-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17beta-HSD12 using cell-based studies. Small interfering RNA-mediated knockdown of 17beta-HSD12 in SK-BR-3 (estrogen receptor-negative breast carcinoma cell line) resulted in significant growth inhibition, which was recovered by the addition of VLCFAs such as arachidonic acid. The status of 17beta-HSD12 immunoreactivity was also correlated with adverse clinical outcome in cyclooxygenase 2 (COX2)-positive breast cancer patients but not in COX2-negative patients. Therefore, these findings indicated that 17beta-HSD12 was not necessarily related to intratumoral E2 biosynthesis, at least in human breast carcinoma, but was rather correlated with production of VLCFAs such as arachidonic acid, which may subsequently be metabolized to prostaglandins by COX2 and result in tumor progression of the patients.

Relationship between levels of Skp2 and P27 in breast carcinomas and possible role of Skp2 as targeted therapy

Estrogen receptor-negative breast carcinomas are more aggressive and are unresponsive to anti-estrogens. Thus, they clearly require new therapies targeted against specific genes and proteins actively engaged in the pathophysiology of cancer. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. We attempted to explore whether Skp2 may be a potential specific therapeutic target in the subset of aggressive breast carcinomas by investigating the possible relationship between expression of Skp2 and p27 proteins and estrogen receptor (ER). Immunohistochemical analysis of tumor tissues was employed to determine the expression of Skp2, p27, and ER proteins in 82 cases of primary breast carcinoma. Higher levels of Skp2 were detected more frequently in ER-negative tumors and tumors metastatic to the axillary lymph nodes. The expression of p27 was inverse with the histologic grade. Statistical analysis showed that the percentage of high Skp2 expressors was significantly greater in the group with low p27 expression than in the group with high p27 expression. The current study, together with the results from a previous study, demonstrated the existence of a subtype of high-grade, negative ER breast carcinomas with high Skp2 and low p27 levels. This implies that Skp2 may be a potential specific therapeutic target in a subset of aggressive breast carcinomas. Thus far, there is no specific therapy for the ER-negative and HER-2/neu resistant groups, which are among the subset of aggressive tumors.

Estrogen receptor-negative breast carcinomas: a review of morphology and immunophenotypical analysis

Estrogen receptor (ER)-negative breast cancers are a group of tumors with poor prognosis and fewer cancer prevention and treatment strategies compared to ER-positive tumors. The aim of this study was to assess the morphological characteristics and immunohistochemical profile of ER-negative tumors and thus to understand the biological behavior and unique nature. In total, 291 consecutive ER-negative cases available from our primary breast cancer series were examined. Hematoxylin- and eosin-stained sections of all the cases were studied for several morphological parameters and their immunophenotype profile. These findings were correlated with patient and tumor characteristics and survival data. ER-negative tumors constituted 30% of the primary operable breast cancer series. The majority of tumors were grade 3 (94%) and the commonest histological types were ductal/no specific type (85%), and atypical medullary carcinoma (8%). High-grade comedo-type necrosis, lymphoid stroma, central necrosis/fibrosis and pushing margins were the most common morphological features. The presence of a pushing margin showed a significant relation to androgen receptor negativity, absence of epidermal growth factor receptor expression and negative lymph nodes. Lymphoid stroma and comedo-necrosis correlated with higher tumor grade. ER-negative breast cancers are a distinct group of tumors with several common morphological features. Grade 3 histology, pushing margin, lymphoid stroma, comedo-type necrosis and central fibrosis/necrosis are the dominant morphological findings. The presence of a pushing margin appears to have a significant correlation with negative lymph node status. ER-negative tumors show a higher expression of p53, CerbB2 and epidermal growth factor receptor compared to ER-positive breast cancer. These unique features support the concept that ER-negative tumors are a morphologically and phenotypically distinct entity and provide a rationale for the study and use of newer promising agents in the treatment of ER-negative breast cancer.

Allelic loss at the 8p22 region as a prognostic factor in large and estrogen receptor negative breast carcinomas

BACKGROUND Allelic losses of tumor suppressor genes, or the chromosomal regions harboring them, in the DNA of tumor cells may become useful postoperative prognostic indicators. The authors studied frequent loss of heterozygosity (LOH) on chromosome 8p22 and its association with disease progression that occurred later in patients with breast carcinoma. METHODS To examine whether allelic losses at 8p22 might correlate with postoperative survival during a 5-year period of prospective follow-up, the authors tested tumors from a cohort of 298 breast carcinoma patients informative for 8p22 markers. The tumors were tested for allelic losses of microsatellite markers D8S136 and D8S1106 located at 8p22, a chromosomal region where genetic alterations are frequent in breast carcinomas. RESULTS Among the 298 breast carcinoma patients, 154 (52%) lost alleles in tumors. Patients whose tumors had lost an allele at 8p22 had a significantly higher risk of postoperative mortality than those whose tumors retained both alleles at those loci; their 5-year mortality rates were 18% (26 patients died among 154 with losses at 8p22) versus 7% (10 patients died among 144 with retentions at 8p22) (P = 0.017). The 8p22 LOH was a significant independent prognostic factor for postoperative survival in a group of patients with large tumors (> 2.1 cm) and in a group of patients with estrogen receptor negative tumors in both univariate and multivariate analyses. These data show that 8p22 LOH was a significant prognostic factor for the postoperative survival of certain clinical groups of patients who underwent surgery for breast carcinoma. CONCLUSIONS Allelic loss on chromosome 8p22 can serve as a negative prognostic indicator to guide the postoperative management of patients, especially patients with large tumors (> 2.1 cm) and those with estrogen receptor negative tumors. Cancer 2000;88:1410–6. © 2000 American Cancer Society.

Allelic loss at the 8p22 region as a prognostic factor in large and estrogen receptor negative breast carcinomas

Allelic losses of tumor suppressor genes, or the chromosomal regions harboring them, in the DNA of tumor cells may become useful postoperative prognostic indicators. The authors studied frequent loss of heterozygosity (LOH) on chromosome 8p22 and its association with disease progression that occurred later in patients with breast carcinoma. To examine whether allelic losses at 8p22 might correlate with postoperative survival during a 5-year period of prospective follow-up, the authors tested tumors from a cohort of 298 breast carcinoma patients informative for 8p22 markers. The tumors were tested for allelic losses of microsatellite markers D8S136 and D8S1106 located at 8p22, a chromosomal region where genetic alterations are frequent in breast carcinomas. Among the 298 breast carcinoma patients, 154 (52%) lost alleles in tumors. Patients whose tumors had lost an allele at 8p22 had a significantly higher risk of postoperative mortality than those whose tumors retained both alleles at those loci; their 5-year mortality rates were 18% (26 patients died among 154 with losses at 8p22) versus 7% (10 patients died among 144 with retentions at 8p22) (P = 0.017). The 8p22 LOH was a significant independent prognostic factor for postoperative survival in a group of patients with large tumors (>2.1 cm) and in a group of patients with estrogen receptor negative tumors in both univariate and multivariate analyses. These data show that 8p22 LOH was a significant prognostic factor for the postoperative survival of certain clinical groups of patients who underwent surgery for breast carcinoma. Allelic loss on chromosome 8p22 can serve as a negative prognostic indicator to guide the postoperative management of patients, especially patients with large tumors (>2.1 cm) and those with estrogen receptor negative tumors.

Expression of human estrogen receptor using an efficient adenoviral gene delivery system is able to restore hormone-dependent features to estrogen receptor-negative breast carcinoma cells

Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat as they do not respond to hormone therapy. In an attempt to determine if expressing the human estrogen receptor in an ectopic manner could restore the hormone responsiveness of these cells, we have expressed the human ER in ER-negative MDA-MB 231 breast cancer cells using a recombinant adenovirus gene delivery system that allows high level expression of ER in essentially all cells. In these cells, the ER was correctly translated, had a wild type hormone binding affinity ( K d=0.6 nM), bound well to estrogen response element-containing DNA, and showed an activation pattern of estrogen response element-reporter gene activity by estrogen and antiestrogens very similar to that observed in MCF-7 breast cancer cells containing endogenous ER (stimulation by estrogen, no stimulation by the antiestrogens trans-hydroxytamoxifen or ICI 164 384, and blockade of estradiol stimulation by trans-hydroxytamoxifen or ICI 164 384). Intriguingly, estradiol stimulation of these cells was also able to induce expression of pS2, an estrogen regulated gene considered to be a favorable prognostic marker for endocrine therapy in ER-positive breast cancer cells. Expression of the ER had no effect by itself on the proliferation rate of MDA-MB 231 cells. However, treatment of the ER-containing cells with estradiol or with the pure antiestrogen ICI 164 384 suppressed proliferation of the cells while the antiestrogen trans-hydroxytamoxifen had little effect on proliferation; and cotreatment with trans-hydroxytamoxifen reversed the estradiol- or ICI 164 384-evoked suppression of proliferation. To understand the mechanism underlying the inhibition of proliferation by estradiol, we examined the expression of several growth related endogenous genes. c- Myc protooncogene expression was strongly inhibited by treatment with estradiol as was expression of BRCA1 and BRCA2 genes, which is in agreement with their mitogenic-dependent expression, while expression of β-actin, a housekeeping gene, was not affected by hormone treatment. Together, these data suggest that reexpressing the human ER in breast cancer cells that no longer express this protein renders them sensitive to hormone treatment. The ability of the antiestrogen ICI 164 384 to suppress the proliferation of ER-negative breast cancer cells that reexpress ER might be useful ultimately as an endocrine gene therapy approach for controlling the growth of ER-negative breast cancer cells. The application of recombinant adenoviruses expressing the human ER presents interesting features which might be used as a basis for designing more powerful and effective treatments for ER-negative breast cancers.

Tamoxifen Modulates Protein Kinase C via Oxidative Stress in Estrogen Receptor-negative Breast Cancer Cells

Nonsteroidal agent tamoxifen (Tam), a therapeutic/chemopreventive agent for breast cancer, inhibits protein kinase C (PKC), which is considered to be one of its extra-estrogen receptor sites of action. This drug is required at higher (>100 microM) concentrations to inhibit PKC in the test tube, whereas it is required at lower (1-10 microM) concentrations to induce inhibition of cell growth in estrogen receptor-negative cell types. To identify additional mechanisms of action of Tam on PKC and cell growth, studies with MDA-MB-231, an estrogen receptor-negative breast carcinoma cell type, have been carried out. Upon treatment with 5-20 microM Tam, a cytosol to membrane translocation of PKC occurred within 30 min, which was then followed by a down-regulation of the enzyme within 2 h. A transient generation of Ca2+/lipid-independent activated form of PKC was observed during this period. Rapidly growing cells require nearly 2-3-fold lower concentrations (2-5 microM) of Tam than do confluent cells to induce changes in PKC. Furthermore, phorbol ester binding observed with intact cells also decreased in Tam-treated cells only under the conditions PKC was inactivated. Unlike phorbol esters, Tam did not directly support the membrane association of PKC. The release of arachidonic acid correlated with the PKC membrane translocation. Studies carried out with [3H]Tam revealed that Tam partitioned into the membrane, and there was no appreciable covalent association of [3H]Tam with cellular proteins within this limited time period (2 h). Various antioxidants (vitamin E, vitamin C, beta-carotene, catalase, and superoxide dismutase) inhibited all these cellular effects of Tam. Moreover, vitamin E strikingly blocked Tam-induced growth inhibition. To determine whether oxymetabolites of Tam can affect PKC permanently, OH-Tam was tested with purified PKC. In contrast to Tam, which reversibly inhibited PKC, OH-Tam permanently inactivated the enzyme by modifying the catalytic domain at lower concentrations. The vicinal thiols present within this domain were found to be required to induce this inactivation. This effect was partially blocked by various antioxidants. This is the first report showing the role of oxidative stress in mediating the actions of Tam. Taken together these results suggest that Tam, by initially partitioning into the membranes, induces a generation of transmembrane signals and an oxidative stress to elicit the membrane association of PKC, followed by an irreversible activation, and subsequent down-regulation of this enzyme, which, in part, may lead to cell growth inhibition.

Inhibition of mammary carcinoma growth by retinoidal benzoic acid derivatives.

The growth of many breast carcinoma cell lines is inhibited by vitamin A, and derivatives as well as synthetic retinoids. New retinoidal derivatives have recently been synthesized. These retinoidal benzoic acid derivatives displayed enhanced potency in their ability to reverse hamster tracheal keratinization and inhibit ornithine decarboxylase induction in mouse epidermis. We therefore screened a series of analogues of these compounds for their ability to inhibit human breast carcinoma cell proliferation utilizing three estrogen receptor-positive and two estrogen receptor-negative cell lines. The compound (E)-4-2-(5,6,7,8)tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)prop enyl benzoic acid (Ro 1374-10) was approximately 2-3 orders of magnitude more potent than all-trans-retinoic acid in inhibiting breast carcinoma cell proliferation while the compound SRI-6409-40, which differs from Ro 1374-10 only by the position of a methyl group, was 50-fold more potent than Ro 1374-10. All of the compounds tested displayed were inactive against the estrogen receptor-negative breast carcinoma lines.

Prognostic factors in patients with stage I, estrogen receptor-negative carcinoma of the breast. A clinicopathologic study.

In order to aid in the identification of a group of breast cancer patients at high risk of recurrence in whom the possible value of adjuvant chemotherapy might be most readily demonstrable, the authors reviewed the clinicopathologic features of 74 women with infiltrating, Stage I, estrogen receptor-negative breast carcinoma. They found significant correlations between short-term recurrence (within 2 years) and extensive necrosis (P less than 0.005), anaplastic tumor nuclear morphology (P less than 0.01), a positive family history of breast carcinoma (P less than 0.05), diffuse hyperplasia of axillary lymph nodes (P less than 0.05), and young age (P less than 0.05). Twenty percent of all patients experienced recurrence within 2 years, while recurrence was noted in 32% of patients with highly pleomorphic nuclei, 38% of those with extensive necrosis, and 45% of those with a positive family history. Recurrence was noted in 55% of the small number of patients with both anaplastic nuclear morphology and a positive family history. These apparent risk factors for early recurrence may be helpful in prospectively selecting patients most eligible to receive adjuvant chemotherapy.

113 Thromboxane and prostacyclin in breast cancer: Effect of medroxyprogesterone acetate (MPA)

Estrogen receptor-negative breast carcinomas are more aggressive and are unresponsive to anti-estrogens. Thus, they clearly require new therapies targeted against specific genes and proteins actively engaged in the pathophysiology of cancer. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. We attempted to explore whether Skp2 may be a potential specific therapeutic target in the subset of aggressive breast carcinomas by investigating the possible relationship between expression of Skp2 and p27 proteins and estrogen receptor (ER). Immunohistochemical analysis of tumor tissues was employed to determine the expression of Skp2, p27, and ER proteins in 82 cases of primary breast carcinoma. Higher levels of Skp2 were detected more frequently in ER-negative tumors and tumors metastatic to the axillary lymph nodes. The expression of p27 was inverse with the histologic grade. Statistical analysis showed that the percentage of high Skp2 expressors was significantly greater in the group with low p27 expression than in the group with high p27 expression. The current study, together with the results from a previous study, demonstrated the existence of a subtype of high-grade, negative ER breast carcinomas with high Skp2 and low p27 levels. This implies that Skp2 may be a potential specific therapeutic target in a subset of aggressive breast carcinomas. Thus far, there is no specific therapy for the ER-negative and HER-2/neu resistant groups, which are among the subset of aggressive tumors.