Deciphering antihormone-induced compensatory mechanisms in breast cancer and their therapeutic implications.

Breast cancer inhibition by antihormones is rarely complete, and our studies using responsive models reveal the remarkable flexibility of breast cancer cells in recruiting alternative signalling to limit maximal anti-tumour effects of oestrogen receptor alpha (ER) blockade. The recruited mechanism involves antihormone-induced expression of oestrogen-repressed signalling genes. For example, epidermal growth factor receptor gene (EGFR) is induced by antioestrogens and maintains residual kinase and ER phosphorylation, cell survival genes, and thereby allows incomplete antihormone response and emergence of resistance. Microarrays are revealing the breadth of antihormone-induced genes that may attenuate growth inhibition, including NFkappaB, Bag1, 14-3-3zeta and tyrosine kinases, such as HER2 and Lyn. Three concepts are emerging: first, some genes are induced exclusively by antioestrogens, while others extend to oestrogen deprivation; secondly, some are transiently induced, while others persist into resistance; finally, some confer additional adverse features when tumour cells are in an appropriate context. Among the latter is CD59 whose antioestrogen induction may permit evasion of immune surveillance in vivo. Also, induction of pro-invasive genes (including NFkappaB, RhoE and delta-catenin) may underlie our findings that antioestrogens can markedly stimulate migratory behaviour when tumour intercellular contacts are compromised. Based on our promising studies selectively inhibiting EGFR (gefitinib), NFkappaB (parthenolide) or CD59 (neutralising antibody) together with antioestrogens, we propose that co-targeting strategies could markedly improve anti-tumour activity (notably enhancing cell kill) during the antihormone-responsive phase. Furthermore, subverting those induced signalling genes that are retained into resistance (e.g. EGFR, NFkappaB, HER2) may prove valuable in this state. Alongside future deciphering and targeting of genes underlying antioestrogen-promoted invasiveness, embracing of intelligent combination strategies could significantly extend patient survival.

Comparison of the effects of a pure steroidal antiestrogen with those of tamoxifen in a model of human breast cancer.

BACKGROUND: Tamoxifen, a nonsteroidal estrogen antagonist, is the most prescribed drug for the treatment of breast cancer. The use of tamoxifen is limited, however, by the development of resistance to this compound in most patients. Although tamoxifen behaves primarily as an estrogen antagonist, it has agonist (or growth-stimulatory) activity as well. ICI 182,780 is a 7 alpha-alkylsulfinyl analogue of estradiol lacking agonist activity. The absence of agonist activity may make this steroidal antiestrogen superior to tamoxifen in suppressing tumor cell growth. PURPOSE: We compared the inhibitory effects of ICI 182,780, tamoxifen, and estrogen withdrawal on the growth of established tumors and on tumorigenesis in a model system that uses estrogen-dependent, human MCF-7 breast tumor cells growing in athymic nude mice. We also studied the hormonal responsiveness of tumors that became resistant to the two estrogen antagonists and the effects of these drugs on estrogen-regulated gene expression. METHODS: MCF-7 cells were injected subcutaneously into the flanks of castrated, female nude mice. The effects of repeated doses of tamoxifen and ICI 182,780 (500 micrograms and 5 mg, respectively) on the growth of established tumors (8-10 mm in size) were determined after supplemental estrogen was removed. The effects of antiestrogen treatments on the process of tumorigenesis, in the absence of estrogen supplementation, were determined by initiating drug administration on the same day as tumor cell inoculation. To evaluate the hormonal responsiveness of tumors resistant to tamoxifen and ICI 182,780, 1-mm3 segments of the tumors were transplanted onto the flanks of new recipient mice, which were then treated with estrogen or the antiestrogens--alone or in combination. Tumor growth was monitored by measuring tumor volumes twice a week. Expression of the estrogen-responsive genes, pLIV1 and pS2, in the tumors of treated animals was analyzed using blots of total cellular RNA and complementary DNA probes. RESULTS: Treatment with ICI 182,780 suppressed the growth of established tumors twice as long as treatment with tamoxifen or estrogen withdrawal. Tumorigenesis, in the absence of supplemental estrogen, was delayed to a greater extent in ICI 182,780-treated mice than in tamoxifen-treated mice. ICI 182,780 was found to be more effective than tamoxifen in reducing the expression of estrogen-regulated genes. Most tumors eventually became resistant to ICI 182,780 and grew independently of estrogen. CONCLUSIONS: ICI 182,780 is a more effective estrogen antagonist than tamoxifen in the MCF-7 tumor cell/nude mouse model system.

Characterization of estrogen receptor messenger RNA in human breast cancer.

The importance of estrogen receptor (ER) determination in breast cancer is well established. Approximately 70% of ER-positive tumors are hormone responsive compared to 5-10% of ER-negative tumors. However, one-third of ER-positive tumors fail to respond, and the reasons for this are unclear. To further investigate these relationships we have determined levels of ER protein and mRNA in a number of human breast cancer biopsies. ER protein was estimated by the dextran-coated charcoal steroid binding method and by an ER immunocytochemical assay using a specific monoclonal antibody. A complimentary DNA clone (lambda OR3) encoding part of the human ER was used to determine mRNA levels. Dot blot analysis of twenty-seven tumors revealed a close agreement between ER mRNA and the dextran-coated charcoal assay (rs = 0.9; P less than 0.001). ER immunocytochemical assay staining also correlated with ER mRNA in twenty-five cases (rs = 0.75; P less than 0.001). Tumors from postmenopausal patients contained much higher levels of ER mRNA and ER protein than their premenopausal counterparts. ER-negative tumors produced no measurable ER mRNA. Northern blot analysis revealed a 6.4- and 3.7-kilobase species in ER-positive tumors and also in the human breast cancer cell line MCF-7. No differences in transcript sizes were found in tumors from hormone-responsive patients compared to nonresponding patients. We have also demonstrated, in tissue sections of normal and malignant breast, localization of ER mRNA by in situ hybridization to the same population of cells which exhibit immunoreactive ER.

Comparison of an immunocytochemical assay for progesterone receptor with a biochemical method of measurement and immunocytochemical examination of the relationship between progesterone and estrogen receptors.

Using a rat monoclonal antibody raised against human progesterone receptor (PR) we have developed an immunocytochemical technique to detect PR in human normal and malignant breast tissue and have compared the distribution of this with that obtained by the conventional dextran-coated charcoal steroid-binding assay. Immunoreactive PR was detected exclusively in the nuclei of epithelial cells in 29/51 (56.9%) of breast cancers studied. There was an excellent correlation between the immunocytochemical and dextran-coated charcoal techniques, with concordance in 43/51 (84.3%) cases [regression coefficient (Spearman) = 0.78; P less than 0.001]. The relationship between PR and estrogen receptor (ER) was also examined immunocytochemically using a monoclonal antiserum to ER. Twenty-eight out of 51 (54.9%) tumors were positive for both receptors and 13/51 (25.5%) negative for both. ER-positive, PR-negative tumors were found in 9/51 (17.6%) cases whereas only one case (2%) was PR-positive, ER-negative.

Immunocytochemical assay for estrogen receptor: relationship to outcome of therapy in patients with advanced breast cancer.

We have used an immunoperoxidase technique utilizing a monoclonal antibody to the estradiol receptor to identify immunoreactive estradiol receptor in breast carcinomas and have examined the relationship between the immunoreactive estradiol receptor and response to therapy in patients with advanced breast cancer. Fifty-six patients were found to be assessable for response to endocrine therapy. Twenty-two showed an objective response to some form of endocrine manipulation, and all these had positively stained carcinomas. None of the 17 patients with negatively stained carcinomas responded to endocrine therapy. We conclude that the monoclonal antibody to estradiol receptor can help identify breast cancer patients who may respond to endocrine therapy.

Immunocytochemical detection of 1,25-dihydroxyvitamin D3 receptor in breast cancer.

We have developed an immunocytochemical technique to visualize the receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cryostat sections of human breast tumors and normal human breast tissue utilizing a monoclonal antibody (9A7 gamma) to chick intestinal receptor which recognizes mammalian 1,25(OH)2D3 receptor. Specific staining was observed in the nuclei of tumor cells. Previous studies by our group have shown that a high proportion of breast tumors bind radiolabeled 1,25(OH)2D3 and we have confirmed this, demonstrating immunocytochemical 1,25(OH)2D3 receptor in 43 of 55 (78%) of breast carcinomas. No correlation with the presence of immunostainable estrogen receptor was found in these breast cancer specimens. Sections of normal breast showed immunoreactivity in the nuclei of epithelial cells of the lobules and ducts. Our results demonstrate that the receptor for 1,25(OH)2D3 resides predominantly in the nucleus of breast carcinoma cells. The reason for its prominent expression in breast cancers is not yet known but may be related to growth regulation.

Presurgical determination of estrogen receptor status using immunocytochemically stained fine needle aspirate smears in patients with breast cancer.

We have developed an immunocytochemical staining procedure (ERICA) using a monoclonal antibody to the estrogen receptor (ER) to determine ER status from samples obtained by fine needle aspiration of primary and recurrent breast cancer tissue (cyto-ERICA). ER status was assessable on 214 of 246 smeared aspirates from breast cancer patients. In 143 (66.8%) assessable smears positive nuclear staining was observed but was completely absent in 71 (33.2%) cases. In 107 cases we were able to compare results with those obtained with the quantifiable dextrancoated charcoal (DCC) radioligand binding technique using surgically excised material. We observed qualitative agreement in 53 of 62 (85.5%) of primary specimens and 16 of 16 (100%) recurrent samples compared to the subsequent DCC result on the same sample. Aspirates obtained from new secondary deposits were also assessed and in 16 of 19 (84.2%) cases results agreed with that established previously by DCC on the primary breast tumor. In a further 6 of 10 (60%) cases the cyto-ERICA result obtained from recurrent samples qualitatively agreed with that determined by DCC on a previous recurrent lesion. A comparison of staining of aspirates was also made against frozen tissue sections stained with the monoclonal antibody (tissue-ERICA). Where comparison was made of primary tumor specimens agreement was observed in 40 of 45 (88.9%) of cases while specimens from secondary lesions agreed qualitatively in 14 of 17 (82.3%) of cases. In a small number of samples where tissue-ERICA was performed on an earlier lesion to that aspirated for cyto-ERICA an agreement of 4 of 5 (80%) was observed. This technique shows good sensitivity in demonstrating ER in aspirate specimens, should therefore permit us to determine ER status before surgery for primary breast cancer, and may also mean that surgery for recurrent disease to determine receptor status is no longer necessary.

Measurement of 1,25-dihydroxyvitamin D3 receptors in breast cancer and their relationship to biochemical and clinical indices.

Both normal and malignant breast tissue contain the specific receptor for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). A recent study has shown its presence in 80% of surgically removed breast tumors, although only at low levels. We have measured the 1,25-(OH)2D3 receptor in breast tumors from 68 patients and have found it at similar frequency (75%) but at much higher concentrations (range, less than 1 to 30 fmol/mg protein). This receptor has the same characteristics as that measured in classic 1,25-(OH)2D3 target tissues and was distinguished by sucrose gradient centrifugation from plasma contaminants. Complete case histories and follow-up were available on 56 of these patients, and 1,25-(OH)2D3 receptor status (less than 8 or greater than or equal to 8 fmol/mg protein) was not related to the level of estrogen receptors, menopausal status, T-stage or histology of tumors, or presence of 99mTc phosphate hot spots on bone scans. The lack of relationship between the level of 1,25-(OH)2D3 receptors and other prognostic indicators suggests its potential as a new independent variable for assessing breast cancer patients. However, at this stage, 1,25-(OH)2D3 receptor status did not result in any significant difference in probability of survival or metastasis-free survival. Assessment of the importance of this variable for treatment or outcome must await an increased number of patients and a longer time since surgery.

Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer.

Frozen sections of breast tumor tissue have been stained using an immunoperoxidase [estrogen receptor (ER)-immunocytochemistry] kit incorporating a monoclonal antiserum [H222] to visualize nuclear human ERs. Quantitation of specific staining has been performed by manual procedures using optical microscopy and by a computer-assisted image analysis system (CAS 100). Initial investigations with a test panel of ER-immunocytochemistry-positive tumors revealed a good qualitative agreement between CAS and manual assessments. Reduced variance was, however, observed between quantified ER-immunocytochemistry results from four experienced investigators using the CAS analysis. An extended study confirmed the relationships between CAS and manual methods of assessment. These findings were evident when studies were scored either by assessment of the percentage of positively stained cells (n = 92; r = 0.919; P less than 0.01) or by H-score calculations (n = 92; r = 0.913; P less than 0.01). A good correlation was also found between CAS quantification and the results of an ER enzyme immunoassay of 48 primary breast cancer specimens (r = 0.715; P less than 0.05). In 49 cases it was possible to relate CAS-defined ER status and levels to the subsequent response of patients to endocrine therapy. ER was assessed on specimens obtained prior to commencement of treatments for recurrent breast cancer. Presuming the presence of ER to be a prerequisite for successful therapy, very good correlations between response and both status and levels of positivity were recorded. None of 16 patients with CAS-ER-negative tumors responded to treatment, while 16 of 33 (48.4%) CAS-ER-positive patients achieved an objective response according to International Union Against Cancer criteria. A relationship between response and the degree of CAS-ER positivity was obtained when the CAS score divisions of 0, 1-100, and greater than 100 (response rates, 0, 41, and 64%, respectively) were used. These data demonstrate that automated image analysis offers a reliable, reproducible procedure for quantifying ER in immunocytochemically stained sections. It has potential advantages over manual procedures, providing less opportunity for subjective influences in scoring sections. Future advances in software design should further reduce elements of subjectivity and increase both the speed and reliability of results. We anticipate image analysis becoming a valuable tool in investigations concerning, for example, the influence of heterogeneity of steroid receptor distribution on the rate of recurrence of breast cancer after mastectomy and in the clinical course of the disease.

Immunocytochemical determination of estrogen receptor, progesterone receptor, and 1,25-dihydroxyvitamin D3 receptor in breast cancer and relationship to prognosis.

We have determined the estrogen receptor, progesterone receptor (PR), and 1,25-dihydroxyvitamin D3 receptor content of 136 breast carcinomas by an immunocytochemical method. The presence of the three receptors was not related to clinical features of presentation such as T-stage or to age or menopausal status. However, each of the three receptors has a different relationship to the course of the disease in these patients. The presence of PR was significantly associated with an improved overall survival (chi 2 = 4.61, P = 0.032). Patients whose tumors contained immunocytochemically detectable 1,25-dihydroxyvitamin D3 receptor had a longer disease-free interval than those patients with negative tumors (chi 2 = 4.01, P = 0.045). The presence of estrogen receptor and PR were found to correlate with an increased survival between relapse and death (P = 0.027 and P = 0.09, respectively). The relationships between estrogen receptor and PR and prognosis are more apparent when the degree of cell staining is considered. Combined receptor analysis improves our ability to predict the course of the disease and may therefore facilitate better management of the patients.

Transforming growth factor-alpha and endocrine sensitivity in breast cancer.

The expression of transforming growth factor-alpha (TGF-alpha) has been evaluated in 51 breast cancers of known responsiveness to endocrine therapy using immunohistochemistry. High levels of TGF-alpha were observed in 65% of tumors and showed no relationship with tumor estrogen receptor or epidermal growth factor receptor status or Ki67 immunostaining. TGF-alpha levels did, however, relate to the endocrine sensitivity of the disease, with unresponsive tumors frequently showing high levels of TGF-alpha immunoreactivity. This relationship was observed in estrogen receptor-positive disease and was independent of the epidermal growth factor receptor status of the tumor. No quantitative association between TGF-alpha and Ki67 immunostaining was observed in any of the subcategories of tumors. These data infer a role for TGF-alpha in the development of endocrine insensitivity in estrogen receptor-positive breast cancer by mechanisms which appear independent of tumor growth fraction, as determined by Ki67 immunostaining.

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival.

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.

Immunocytochemical assay for estrogen receptor in patients with breast cancer: relationship to a biochemical assay and to outcome of therapy.

We developed an immunoperoxidase technique using a monoclonal antibody to the estradiol receptor (ER) to identify immunoreactive ER (iER) in breast carcinomas and compared this with the conventional dextran-coated charcoal (DCC) steroid binding assay. We also examined the relationship between the iER and response to therapy in patients with advanced breast cancer. We found iER-positive cells in 60 of 90 carcinomas (66.7%); this correlated with the DCC assay (r = 0.76; P less than .001). Of these, 56 patients were found to be assessable for response to endocrine therapy. Twenty-two showed an objective response to some form of endocrine manipulation, and all these had positively stained carcinomas. By deriving a staining intensity index (SII) we observed that 21 of 22 responders (95%) had an SII of greater than or equal to 0.5, whereas only 8 of 34 nonresponders (24%) had an SII of greater than or equal to 0.5. This difference is highly significant (P less than .001). None of the 17 patients with negatively stained carcinomas responded to endocrine therapy. We conclude that the monoclonal antibody to ER can help identify breast cancer patients who may respond to endocrine therapy.

Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer.

The CCND1 gene, encoding the cell cycle regulatory protein cyclin D1, maps to chromosome 11q13, a locus that is amplified in about 13% of breast cancers. Because several studies have indicated a relationship between 11q13 amplification and markers of phenotype including estrogen receptor (ER) status, we tested the relationship between CCND1 and ER gene expression in 364 primary breast cancers using Northern blot analysis. Seventy-three % of samples were positive for ER mRNA, and cyclin D1 mRNA levels in the ER-positive group were significantly higher than those in the ER-negative group (P = 0.0001). When the samples were divided into quartiles of cyclin D1 expression, 58% of samples were ER positive in the lowest quartile and 87% in the highest quartile. The tumors expressing the highest levels of cyclin D1 (7%) were all ER positive. Furthermore, ER mRNA levels in the half with lower cyclin D1 mRNA were significantly less than in the half with higher cyclin D1 levels (P = 0.0001). Using simple regression analysis, there was a significant positive correlation between cyclin D1 and ER mRNA levels in the total population (P = 0.0001). This study demonstrates that cyclin D1 mRNA and ER mRNA are positively correlated in primary breast cancer, but the functional relationship between these genes remains to be elucidated.

Use of reverse transcription-polymerase chain reaction methodology to detect estrogen-regulated gene expression in small breast cancer specimens.

We describe the development and use of a sensitive reverse transcription-PCR (RT-PCR) procedure to detect novel estrogen-regulated gene expression in small clinical breast cancer samples, in which such study would be extremely difficult by any other molecular or immunocytochemical means. Assay optimization for pLIV1, estrogen receptors (ERs), progesterone receptors, and pS2 gene products was carried out on 50 primary breast cancers for which comparative Northern analysis and immunocytochemical data were available. Using 27 amplification cycles and a 0.5 microM primer concentration, varying expressions of the gene products were recorded simultaneously with a constant densitometric signal for a coamplified endogenous control gene (alpha-actin). Good concordances were subsequently observed between pLIV1 status generated by RT-PCR and both Northern analysis (P = 0.002) and ER status by immunocytochemistry (P = 0.0244). Agreement was also noted between ER (P = 0.002), progesterone receptor (P = 0.0005), and pS2 (P = 0. 0023) RT-PCR and immunocytochemical methodologies. The RT-PCR assays were then applied to 10 needle core trucut biopsies in which similar relationships were obtained. Our results justify the future use of this RT-PCR methodology to examine new estrogen-regulated genes in small breast cancer samples, and it is envisaged that this technology will prove invaluable in many future breast cancer studies.

Involvement of steroid hormone and growth factor cross-talk in endocrine response in breast cancer.

Multiple lines of evidence implicate steroid hormone and growth factor cross-talk as a modulator of endocrine response in breast cancer and that aberrations in growth factor signaling pathways are a common element in the endocrine resistant phenotype. Delineation of these relationships is thus an important diagnostic goal in cancer research, while the targeting of aberrant growth factor signaling holds the promise of improving therapeutic response rates.

Modulation of epidermal growth factor receptor in endocrine-resistant, oestrogen receptor-positive breast cancer.

There is an increasing body of evidence demonstrating that growth factor networks are highly interactive with oestrogen receptor (ER) signalling in the control of breast cancer growth. As such, tumour responses to anti- hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents. The current article examines the modulation of growth factor networks during endocrine response, and presents in vitro and clinical evidence that epidermal growth factor receptor signalling, maintained in either an ER-dependent or -independent manner, is critical to anti- hormonal-resistant breast cancer cell growth. The considerable potential of the epidermal growth factor receptor-selective tyrosine kinase inhibitor, ZD 1839 (Iressa; AstraZeneca) to efficiently treat, and perhaps even prevent, endocrine-resistant breast cancer is highlighted.

Enhanced epidermal growth factor receptor signaling in MCF7 breast cancer cells after long-term culture in the presence of the pure antiestrogen ICI 182,780 (Faslodex).

This paper describes the establishment of an antiestrogen-resistant MCF7 breast cancer cell subline (FASMCF) by continuous culture of the estrogen-responsive parental line in steroid-depleted, ICI 182,780 (Faslodex; 10(-7) M)-supplemented medium. After a 3-month period of growth suppression, cells began to proliferate in ICI 182,780 at rates similar to those of untreated wild-type cells. Immunocytochemistry showed these cells to have reduced estrogen receptor and an absence of progesterone receptor proteins. RT-PCR and transient transfection studies with estrogen response element-reporter constructs confirmed that ICI 182,780-suppressed estrogen response element-mediated signaling. FASMCF cells show increased dependence upon epidermal growth factor receptor (EgfR)/mitogen-activated protein kinase (MAPK)-mediated signaling. Thus, EgfR protein and messenger RNA, growth responses to transforming growth factor-alpha, and extracellular signal-regulated kinase 1/2 MAPK activation levels are all increased. Unlike wild-type cells, FASMCF cells are highly sensitive to growth inhibition by an EgfR-specific tyrosine-kinase inhibitor (TKI), ZD1839 (Iressa), and an inhibitor of the activation of MEK1 (MAPKK), PD098059. Short-term ( approximately 3 weeks) withdrawal of cells from antiestrogen had no effect on growth or phenotype, whereas longer withdrawal (>10 weeks) appeared to partially reverse the cellular phenotype with increasing estrogen receptor and decreasing EgfR levels. In subsequent studies FASMCF cells were maintained in TKI, where their growth was again suppressed and secondary TKI resistance failed to develop within the 3-month period in which initial ICI 182,780 resistance arose. Furthermore, wild-type cells similarly maintained in combination ICI 182,780 and TKI treatment conditions remained growth arrested (>6 months), with notable cell loss through both reduced rates of cellular proliferation and increased cell death.

The antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro.

Although many estrogen receptor-positive breast cancers initially respond to antihormones, responses are commonly incomplete with resistance ultimately emerging. Delineation of signaling mechanisms underlying these phenomena would allow development of therapies to improve antihormone response and compromise resistance. This in vitro investigation in MCF-7 breast cancer cells examines whether epidermal growth factor receptor (EGFR) signaling limits antiproliferative and proapoptotic activity of antihormones and ultimately supports development of resistance. It addresses whether the anti-EGFR agent gefitinib (ZD1839/Iressa; TKI: 1 mum) combined with the antihormones 4-hydroxytamoxifen (TAM: 0.1 mum) or fulvestrant (Faslodex; 0.1 mum) enhances growth inhibition and prevents resistance. TAM significantly suppressed MCF-7 growth over wk 2-5, reducing proliferation detected by immunocytochemistry and fluorescence-activated cell sorter cell cycle analysis. A modest apoptotic increase was observed by fluorescence-activated cell sorter and fluorescence microscopy, with incomplete bcl-2 suppression. EGFR induction occurred during TAM response, as measured by immunocytochemistry and Western blotting, with EGFR-positive, highly proliferative resistant growth subsequently emerging. Although TKI alone was ineffective on growth, TAM plus TKI cotreatment exhibited superior antigrowth activity vs. TAM, with no viable cells by wk 12. Cotreatment was more effective in inhibiting proliferation, promoting apoptosis, and eliminating bcl-2. Cotreatment blocked EGFR induction, markedly depleted ERK1/2 MAPK and protein kinase B phosphorylation, and prevented emergence of EGFR-positive resistance. Faslodex plus TKI cotreatment was also a superior antitumor strategy. Thus, increased EGFR evolves during treatment with antihormones, limiting their efficacy and promoting resistance. Gefitinib addition to antihormonal therapy could prove more effective in treating estrogen receptor-positive breast cancer and may combat development of resistance.

Immunocytochemical detection of 1,25-dihydroxyvitamin D receptors in normal human tissues.

We developed an immunocytochemical technique to visualize the receptors for 1,25-dihydroxyvitamin D [1,25-(OH)2D receptor] in cryostat sections of normal human tissues, using a rat monoclonal antibody (9A7 gamma) to the chick intestinal receptor, which has been found to react with mammalian 1,25-(OH)2D receptors. Localization of the antigen was predominantly nuclear, with little cytoplasmic immunoreactivity. Specific staining was seen in the nuclei of many normal epithelial tissues, including liver, kidney, thyroid, adrenal, gastrointestinal tract, breast, and skin. No nuclear staining was seen when tissue sections were incubated with normal rat immunoglobulin G or when the monoclonal antibody was preincubated with a receptor-enriched chick intestinal cytosol preparation. Our results demonstrate that the receptor for 1,25-(OH)2D is localized in the nucleus and widely distributed in normal human tissues.