A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.

BACKGROUND: HER2-positive (+) breast cancers, defined by HER2 overexpression and/or amplification, are often addicted to HER2 to maintain their malignant phenotype. Yet, some HER2+ tumors do not benefit from anti-HER2 therapy. We hypothesize that HER2 amplification levels and PI3K pathway activation are key determinants of response to HER2-targeted treatments without chemotherapy. PATIENTS AND METHODS: Baseline HER2+ tumors from patients treated with neoadjuvant lapatinib plus trastuzumab [with endocrine therapy for estrogen receptor (ER)+ tumors] in TBCRC006 (NCT00548184) were evaluated in a central laboratory for HER2 amplification by fluorescence in situ hybridization (FISH) (n = 56). HER2 copy number (CN) and FISH ratios, and PI3K pathway status, defined by PIK3CA mutations or PTEN levels by immunohistochemistry were available for 41 tumors. Results were correlated with pathologic complete response (pCR; no residual invasive tumor in breast). RESULTS: Thirteen of the 56 patients (23%) achieved pCR. None of the 11 patients with HER2 ratio <4 and/or CN <10 achieved pCR, whereas 13/45 patients (29%) with HER2 ratio ≥4 and/or CN ≥10 attained pCR (P = 0.0513). Of the 18 patients with tumors expressing high PTEN or wild-type (WT) PIK3CA (intact PI3K pathway), 7 (39%) achieved pCR, compared with 1/23 (4%) with PI3K pathway alterations (P = 0.0133). Seven of the 16 patients (44%) with HER2 ratio ≥4 and intact PI3K pathway achieved pCR, whereas only 1/25 (4%) patients not meeting these criteria achieved pCR (P = 0.0031). CONCLUSIONS: Our findings suggest that there is a clinical subtype in breast cancer with high HER2 amplification and intact PI3K pathway that is especially sensitive to HER2-targeted therapies without chemotherapy. A combination of HER2 FISH ratio and PI3K pathway status warrants validation to identify patients who may be treated with HER2-targeted therapy without chemotherapy.

CYP2D6 genotype is not associated with survival in breast cancer patients treated with tamoxifen: results from a population-based study.

PURPOSE: A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. METHODS: Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41, and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (AS = 0) phenotype were compared to patients with AS > 0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). RESULTS: CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p > 0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio 1.43, 95% confidence interval 1.00-2.04, p = 0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS > 0, were associated with superior RFS (each p = 0.0015). CONCLUSIONS: This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice.

Clinical and biologic features of triple-negative breast cancers in a large cohort of patients with long-term follow-up.

Studies on well characterized, large populations of estrogen receptor (ER)/progesterone receptor (PgR)/HER2-negative [triple-negative (TN)] breast cancer (BC) patients with long-term follow-up are lacking. In this study, we analyze clinical outcomes of TN BC and implications of epidermal growth factor receptor (EGFR) expression. Clinical and biologic features, time to first recurrence (TTFR), and overall survival (OS) were compared in 253 TN versus 1,036 ER positive, PgR positive, HER2-negative [estrogen-driven (ED)] BC. Compared to ED, TN tumors were larger (p = 0.02), more proliferative (high S-phase 54 vs. 17 %, p < 0.0001), more aneuploid (64 vs. 43 %, p < 0.0001) and more likely EGFR positive (≥10 fmol/mg by radioligand-binding assay, 49 vs. 7 %, p < 0.0001). Among TN, EGFR-positive BC were larger (p = 0.0018), more proliferative (p < 0.0001), and more aneuploid, (p < 0.0001) than EGFR-negative BC. Adjuvant-treated TN patients had shorter TTFR (p = 0.0003), and OS (p = 0.0017), than ED patients. However, in untreated patients, no differences in TTFR and OS were observed at 8 years median follow-up. Among TN patients, EGFR expression was not associated with worse outcome. TN tumors have a worse outcome in systemically treated patients but not in untreated patients. EGFR expression, does not predict for worse long-term survival.

Systemic mirnas as potential biomarkers for malignancy.

MiRNAs are a class of short, endogenous, single-stranded RNA molecules that play a role in the regulation of gene expression. They have been shown to modulate a number of cellular processes including cell differentiation, growth and apoptosis and as a result have been implicated in carcinogenesis. They are detectable in tumour tissue, and altered expression levels have been identified in various cancer types. Of interest, miRNAs have recently been detected and identified to be dysregulated in the circulation of patients with breast cancer. The fact that a minimally invasive test can distinguish the presence or absence of disease illustrates the immense potential these molecules hold as predictive markers. This review serves to identify those systemic miRNAs that are upregulated or downregulated in malignancy and how treatment impacts on their circulating levels. In addition, this review questions the source of these small molecules in the bloodstream and how they may possibly play a role in the future detection of cancer as either prognostic or predictive markers.

A SNP in steroid receptor coactivator-1 disrupts a GSK3ß phosphorylation site and is associated with altered tamoxifen response in bone.

The coregulator steroid receptor coactivator (SRC)-1 increases transcriptional activity of the estrogen receptor (ER) in a number of tissues including bone. Mice deficient in SRC-1 are osteopenic and display skeletal resistance to estrogen treatment. SRC-1 is also known to modulate effects of selective ER modulators like tamoxifen. We hypothesized that single nucleotide polymorphisms (SNP) in SRC-1 may impact estrogen and/or tamoxifen action. Because the only nonsynonymous SNP in SRC-1 (rs1804645; P1272S) is located in an activation domain, it was examined for effects on estrogen and tamoxifen action. SRC-1 P1272S showed a decreased ability to coactivate ER compared with wild-type SRC-1 in multiple cell lines. Paradoxically, SRC-1 P1272S had an increased protein half-life. The Pro to Ser change disrupts a putative glycogen synthase 3 (GSK3)ß phosphorylation site that was confirmed by in vitro kinase assays. Finally, knockdown of GSK3ß increased SRC-1 protein levels, mimicking the loss of phosphorylation at P1272S. These findings are similar to the GSK3ß-mediated phospho-ubiquitin clock previously described for the related coregulator SRC-3. To assess the potential clinical significance of this SNP, we examined whether there was an association between SRC-1 P1272S and selective ER modulators response in bone. SRC-1 P1272S was associated with a decrease in hip and lumbar bone mineral density in women receiving tamoxifen treatment, supporting our in vitro findings for decreased ER coactivation. In summary, we have identified a functional genetic variant of SRC-1 with decreased activity, resulting, at least in part, from the loss of a GSK3ß phosphorylation site, which was also associated with decreased bone mineral density in tamoxifen-treated women.

DNA repair signature is associated with anthracycline response in triple negative breast cancer patients.

We hypothesized that a subset of sporadic triple negative (TN) breast cancer patients whose tumors have defective DNA repair similar to BRCA1-associated tumors are more likely to exhibit up-regulation of DNA repair-related genes, anthracycline-sensitivity, and taxane-resistance. We derived a defective DNA repair gene expression signature of 334 genes by applying a previously published BRCA1-associated expression pattern to three datasets of sporadic TN breast cancers. We confirmed a subset of 69 of the most differentially expressed genes by quantitative RT-PCR, using a low density custom array (LDA). Next, we tested the association of this DNA repair microarray signature expression with pathologic response in neoadjuvant anthracycline trials of FEC (n = 50) and AC (n = 16), or taxane-based TET chemotherapy (n = 39). Finally, we collected paraffin-fixed, formalin-embedded biopsies from TN patients who had received neoadjuvant AC (n = 28), and tested the utility of the LDA to discriminate response. Correlation between RNA expression measured by the microarrays and 69-gene LDA was ascertained. This defective DNA repair microarray gene expression pattern was significantly associated with anthracycline response and taxane resistance, with the area under the ordinary receiver operating characteristic curve (AUC) of 0.61 (95% CI = 0.45-0.77), and 0.65 (95% CI = 0.46-0.85), respectively. From the FFPE samples, the 69-gene LDA could discriminate AC responders, with AUC of 0.79 (95% CI = 0.59-0.98). In conclusion, a promising defective DNA repair gene expression signature appears to differentiate TN breast cancers that are sensitive to anthracyclines and resistant to taxane-based chemotherapy, and should be tested in clinical trials with other DNA-damaging agents and PARP-1 inhibitors.

Growth factor signalling and response to endocrine therapy: the Royal Marsden Experience.

De novo resistance to endocrine therapy is a near-universal feature of oestrogen receptor (ER)- negative breast cancer. Although many ER-positive breast cancers also show no response to tamoxifen or aromatase inhibitors on objective clinical grounds the large majority show reduced proliferation indicating that some oestrogen dependence is present in almost all ER-positive breast cancer. In neoadjuvant studies HER2 positivity is associated with poor response rates to tamoxifen but not aromatase inhibitors, consistent with preclinical models. Acquired resistance to tamoxifen is associated with decreases in ER positivity but most recurrent lesions remain ER-positive. A small proportion of these show increased HER2 expression and in these patients increased phospho-p38 may contribute to the tamoxifen-resistant phenotype. There is an unfortunate paucity of clinical and biological data on acquired resistance to aromatase inhibitors.

Hormone receptor status of a contralateral breast cancer is independent of the receptor status of the first primary in patients not receiving adjuvant tamoxifen.

PURPOSE: To determine whether the hormone receptor status of the primary breast cancer (PBC) is predictive of the hormone receptor status of the subsequent contralateral breast cancer (CBC). PATIENTS AND METHODS: We identified patients in our database with known estrogen receptor (ER; n = 193) and/or progesterone receptor (PgR; n = 178) status in their PBC and in their subsequent CBC. One hundred twenty-six of these patients had received no adjuvant therapy, 34 had received adjuvant tamoxifen, and 33 had received adjuvant chemotherapy alone. The median interval between the first diagnosis of PBC and the development of the subsequent CBC was 3 years. ER and PgR assays were assessed biochemically in two central reference laboratories using identical quality-controlled ligand-binding methods. RESULTS: Among systemically untreated patients (n = 126), 88% of patients with ER-positive PBC and 75% of patients with ER-negative PBC developed an ER-positive CBC (P = .11). Among the tamoxifen-treated patients, those with an ER-positive PBC were almost equally likely to develop an ER-positive (47%) or ER-negative (53%) CBC (P = .99). PgR status was similar. In the untreated group (n = 112), 59% of patients with a PgR-positive PBC and 66% with a PgR-negative PBC developed a PgR-positive CBC (P = .48). Among tamoxifen-treated patients (n = 33), 50% of patients with a PgR-positive PBC versus 27% of patients with a PgR-negative PBC developed a PgR-positive CBC (P = .28). CONCLUSION: ER and PgR status of the primary tumor does not predict the hormone receptor status of the subsequent CBC in the absence of selective pressure of adjuvant therapy. Thus, other reasons should be considered to clarify the failure of tamoxifen to reduce the incidence of CBC in patients with a receptor-negative PBC.

Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action.

Due to their favourable tolerability profiles, endocrine therapies have long been considered the treatment of choice for hormone-sensitive metastatic breast cancer. However, the oestrogen agonist effects of the available selective oestrogen receptor modulators, such as tamoxifen, and the development of cross-resistance between endocrine therapies with similar modes of action have led to the need for new treatments that act through different mechanisms. Fulvestrant ('Faslodex') is the first of a new type of endocrine treatment--an oestrogen receptor (ER) antagonist that downregulates the ER and has no agonist effects. This article provides an overview of the current understanding of ER signalling and illustrates the unique mode of action of fulvestrant. Preclinical and clinical study data are presented in support of the novel mechanism of action of this new type of ER antagonist.

Postmenopausal women who progress on fulvestrant ('Faslodex') remain sensitive to further endocrine therapy.

PURPOSE: This retrospective evaluation of data from two randomized, multicenter trials examined whether tumor responses to further endocrine therapy were seen in postmenopausal women with advanced breast cancer who had progressed on both initial endocrine therapy, usually tamoxifen, and on the estrogen receptor (ER) antagonist fulvestrant ('Faslodex'). PATIENTS AND METHODS: A combined total of 423 patients received fulvestrant 250 mg as a monthly intramuscular injection. After progression on fulvestrant, some patients received another endocrine therapy. Responses to subsequent endocrine therapy were assessed using a questionnaire sent to the trial investigators. Best responses were classified as a complete or partial response (CR or PR), stable disease (SD) lasting > or = 24 weeks, or disease progression. RESULTS: Follow-up data were available for 54 patients who derived clinical benefit (CB, defined as CR, PR or SD) from fulvestrant and who received subsequent endocrine therapy, resulting in a PR in 4 patients, SD in 21 patients, and disease progression in 29 patients. Data were available for 51 patients who derived no CB from fulvestrant and who received further endocrine therapy, resulting in a PR in 1 patient, SD in 17 patients, and disease progression in 33 patients. Aromatase inhibitors were used as subsequent endocrine therapy in > 80% of patients. CONCLUSIONS: After progression on fulvestrant, patients may retain sensitivity to other endocrine agents. Fulvestrant provides an additional option to existing endocrine therapies for the treatment of advanced or metastatic breast cancer in postmenopausal women, and may provide the opportunity to extend the sequence of endocrine regimens before cytotoxic chemotherapy is required.

Fulvestrant (Faslodex) versus anastrozole for the second-line treatment of advanced breast cancer in subgroups of postmenopausal women with visceral and non-visceral metastases: combined results from two multicentre trials.

The efficacy of fulvestrant (Faslodex), a novel oestrogen receptor (ER) antagonist that downregulates the ER and has no known agonist effects, was compared with the aromatase inhibitor anastrozole (Arimidex) for the second-line treatment of advanced breast cancer in postmenopausal women with visceral and non-visceral metastases. Assessment was by means of a retrospective subgroup analysis of combined data from two randomised, phase III trials. Objective response (OR) rates were similar in patients treated with fulvestrant and anastrozole, respectively (21.9% versus 19.3%-patients with no visceral metastases; 15.7% versus 13.2%-all of the patients with visceral metastases; 18.8% versus 14.0%-patients with visceral metastases only). The proportion of patients with clinical benefit (CB) was also similar between treatments and between subgroups with and without visceral disease. Fulvestrant is at least as effective as anastrozole, providing a valuable treatment option for advanced breast cancer in postmenopausal women with visceral metastases who have failed on prior endocrine therapy.

Double-blind, randomized trial comparing the efficacy and tolerability of fulvestrant versus anastrozole in postmenopausal women with advanced breast cancer progressing on prior endocrine therapy: results of a North American trial.

PURPOSE: To compare the efficacy and tolerability of fulvestrant (formerly ICI 182,780) with anastrozole in the treatment of advanced breast cancer in patients whose disease progresses on prior endocrine treatment. PATIENTS AND METHODS: In this double-blind, double-dummy, parallel-group study, postmenopausal patients were randomized to receive either an intramuscular injection of fulvestrant 250 mg once monthly or a daily oral dose of anastrozole 1 mg. The primary end point was time to progression (TTP). Secondary end points included objective response (OR) rate, duration of response (DOR), and tolerability. RESULTS: Patients (n = 400) were followed for a median period of 16.8 months. Fulvestrant was as effective as anastrozole in terms of TTP (hazard ratio, 0.92; 95.14% confidence interval [CI], 0.74 to 1.14; P =.43); median TTP was 5.4 months with fulvestrant and 3.4 months with anastrozole. OR rates were 17.5% with both treatments. Clinical benefit rates (complete response + partial response + stable disease > or = 24 weeks) were 42.2% for fulvestrant and 36.1% for anastrozole (95% CI, -4.00% to 16.41%; P =.26). In responding patients, median DOR (from randomization to progression) was 19.0 months for fulvestrant and 10.8 months for anastrozole. Using all patients, DOR was significantly greater for fulvestrant compared with anastrozole; the ratio of average response durations was 1.35 (95% CI, 1.10 to 1.67; P < 0.01). Both treatments were well tolerated. CONCLUSION: Fulvestrant was at least as effective as anastrozole, with efficacy end points slightly favoring fulvestrant. Fulvestrant represents an additional treatment option for postmenopausal women with advanced breast cancer whose disease progresses on tamoxifen therapy.

Re-expression of estrogen receptor alpha in estrogen receptor alpha-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth.

Estrogen can increase insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) expression, two key components of IGF-I-mediated signaling. The result is sensitization of breast cancer cells to IGF-I and synergistic growth in the presence of estrogen and IGF-I. We hypothesized that loss of estrogen receptor alpha (ERalpha) would result in reduced IGF-mediated signaling and growth. To test this hypothesis, we examined IGF-I effects in MCF-7 breast cancer cell sublines that have been selected for loss of ERalpha (C4 and C4-12 cells are ERalpha-negative) by long-term estrogen withdrawal. C4 and C4-12 cells had reduced IGF-IR and IRS-1 mRNA and protein expression (compared with MCF-7 cells) that was not inducible by estrogen. Furthermore, C4 and C4-12 cells showed reduced IGF-I signaling and failed to show any growth response to either estrogen or IGF-I. To prove that loss of IGF and estrogen-mediated signaling and growth was a consequence of loss of ERalpha, we re-expressed ERalpha in C4-12 cells by stable transfection with HA-tagged ERalpha. Three independent C4-12 ERalpha-HA clones expressed a functional ERalpha that (a) was down-regulated by estrogen, (b) conferred estrogen-induction of cyclin D1 expression, and (c) caused estrogen-mediated increase in the number of cells in S phase. All of the effects were completely blocked by antiestrogens. Interestingly, ERalpha-HA expression in C4-12 cells did not restore estrogen induction of progesterone receptor expression. However, ERalpha-positive C4-12 cells now exhibited estrogen-induction of IGF-IR and IRS-1 levels and responded mitogenically to both estrogen and IGF-I. These data show that ERalpha is a critical requirement for IGF signaling, and to our knowledge this is the first report of functional ERalpha expression that confers estrogen-mediated growth of an ER-negative breast cancer cell line.

Forkhead homologue in rhabdomyosarcoma functions as a bifunctional nuclear receptor-interacting protein with both coactivator and corepressor functions.

In a search for novel transcriptional intermediary factors for the estrogen receptor (ER), we used the ligand-binding domain and hinge region of ER as bait in a yeast two-hybrid screen of a cDNA library derived from tamoxifen-resistant MCF-7 human breast tumors from an in vivo athymic nude mouse model. Here we report the isolation and characterization of the forkhead homologue in rhabdomyosarcoma (FKHR), a recently described member of the hepatocyte nuclear factor 3/forkhead homeotic gene family, as a nuclear hormone receptor (NR) intermediary protein. FKHR interacts with both steroid and nonsteroid NRs, although the effect of ligand on this interaction varies by receptor type. The interaction of FKHR with ER is enhanced by estrogen, whereas its interaction with thyroid hormone receptor and retinoic acid receptor is ligand-independent. In addition, FKHR differentially regulates the transactivation mediated by different NRs. Transient transfection of FKHR into mammalian cells dramatically represses transcription mediated by the ER, glucocorticoid receptor, and progesterone receptor. In contrast, FKHR stimulates rather than represses retinoic acid receptor- and thyroid hormone receptor-mediated transactivation. Most intriguingly, overexpression of FKHR dramatically inhibits the proliferation of ER-dependent MCF-7 breast cancer cells. Therefore, FKHR represents a bifunctional NR intermediary protein that can act as either a coactivator or corepressor, depending on the receptor type.

Loss of heterozygosity events impeding breast cancer metastasis contain the MTA1 gene.

Breast cancer mortality is seldom attributable to the primary tumor, but rather to the presence of systemic (metastatic) disease. Axillary lymph node dissection can identify the presence of metastatic breast cancer cells and serves as a marker for systemic disease. Previous work in our laboratory determined that rates of loss of heterozygosity (LOH) of a 1.6-Mb region of chromosome 14q 31.2 is much higher in axillary lymph node-negative primary breast tumors than in axillary lymph node-positive primary breast tumors (P. O'Connell et al., J. NATL: Cancer INST:, 91: 1391-1397, 1999.). This unusual observation suggests that, whereas the LOH of this region promotes primary breast cancer formation, some gene(s) mapping to this 1.6-Mb region is rate-limiting for breast cancer metastasis. Thus, if primary breast cancers delete this region, their ability to metastasize decreases. To identify this gene(s), we have physically mapped this area of chromosome 14q, confirmed the position of two known genes and 13 other expressed sequence tags into this 1.6-Mb region. One of these, the metastasis-associated 1 (MTA1) gene, previously identified as a metastasis-promoting gene (Y. Toh et al., J. BIOL: CHEM:, 269: 22958-22963, 1994.), mapped to the center of our 1.6-Mb target region. Thus, MTA1 represents a strong candidate for this breast cancer metastasis-promoting gene.

Elevation of breast carcinoma Nm23-H1 metastasis suppressor gene expression and reduced motility by DNA methylation inhibition.

We hypothesize that elevation of Nm23-H1 expression in micrometastatic breast cancer cells may inhibit their metastatic colonization and further invasion, and induce differentiation, thus resulting in a clinical benefit. The current study investigated the possible contribution of DNA methylation to the regulation of Nm23-H1 expression, based on the observation that two CpG islands are present in its promoter. 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor, increased the Nm23-H1 expression of 5 of 11 human breast carcinoma cell lines in vitro, including 3 of 3 metastatically competent lines. Increased Nm23-H1 expression was accompanied by a reduction in motility in vitro, with minimal effect on proliferation. Both increased Nm23-H1 expression and decreased motility were observed using low (75 nM) concentrations of 5-Aza-CdR. Array analysis of MDA-MB-231 breast carcinoma cells treated with 5-Aza-CdR confirmed the elevation of nm23-H1 mRNA, whereas relatively few other genes exhibited altered expression. Bisulfite sequencing of the two CpG islands in a panel of cell lines and in 20 infiltrating ductal carcinomas revealed that one island (-3090 bp to -3922 bp) exhibited infrequent differential methylation. The data indicate that DNA methylation inhibitors can directly or indirectly cause both elevation of Nm23-H1 expression and decreased function in one aspect of metastasis, motility.

High rates of loss of heterozygosity on chromosome 19p13 in human breast cancer.

We have recently discovered that the nuclear matrix protein SAFB is an oestrogen receptor corepressor. Since it has become clear that many steroid receptor cofactors play important roles in breast tumorigenesis, we investigated whether SAFB could also be involved in breast cancer. To address this question, the gene locus was examined for structural alterations in breast cancer tissue. Laser capture microdissection was used for isolating DNA from paired primary breast tumour and normal tissue specimens, and the loss of heterozygosity (LOH) at chromosome 19p13.2-3 was determined by use of microsatellite markers. LOH was detected at the marker D19S216, which colocalizes with the SAFB locus, in specimens from 29 (78.4%) of 37 informative patients. The peak LOH rate occurred at D19S216 near the SAFB locus, with LOH frequencies ranging from 21.6% to 47.2% at other markers. The finding of a very high LOH rate at the marker D19S216 strongly indicates the presence of a breast tumour-suppressor gene locus. While preliminary findings of mutations in SAFB suggest that this indeed may be a promising candidate, other potential candidate genes are located at this locus.

Estrogen receptor: current understanding of its activation and modulation.

Breast cancer development and progression are directly related to the effects of the female hormone estrogen. The nuclear receptor for estrogen (ER) functions as a transcription factor controlling estrogen-regulated genes. Receptor conformation on ligand binding, its interaction with various coregulators, and response elements in the promoter region of target genes all contribute to the net estrogenic effects in a cell. ER is an important diagnostic and therapeutic target in breast cancer. Various polypeptide growth factors and their membrane receptors also contribute to breast cancer development and progression. Pathways mediating cell survival, cell proliferation, and response to stress not only generate signals through various protein kinase pathways to enhance cell survival and proliferation, but these pathways also interact with ERs. Kinases in the growth factor cascade can phosphorylate and activate ER, and ER in turn activates and augments signaling through the growth factor pathways. Signaling through the growth factor pathways may contribute to hormonal resistance states by ligand-independent activation of ER. Targeting growth factor pathways, in addition to ER, is a developing strategy that hypothetically may represent optimal therapy by preventing the development of resistance to endocrine therapy.

Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo.

BACKGROUND: Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth. METHODS: Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun NH(2)-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided. RESULTS: Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P=.004) and GST (twofold; P=.004) activity and statistically significantly reduced glutathione levels (greater than twofold; P<.001) and HMS activity (10-fold; P<.001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P=.04) and phosphorylated c-Jun and JNK levels (P<.001) were statistically significantly increased in the tamoxifen-resistant tumors. CONCLUSION: Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth.