· About 75% of breast tumors are positive for the estrogen receptor (ER) or progesterone receptor (PgR) or both, and estrogen is the main stimulant in the development and growth of these tumors. Tamoxifen, an estrogen receptor antagonist has been the endocrine treatment for hormone-sensitive breast cancer for more than 20 years. However, the underlying cause of treatment failure in many breast cancer patients receiving tamoxifen is resistance to tamoxifen. (Altundag et al 2004)

· Unfortunately, up to 50% of ER+ breast cancers donot respond to endocrine therapies, displaying de novo or intrinsicresistance, while breast tumors that initially respond to antiestrogensfrequently acquire resistance to these treatments. (Gomez et al 2007)

· Despite advances in early detection and understanding of the molecular bases of breast cancer biology, approximately 30% of all patients with early-stage breast cancer have recurrent disease, which is metastatic in most cases. (Gonzalez-Angulo et al 2007) The metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. (Figueira et al 2009)

· Decreased BRCA1 confers tamoxifen resistance in breast cancer cells by altering estrogen receptor-coregulator interactions. (Wen et al 2009)

· Genetic screen identifies insulin-like growth factor binding protein 5 as a modulator of tamoxifen resistance in breast cancer. (Ahn et al 2010)

· Patients with tumors expressing Pak1 and pERalpha(ser305) in combination are a group in which tamoxifen treatment is insufficient. (Bostner et al 2010)

· Over-expression of A1B1 is associated with resistance to tamoxifen: AIB1 (amplified in breast cancer 1), also called SRC-3 and NCoA-3, is a member of the p160 nuclear receptor co-activator family and is considered an important oncogene in breast cancer. Increased AIB1 levels in human breast cancer have been correlated with poor clinical prognosis. Over-expression of AIB1 in conjunction with members of the epidermal growth factor receptor (EGF/HER) tyrosine kinase family, such as HER2, is associated with resistance to tamoxifen therapy and decreased disease-free survival. (Lahusen et al 2009)

· When expression of AIB1 and HER-2 were considered together, patients whose tumors expressed high levels of both AIB1 and HER-2 had worse outcomes with tamoxifen therapy than all other patients combined. (Osborne et al 2003)

· The detection of CK-19 mRNA+ cells throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer. (Xenidis et al 2007)

· Tamoxifen behaves as an estrogen agonist (promotes estrogen) in breast cancer cells that express high levels of AIB1 and HER2, resulting in de novo resistance.

Patients receiving adjuvant tamoxifen whose tumors express high levels of both HER2/neu (HER2) and the estrogen receptor (ER) coactivator AIB1 often develop tamoxifen resistance. (Shou et al 2004)

· The lysosomal protease Cathepsin D (Cath D) is associated with increased invasiveness and metastasis in breast cancer.

Immunofluorescence showed that estradiol located Cath D to the cell surface, while tamoxifen accumulated Cath D to dense lysosomes in perinuclear regions. Moreover, tamoxifen increased the intracellular transporter of Cath D, the mannose 6-phosphate/IGF-II receptor (M6P/IGF2R. (Dabrosin et al 2004)

· Tamoxifen and CYP2D6: Impaired tamoxifen metabolism reduces survival in familial breast cancer patients.

The clinical outcomes of breast cancer patients treated with tamoxifen may be influenced by the activity of cytochrome P450 2D6 (CYP2D6) enzyme. Among 67 patients examined, those homozygous for the CYP2D6*10 alleles revealed a significantly higher incidence of recurrence within 10 years after the operation (P = 0.0057; odds ratio, 16.63; 95% confidence interval, 1.75-158.12), compared with those homozygous for the wild-type CYP2D6*1 alleles. Patients with the CYP2D6*10/*10 genotype showed a significantly shorter recurrence-free survival period (P = 0.036; adjusted hazard ratio, 10.04; 95% confidence interval, 1.17-86.27) compared to patients with CYP2D6*1/*1 after adjustment of other prognosis factors. The present study suggests that the CYP2D6 genotype should be considered when selecting adjuvant hormonal therapy for breast cancer patients. (Kiyotani et al 2008)

· Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes. (Schroth et al 2009)

· Nonfunctional and severely impaired CYP2D6 alleles are associated with higher recurrence rates:

Recent mechanistic, pharmacologic, and clinical evidence suggests that genetic variants and drug interaction by CYP2D6 inhibitors influence the plasma concentrations of active tamoxifen metabolites and the outcomes of tamoxifen-treated patients. In particular, nonfunctional (poor metabolizer) and severely impaired (intermediate metabolizer) CYP2D6 alleles are associated with higher recurrence rates. (Brauch et al 2009)

· Polymorphisms in CYP2D6 and ABCC2 are important predictors for the prognosis of patients with breast cancer treated with tamoxifen. (Kiytani et al 2010)

· Clinical studies demonstrate that breast cancer patients treated with adjuvant tamoxifen who have decreased CYP2D6 due to genetic polymorphisms or drug interactions may have an increased risk of recurrence and reductions in disease-free survival. (Gaston & Kolesar 2008)

· Although prospective data are lacking, the balance of evidence available currently suggests that a single nucleotide polymorphism in the CYP2D6 gene, particularly the presence of 2 null alleles, predicts for reduced tamoxifen metabolism and possibly poorer outcome than expected in patients with a wild-type genotype. (Higgins et al 2009)

· Poor metabolizer status for CYP2D6 predicts worse overall survival in patients with familial breast cancer. Therefore, CYP2D6 inhibitor drugs should not be prescribed concomitantly with tamoxifen. (Newman et al, 2008)

· Pin1 overexpression is closely associated with VEGF-mediated angiogenesis and is a potential therapeutic target of excessive angiogenesis in tamoxifen-resistant breast cancer cases. (Kim et al 2009)

· Tamoxifen actually activates the AP-1 transcription factor; and increased AP-1 activity in breast cancer cells can lead to tamoxifen resistance. (Altundag et al 2004)

· The present data suggest that genetic variation in CYP3A5 may predict response to tamoxifen therapy. (Wegman et al 2007)

· Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen. (Hurtado et al 2008)

· X-box-binding protein-1, or the XPB1 gene is an alternatively spliced transcription factor that participates in a stress-signaling pathway to protect cells from damage. Over-expression of the spliced variant of the gene in estrogen receptor-positive breast cancer cells led to reduced sensitivity to Tamoxifen and Faslodex. (Gomez et al 2007)

Oral administration of tamoxifen often induces hepatic steatosis (THS, tamoxifen-induced hepatic steatosis) as a complication, which can progress to non-alcoholic steatohepatitis (NASH). The development of this complication is strongly associated with three clinical risk factors; specifically, insulin resistance, central obesity, and hypertriglyceridemia, however a genetic predisposition to THS has yet to be investigated. Our study provides the first evidence that CYP17 polymorphism participates in the development of THS, and sheds light on the genetic causes of this side effect and genetic differences between tamoxifen-treated individuals. (Ohnishi et al 2005)

· In patients treated with tamoxifen there is substantial interindividual variability in the development of resistance to tamoxifen therapy, and in the incidence of tamoxifen-induced adverse events, including deep vein thrombosis, hot flashes, and the development of endometrial cancer. (Lazarus et al 2009)

· In addition to its antiestrogenic properties, which have been related to menopause-like symptoms including hot flashes and vaginal bleeding, tamoxifen may also increase the risk for endometrial cancer and venous thromboembolism. (Lazarus et al 2009)

· Although tamoxifen is generally well tolerated, significant interindividual variability has been observed in the clinical efficacy as well as toxicity of tamoxifen. For instance, about 30% of patients acquire tamoxifen resistance and relapse. In addition, the relative risk of endometrial cancers in patients treated with tamoxifen is estimated to be 2- to 3-fold that of controls, and the risk increases with both the duration and cumulative dose of tamoxifen treatment. (Lazarus et al 2009)

· Breast cancer recurrence risk related to concurrent use of SSRI antidepressants and tamoxifen. (Lash et al 2010)

· Paroxetine (Paxil – a selective serotonin reuptake inhibitor – SSRI) use during tamoxifen treatment is associated with an increased risk of death from breast cancer. (Kelly et al 2010)

· Tamoxifen use has been associated with increased risk of thromboembolicevents in women with breast cancer and women at high riskfor the disease. Factor V Leiden is the most common inheritedclotting factor mutation and also confers increased thrombosisrisk. (Garber et al 2010)

· After five years, the incidence of gallstone formation was 37.4% in tamoxifen-treated patients compared with 2.0% in patients who did not receive tamoxifen.

The incidences of gallstones being detected in 171 tamoxifen-treated patients were 0.4%, 3.7%, 24.4%, 33.1%, and 37.4% cumulatively during the first, second, third, fourth, and fifth years, respectively. Hence adjuvant tamoxifen therapy leads to gallstone formation in postmenopausal breast cancer patients and is most apparent after 3 years of treatment. (Akin et al 2003)

· Switching from tamoxifen to exemestane (an aromatase inhibitor) in post-menopausal women significantly reverses endometrial thickening associated with continued tamoxifen. (Bertelli et al 2010)

· Adjuvant endocrine treatment with aromatase inhibitors improves disease-free survival compared with tamoxifen in postmenopausal women with estrogen receptor-positive breast cancer. This difference could be due to differences in tamoxifen metabolism because levels of endoxifen, the active tamoxifen metabolite, vary with the number of mutant alleles, including the *4 allele, of the gene encoding cytochrome P450 2D6 (CYP2D6). (Punglia et al 2008)

· These data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate. (Borley et al 2008)

· The third generation aromatase inhibitors (AIs), letrozole, anastrozole and exemestane, have demonstrated superior efficacy to tamoxifen in large, randomized clinical trials in postmenopausal women with HR+ early breast cancer. (Perez 2007)

· The results demonstrate that tamoxifen at realistic doses can suppress the growth of ER-negative breast cancer when combined with EGCG (the most active component in green tea). In addition, the dominant mechanism for tumour suppression is the concomitant decrease in tumour protein expressions of mTOR and the EGFR. (Scandlyn et al 2008)

· A combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers. (Nehra et al 2010)

· The data obtained from this study speculated that 1.5 % green tea extract has the capacity to scavenge free radical and can protect against oxidative stress induced by tamoxifen intoxication. Supplementation of green tea extract could be useful in alleviating tamoxifen-induced liver injury in rats. (El-Beshbishy HA 2005)

· Tamoxifen has estrogenic activity on liver and endometrium causing severe oxidative stress with various biochemical derangements. Coenzyme Q(10), Riboflavin and Niacin (CoRN) are well-known potent antioxidants and protective agents against many diseases including cancer. Tamoxifen on co-administration with CoRN has a favorable impact on various blood chemistry profiles. (Yuvaraj et al 2008)

· To our knowledge, this study is the first to show the interaction of green tea with the ER pathway, as well as provide mechanistic evidence that the combination of green tea and tamoxifen is more potent than either agent alone in suppressing breast cancer growth. These results may lead to future improvements in breast cancer treatment and prevention. (Sartippour et al 2006)

· These findings demonstrate synergistic growth inhibition of ERalpha positive breast cancer xenografts by combination of tamoxifen with organic selenium compounds. Organic selenium may provide added benefit when combined with tamoxifen in adjuvant therapy or prevention. (Li et al 2008)

· Altogether the present data demonstrated that EGCG (the most active component in green tea) could attenuate the tamoxifen-resistant phenotype of MCF-7Tam cells. EGCG could contrast MCF-7Tam cell growth and in vitro invasion through downregulation of EGFR and other molecules implicated in aggressive biologic behavior. The present data support the hypothesis that EGCG is an interesting molecule to be investigated in tamoxifen-resistant breast carcinoma. (Farabegoli et al 2007)

· Flaxseeds inhibited MCF-7 tumor growth in a dose-dependent manner and enhanced the inhibitory effect of tamoxifen due to the modulation of ER and growth factor signal transduction pathways. (Chen et al 2007)

· Treatment with EGCG (the most active component in green tea) inhibited telomerase expression significantly (P < 0.01) and enhanced the effect of cisplatin and tamoxifen in both 1321N1 (P < 0.01) and U87-MG (P < 0.001) cells. (Shervington et al 2009)

· The present study shows that the organic selenium compound methylseleninic acid can potentiate growth inhibition of 4-hydroxytamoxifen (10(-7) mol/L) in tamoxifen-sensitive MCF-7 and T47D breast cancer cell lines. (Shah et al 2005)

· As higher EGCG (the most active component in green tea) concentrations also killed cells resistant to tamoxifen or treated by 10(-7)M ICI 182,780, EGCG ought to be better investigated in breast carcinoma cells treated with drugs targeted to steroid receptors, as a potential complement of therapy. (Farabegoli et al 2007)

Decreased BRCA1 confers tamoxifen resistance in breast cancer cells by altering estrogen receptor-coregulator interactions.

The breast cancer susceptibility gene 1 (BRCA1) is mutated in approximately 50% of hereditary breast cancers, and its expression is decreased in 30-40% of sporadic breast cancers, suggesting a general role in breast cancer development. BRCA1 physically and functionally interacts with estrogen receptor-alpha (ERalpha) and several transcriptional regulators. We investigated the relationship between cellular BRCA1 levels and tamoxifen sensitivity. Decreasing BRCA1 expression in breast cancer cells by small interfering RNA alleviated tamoxifen-mediated growth inhibition and abolished tamoxifen suppression of several endogenous ER-targeted genes. ER-stimulated transcription and cytoplasmic signaling was increased without detectable changes in ER or ER coregulator expression. Co-immunoprecipitation studies showed that with BRCA1 knockdown, tamoxifen-bound ERalpha was inappropriately associated with coactivators, and not effectively with corepressors. Chromatin immunoprecipitation studies demonstrated that with tamoxifen, BRCA1 knockdown did not change ERalpha promoter occupancy, but resulted in increased coactivator and decreased corepressor recruitment onto the endogenous cyclin D1 promoter. Our results suggest that decreased BRCA1 levels modify ERalpha-mediated transcription and regulation of cell proliferation in part by altering ERalpha-coregulator association. In the presence of tamoxifen, decreased BRCA1 expression results in increased coactivator and decreased corepressor recruitment on ER-regulated gene promoters. Oncogene. 2009 Jan 29;28(4):575-86. Epub 2008 Nov 10. Wen J, Li R, Lu Y, Shupnik MA.

Genetic screen identifies insulin-like growth factor binding protein 5 as a modulator of tamoxifen resistance in breast cancer.

Tamoxifen resistance is one of the overarching challenges in the treatment of patients with estrogen receptor (ER)-positive breast cancer. Through a genome-wide RNA interference screen to discover genes responsible for tamoxifen resistance in vitro, we identified insulin-like growth factor binding protein 5 (IGFBP5) as a determinant of drug sensitivity. Specific knockdown of IGFBP5 by retroviral infection with short hairpin RNA-expressing cassette in MCF7 human breast cancer cells (pRS-shIGFBP5) conferred tamoxifen resistance in vitro due to concomitant loss of ERalpha expression and signaling. IGFBP5 expression was also reduced in MCF7 cells selected for tamoxifen resistance in culture (TAMR). Both tamoxifen-resistant MCF7-TAMR and MCF7-pRS-shIGFBP5 cells could be resensitized to drug by treatment with exogenous recombinant IGFBP5 (rIGFBP5) protein. Treatment with rIGFBP5 protein in mouse tumor xenografts reversed the in vivo tamoxifen resistance of MCF7-pRS-shIGFBP5 cell-derived tumors by reducing tumor cell proliferation. IGFBP5 immunohistochemical staining in a cohort of 153 breast cancer patients showed that low IGFBP5 expression was associated with shorter overall survival after tamoxifen therapy. Thus, IGFBP5 warrants investigation as an agent to reverse tamoxifen resistance. Ahn BY, Elwi AN, Lee B, Trinh DL, Klimowicz AC, Yau A, Chan JA, Magliocco A, Kim SW. Cancer Res. 2010 Apr 15;70(8):3013-9. Epub 2010 Mar 30.

Estrogen receptor-alpha phosphorylation at serine 305, nuclear p21-activated kinase 1 expression, and response to tamoxifen in postmenopausal breast cancer.

PURPOSE: In vitro, p21-activated kinase 1 (Pak1) phosphorylates the serine 305 residue of the estrogen receptor alpha (ERalpha) and influences the response of breast cancer cells to tamoxifen. We investigated the influence of Pak1 and pERalpha(ser305) on breast cancer prognosis and results of tamoxifen therapy. EXPERIMENTAL DESIGN: We examined Pak1 and pERalpha(ser305) protein by immunohistochemistry in a series of 912 tumors from node-negative breast cancer patients randomized to tamoxifen or no adjuvant endocrine treatment. RESULTS: Cytoplasmic Pak1 correlated to large tumors and ER negativity, whereas nuclear Pak1 and pERalpha(ser305) correlated to small tumors and ER positivity. Nuclear expression of Pak1 and pERalpha(ser305) predicted reduced response to tamoxifen in patients with ERalpha-positive tumors (tamoxifen versus no tamoxifen: hazard ratio (HR), 1.33; 95% confidence interval (95% CI), 0.42-4.2; P = 0.63), whereas patients lacking this combination benefitted significantly from tamoxifen (HR, 0.43; 95% CI, 0.30-0.62; P < 0.0001). Similar nonsignificant trends were detected in analyses of the proteins separately. Pak1 in the cytoplasm was an independent prognostic marker, indicating increased recurrence rate (HR, 1.79; 95% CI, 1.17-2.74; P = 0.0068) and breast cancer mortality (HR, 1.98; 95% CI, 1.14-3.46; P = 0.016) for patients randomized to no adjuvant treatment. CONCLUSION: Our results suggest that patients with tumors expressing Pak1 and pERalpha(ser305) in combination are a group in which tamoxifen treatment is insufficient. In addition, the pathway may be of interest as a drug target in breast cancer. Furthermore, the findings support previous studies showing that Pak1 has differential roles in the cytoplasm and the nucleus Bostner J, Skoog L, Fornander T, Nordenskjöld B, Stål O. Clin Cancer Res. 2010 Mar 1;16(5):1624-33. Epub 2010 Feb 23.

Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen.

Crosstalk between the oestrogen receptor (ER) and ERBB2/HER-2 pathways has long been implicated in breast cancer aetiology and drug response, yet no direct connection at a transcriptional level has been shown. Here we show that oestrogen-ER and tamoxifen-ER complexes directly repress ERBB2 transcription by means of a cis-regulatory element within the ERBB2 gene in human cell lines. We implicate the paired box 2 gene product (PAX2), in a previously unrecognized role, as a crucial mediator of ER repression of ERBB2 by the anti-cancer drug tamoxifen. We show that PAX2 and the ER co-activator AIB-1/SRC-3 compete for binding and regulation of ERBB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ERBB2 by ER-PAX2 links these two breast cancer subtypes and suggests that aggressive ERBB2-positive tumours can originate from ER-positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer Hurtado A, Holmes KA, Geistlinger TR, Hutcheson IR, Nicholson RI, Brown M, Jiang J, Howat WJ, Ali S, Carroll JS. Nature. 2008 Dec 4;456(7222):663-6. Epub 2008 Nov 12.

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells.

INTRODUCTION: Anti-oestrogens have been the mainstay of therapy in patients with oestrogen-receptor (ER) positive breast cancer and have provided significant improvements in survival. However, their benefits are limited by tumour recurrence in a significant proportion of initially drug-responsive breast cancer patients because of acquired anti-oestrogen resistance. Relapse on such therapies clinically presents as local and/or regional recurrences, frequently with distant metastases, and the prognosis for these patients is poor. The selective ER modulator, tamoxifen, classically exerts gene inhibitory effects during the drug-responsive phase in ER-positive breast cancer cells. Paradoxically, this drug is also able to induce the expression of genes, which in the appropriate cell context may contribute to an adverse cell phenotype. Here we have investigated the effects of tamoxifen and fulvestrant treatment on invasive signalling and compared this with the direct effects of oestrogen withdrawal to mimic the action of aromatase inhibitors. METHODS: The effect of oestrogen and 4-hydroxy-tamoxifen on the invasive capacity of endocrine-sensitive MCF-7 cells, in the presence or absence of functional E-cadherin, was determined by Matrigel invasion assays. Studies also monitored the impact of oestrogen withdrawal or treatment with fulvestrant on cell invasion. Western blotting using phospho-specific antibodies was performed to ascertain changes in invasive signalling in response to the two anti-oestrogens versus both oestradiol treatment and withdrawal. RESULTS: To the best of our knowledge, we report for the first time that tamoxifen can promote an invasive phenotype in ER-positive breast cancer cells under conditions of poor cell-cell contact and suggest a role for Src kinase and associated pro-invasive genes in this process. Our studies revealed that although this adverse effect is also apparent for further classes of anti-oestrogens, exemplified by the steroidal agent fulvestrant, it is absent during oestrogen withdrawal. CONCLUSIONS: These data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate. Borley AC, Hiscox S, Gee J, Smith C, Shaw V, Barrett-Lee P, Nicholson RI. Breast Cancer Res. 2008;10(6):R103. Epub 2008 Dec 4.

Association between CYP2D6 polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen.

CONTEXT: The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor-positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. The formation of active metabolites is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. OBJECTIVE: To determine whether CYP2D6 variation is associated with clinical outcomes in women receiving adjuvant tamoxifen. DESIGN, SETTING, AND PATIENTS: Retrospective analysis of German and US cohorts of patients treated with adjuvant tamoxifen for early stage breast cancer. The 1325 patients had diagnoses between 1986 and 2005 of stage I through III breast cancer and were mainly postmenopausal (95.4%). Last follow-up was in December 2008; inclusion criteria were hormone receptor positivity, no metastatic disease at diagnosis, adjuvant tamoxifen therapy, and no chemotherapy. DNA from tumor tissue or blood was genotyped for CYP2D6 variants associated with reduced (*10, *41) or absent (*3, *4, *5) enzyme activity. Women were classified as having an extensive (n=609), heterozygous extensive/intermediate (n=637), or poor (n=79) CYP2D6 metabolism. MAIN OUTCOME MEASURES: Time to recurrence, event-free survival, disease-free survival, and overall survival. RESULTS: Median follow-up was 6.3 years. At 9 years of follow-up, the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and 29.0% for poor metabolizers, and all-cause mortality rates were 16.7%, 18.0%, and 22.8%, respectively. Compared with extensive metabolizers, there was a significantly increased risk of recurrence for heterozygous extensive/intermediate metabolizers (time to recurrence adjusted hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.04-1.90) and for poor metabolizers (time to recurrence HR, 1.90; 95% CI, 1.10-3.28). Compared with extensive metabolizers, those with decreased CYP2D6 activity (heterozygous extensive/intermediate and poor metabolism) had worse event-free survival (HR, 1.33; 95% CI, 1.06-1.68) and disease-free survival (HR, 1.29; 95% CI, 1.03-1.61), but there was no significant difference in overall survival (HR, 1.15; 95% CI, 0.88-1.51). CONCLUSION: Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes Schroth W, Goetz MP, Hamann U, Fasching PA, Schmidt M, Winter S, Fritz P, Simon W, Suman VJ, Ames MM, Safgren SL, Kuffel MJ, Ulmer HU, Boländer J, Strick R, Beckmann MW, Koelbl H, Weinshilboum RM, Ingle JN, Eichelbaum M, Schwab M, Brauch H. JAMA. 2009 Oct 7;302(13):1429-36.

Genetic variants of CYP3A5, CYP2D6, SULT1A1, UGT2B15 and tamoxifen response in postmenopausal patients with breast cancer.

INTRODUCTION: Tamoxifen therapy reduces the risk of recurrence and prolongs the survival of oestrogen-receptor-positive patients with breast cancer. Even if most patients benefit from tamoxifen, many breast tumours either fail to respond or become resistant. Because tamoxifen is extensively metabolised by polymorphic enzymes, one proposed mechanism underlying the resistance is altered metabolism. In the present study we investigated the prognostic and/or predictive value of functional polymorphisms in cytochrome P450 3A5 CYP3A5 (*3), CYP2D6 (*4), sulphotransferase 1A1 (SULT1A1; *2) and UDP-glucuronosyltransferase 2B15 (UGT2B15; *2) in tamoxifen-treated patients with breast cancer. METHODS: In all, 677 tamoxifen-treated postmenopausal patients with breast cancer, of whom 238 were randomised to either 2 or 5 years of tamoxifen, were genotyped by using PCR with restriction fragment length polymorphism or PCR with denaturing high-performance liquid chromatography. RESULTS: The prognostic evaluation performed in the total population revealed a significantly better disease-free survival in patients homozygous for CYP2D6*4. For CYP3A5, SULT1A1 and UGT2B15 no prognostic significance was observed. In the randomised group we found that for CYP3A5, homozygous carriers of the *3 allele tended to have an increased risk of recurrence when treated for 2 years with tamoxifen, although this was not statistically significant (hazard ratio (HR) = 2.84, 95% confidence interval (CI) = 0.68 to 11.99, P = 0.15). In the group randomised to 5 years' tamoxifen the survival pattern shifted towards a significantly improved recurrence-free survival (RFS) among CYP3A5*3-homozygous patients (HR = 0.20, 95% CI = 0.07 to 0.55, P = 0.002). No reliable differences could be seen between treatment duration and the genotypes of CYP2D6, SULT1A1 or UGT2B15. The significantly improved RFS with prolonged tamoxifen treatment in CYP3A5*3 homozygotes was also seen in a multivariate Cox model (HR = 0.13, CI = 0.02 to 0.86, P = 0.03), whereas no differences could be seen for CYP2D6, SULT1A1 and UGT2B15. CONCLUSION: The metabolism of tamoxifen is complex and the mechanisms responsible for the resistance are unlikely to be explained by a single polymorphism; instead it is a combination of several mechanisms. However, the present data suggest that genetic variation in CYP3A5 may predict response to tamoxifen therapy. Wegman P, Elingarami S, Carstensen J, Stål O, Nordenskjöld B, Wingren S. Breast Cancer Res. 2007;9(1):R7.

Pharmacogenetics of tamoxifen: who should undergo CYP2D6 genetic testing?

Many women with hormone receptor-positive breast cancer will receive tamoxifen at some point in their treatment course. Tamoxifen is biotransformed to the potent antiestrogen endoxifen almost exclusively through the cytochrome P450 (CYP) 2D6 isoform. Although prospective data are lacking, the balance of evidence available currently suggests that a single nucleotide polymorphism in the CYP2D6 gene, particularly the presence of 2 null alleles, predicts for reduced tamoxifen metabolism and possibly poorer outcome than expected in patients with a wild-type genotype. Studies evaluating the impact of genetic polymorphisms that result in CYP2D6 with reduced or no activity on long-term outcome have been mostly retrospective and conducted on archival tissues or those obtained previously in prospective studies of tamoxifen. Until data are available from retrospective examinations of the large prospective trials already conducted, or adequately powered prospective analyses, transforming this information into guidelines for individual patients remains challenging. The authors do not currently recommend routine testing for CYP2D6 genotype for making clinical decisions regarding tamoxifen. Use of concomitant strong or intermediate inhibitors of CYP2D6 should be avoided when alternate medications are available. Ongoing research is directed toward identifying other polymorphisms that may influence the efficacy and safety of tamoxifen, other hormonal agents, and chemotherapies used to treat breast cancer. The hope is that in the future, not only tumor-associated factors but also germ-line host genetics can be used to determine whether a woman should receive treatment, and with which specific agents, to prevent breast cancer recurrence or death or avoid drug-related toxicities. Higgins MJ, Rae JM, Flockhart DA, Hayes DF, Stearns V. J Natl Compr Canc Netw. 2009 Feb;7(2):203-13.

BACKGROUND: Tamoxifen is a standard endocrine therapy for the prevention and treatment of steroid hormone receptor-positive breast cancer. CONTENT: Tamoxifen requires enzymatic activation by cytochrome P450 (CYP) enzymes for the formation of active metabolites 4-hydroxytamoxifen and endoxifen. As compared with the parent drug, both metabolites have an approximately 100-fold greater affinity for the estrogen receptor and the ability to inhibit cell proliferation. The polymorphic CYP2D6 is the key enzyme in this biotransformation, and recent mechanistic, pharmacologic, and clinical evidence suggests that genetic variants and drug interaction by CYP2D6 inhibitors influence the plasma concentrations of active tamoxifen metabolites and the outcomes of tamoxifen-treated patients. In particular, nonfunctional (poor metabolizer) and severely impaired (intermediate metabolizer) CYP2D6 alleles are associated with higher recurrence rates. SUMMARY: Accordingly, CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6) genotyping before treatment to predict metabolizer status may open new avenues for individualizing endocrine treatment, with the maximum benefit being expected for extensive metabolizers. Moreover, strong CYP2D6 inhibitors such as the selective serotonin reuptake inhibitors paroxetine and fluoxetine, which are used to treat hot flashes, should be avoided because they severely impair formation of the active metabolites. Brauch H, Mürdter TE, Eichelbaum M, Schwab M. Clin Chem. 2009 Oct;55(10):1770-82. Epub 2009 Jul 2.

Clinical relevance of circulating CK-19 mRNA-positive cells detected during the adjuvant tamoxifen treatment in patients with early breast cancer.

BACKGROUND: The purpose of this study was to evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ cells in patients with early-stage breast cancer. PATIENTS AND METHODS: CK-19 mRNA+ cells were prospectively and longitudinally detected using a specific real-time PCR assay for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration. RESULTS: Twenty-two (18.5%) patients had detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients, no CK-19 mRNA+ cells could be detected throughout the follow-up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (P = 0.00026). Persistency of CK-19 mRNA+ cells was associated with a significantly lower median disease-free interval (P = 0.0001) and overall survival (P = 0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ cells during the administration of tamoxifen was associated with an increased risk of relapse [hazard ratio (HR) = 22.318, P = 0.00006] and death (HR = 13.954, P < 0.00001). CONCLUSIONS: The detection of CK-19 mRNA+ cells throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer. Xenidis N, Markos V, Apostolaki S, Perraki M, Pallis A, Sfakiotaki G, Papadatos-Pastos D, Kalmanti L, Kafousi M, Stathopoulos E, Kakolyris S, Mavroudis D, Georgoulias V. Ann Oncol. 2007 Oct;18(10):1623-31. Epub 2007 May 21.

Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation.

We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell-cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated beta-catenin was decreased. These cells also displayed loss of association between beta-catenin and E-cadherin, increased cytoplasmic and nuclear beta-catenin and elevated transcription of beta-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced beta-catenin tyrosine phosphorylation, increased beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such treated cells, the association of beta-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of beta-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro. Hiscox S, Jiang WG, Obermeier K, Taylor K, Morgan L, Burmi R, Barrow D, Nicholson RI. Int J Cancer. 2006 Jan 15;118(2):290-301.

Factor V Leiden Mutation and Thromboembolism Risk in Women Receiving Adjuvant Tamoxifen for Breast Cancer

Background: Tamoxifen use has been associated with increased risk of thromboembolicevents (TEs) in women with breast cancer and women at high riskfor the disease. Factor V Leiden (FVL) is the most common inheritedclotting factor mutation and also confers increased thrombosisrisk. We investigated whether FVL was associated with TE riskin women with early-stage breast cancer who took adjuvant tamoxifen.

Methods: A case–control study was conducted among 34 Cancer andLeukemia Group B (CALGB) institutions. We matched each of 124women who had experienced a documented TE while taking adjuvanttamoxifen for breast cancer (but who were not necessarily ona CALGB treatment trial) to two control subjects (women who took adjuvant tamoxifen but did not experience TE) by age at diagnosis (±5 years). DNA from blood was analyzed forFVL mutations. Conditional logistic regression was used to estimateodds ratios (ORs) and to evaluate other potential factors associatedwith TE and tamoxifen use. All P values are based on two-sidedtests.

Results: FVL mutations were identified in 23 (18.5%) case and 12 (4.8%)control subjects (OR = 4.66, 95% confidence interval = 2.14to 10.14, P < .001). In the multivariable model, FVL mutationwas associated with TE (OR = 4.73, 95% confidence interval =2.10 to 10.68, P < .001). Other statistically significantfactors associated with TE risk were personal history of TEand smoking.

Conclusions: Among women taking adjuvant tamoxifen for early-stage breastcancer, those who had a TE were nearly five times more likely to carry a FVL mutation than those who did not have a TE. Postmenopausalwomen should be evaluated for the FVL mutation before prescriptionof adjuvant tamoxifen if a positive test would alter therapeuticdecision making.

Judy E. Garber, Susan Halabi, Sara M. Tolaney, Ellen Kaplan, Laura Archer, James N. Atkins, Stephen Edge, Charles L. Shapiro, Lynn Dressler, Electra M. Paskett, Gretchen Kimmick, James Orcutt, Anthony Scalzo, Eric Winer, Ellis Levine, Nasir Shahab, Nancy Berliner, for the Cancer and Leukemia Group B Journal of the National Cancer Institute Advance Access published online on June 16, 2010
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djq211

Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.

BACKGROUND: Patients receiving adjuvant tamoxifen whose tumors express high levels of both HER2/neu (HER2) and the estrogen receptor (ER) coactivator AIB1 often develop tamoxifen resistance. We used a breast cancer model system with high expression of AIB1 and HER2 to investigate the possible mechanisms underlying this resistance. METHODS: MCF-7 breast cancer cells, which express high levels of AIB1, and a tamoxifen-resistant derivative cell line engineered to overexpress HER2 (MCF-7/HER2-18) were treated with estrogen, tamoxifen, epidermal growth factor (EGF), or heregulin in the absence or presence of the EGF receptor (EGFR) tyrosine kinase inhibitor gefitinib. We analyzed phosphorylation of signaling intermediates by immunoblotting, ER transcriptional activity with reporter gene constructs and immunoblot analysis of endogenous gene products, promoter assembly by chromatin immunoprecipitation (ChIP) assay, and tumor cell growth in vitro by anchorage-independent colony formation and in vivo using xenografts in nude mice. RESULTS: MCF-7/HER2-18 tumors were completely growth inhibited by estrogen deprivation but were growth stimulated by tamoxifen. Molecular cross-talk between the ER and HER2 pathways was increased in the MCF-7/HER-2 cells compared with MCF-7 cells, with cross-phosphorylation and activation of both the ER and the EGFR/HER2 receptors, the signaling molecules AKT and ERK 1,2 mitogen-activated protein kinase (MAPK), and AIB1 itself with both estrogen and tamoxifen treatment. Tamoxifen recruited coactivator complexes (ER, AIB1, CBP, p300) to the ER-regulated pS2 gene promoter in MCF-7/HER2-18 cells and corepressor complexes (NCoR, histone deacetylase 3) in MCF-7 cells. Gefitinib pretreatment blocked receptor cross-talk, reestablished corepressor complexes with tamoxifen-bound ER on target gene promoters, eliminated tamoxifen's agonist effects, and restored its antitumor activity both in vitro and in vivo in MCF-7/HER2-18 cells. CONCLUSIONS: Tamoxifen behaves as an estrogen agonist in breast cancer cells that express high levels of AIB1 and HER2, resulting in de novo resistance. Gefitinib's ability to eliminate this cross-talk and to restore tamoxifen's antitumor effects should be tested in the clinic Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H, Schiff R. J Natl Cancer Inst. 2004 Jun 16;96(12):926-35.

Enhancement of vascular endothelial growth factor-mediated angiogenesis in tamoxifen-resistant breast cancer cells: role of Pin1 overexpression.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. Here, we found that TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) produced higher levels of vascular endothelial growth factor (VEGF) than control MCF-7 cells. Molecular analyses using reporter genes and Western blots supported the involvement of c-Jun/activator protein-1 and hypoxia-inducible factor 1alpha in enhanced VEGF transcription in TAMR-MCF-7 cells. Pin1, a peptidyl prolyl isomerase, was consistently overexpressed in TAMR-MCF-7 cells, and c-Jun/activator protein-1-dependent VEGF transcription in TAMR-MCF-7 cells was almost completely inhibited by Pin1 siRNA and by the Pin1 inhibitor juglone. Chick chorioallantoic membrane assays confirmed that the increased angiogenic intensity of TAMR-MCF-7 cells was significantly suppressed by Pin1 inhibition. These results show that Pin1 overexpression is closely associated with VEGF-mediated angiogenesis and suggest that Pin1 is a potential therapeutic target of excessive angiogenesis in TAM-resistant breast cancer cases.Kim MR, Choi HS, Yang JW, Park BC, Kim JA, Kang KW. Mol Cancer Ther. 2009 Aug;8(8):2163-71. Epub 2009 Aug 11.

Potential Role of UGT Pharmacogenetics in Cancer Treatment and Prevention: Focus on Tamoxifen

Tamoxifen (TAM) is a selective estrogen receptor modulator that is widely used in the prevention and treatment of estrogen receptor-positive (ER⁺) breast cancer. Its use has significantly contributed to a decline in breast cancer mortality, since breast cancer patients treated with TAM for 5 years exhibit a 30–50% reduction in both the rate of disease recurrence after 10 years of patient follow-up and occurrence of contralateral breast cancer. However, in patients treated with TAM there is substantial interindividual variability in the development of resistance to TAM therapy, and in the incidence of TAM-induced adverse events, including deep vein thrombosis, hot flashes, and the development of endometrial cancer. This article will focus on the UDP glucuronosyltransferases, a family of metabolizing enzymes that are responsible for the deactivation and clearance of TAM and TAM metabolites, and how interindividual differences in these enzymes may play a role in patient response to TAM.

Tamoxifen (TAM) (1-[4-(2-dimethylamino-ethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) is a nonsteroidal antiestrogen that has been commonly used for the treatment and prevention of estrogen-dependent breast cancer.^1–4 First approved in 1977 by the FDA (U.S.) for the treatment of women with metastatic breast cancer, TAM is currently an established hormonal treatment for all stages of estrogen receptor (ER)-positive breast cancer. Adjuvant TAM treatment increases recurrence-free survival and overall survival in breast cancer patients with hormone receptor-positive tumors, irrespective of these patients’ nodal status, menopausal status, or age.^3,4 TAM is also widely used as a chemopreventive agent in women at risk for developing breast cancer.^1,2

In addition to its antiestrogenic properties, which have been related to menopause-like symptoms including hot flashes and vaginal bleeding,⁴ TAM also has seemingly tissue-dependent partial estrogen-agonistic effects that may be linked to reduced risk for is-chemic heart disease and osteoporosis,^5,6 but may also increase the risk for endometrial cancer^7,8 and venous thromboembolism.⁹ Although TAM is generally well tolerated, significant interindividual variability has been observed in the clinical efficacy as well as toxicity of TAM. For instance, about 30% of patients acquire TAM resistance and relapse.¹⁰ In addition, the relative risk of endometrial cancers in patients treated with TAM is estimated to be 2- to 3-fold that of controls, and the risk increases with both the duration and cumulative dose of TAM treatment. Philip Lazarus,^ab Andrea S. Blevins-Primeau,^a Yan Zheng,^ac and Dongxiao Sun^aAnn N Y Acad Sci. 2009 February; 1155: 99–111.

Possible interaction between activator protein-1 and proto-oncogene B-cell lymphoma gene 6 in breast cancer patients resistant to tamoxifen.

About 75% of breast tumors are positive for the estrogen receptor (ER) or progesterone receptor (PgR) or both, and estrogen is the main stimulant in the development and growth of these tumors. Tamoxifen, an estrogen receptor antagonist has been endocrine treatment for hormone-sensitive breast cancer for more than 20 years. However, the underlying cause of treatment failure in many breast cancer patients receiving tamoxifen is resistance to tamoxifen. The mechanisms of tamoxifen and the molecular events responsible for resistance to tamoxifen are not fully understood. Two ER subtypes, ERalpha and ERbeta, activate the Activator Protein-1 (AP-1) response elements, and through interactions between ERs and the AP-1 transcription factors c-fos and c-jun, these transcription factors regulate the genes involved in many cellular processes, including proliferation, differentiation, cell motility, and apoptosis. Thus, the interaction between ERs and AP-1 could be important clinically and could have bearing on the response to tamoxifen. Tamoxifen acts as an agonist on genes under the control of an AP-1 response elements when ERalpha or ERbeta is expressed. AP-1 blockade suppresses mitogenic signals from multiple different peptide growth factors as well as estrogen, and inhibits the growth of MCF-7 breast cancer cells both in vitro and in vivo. Tamoxifen actually activate the AP-1 transcription factor. Increased AP-1 activity in breast cancer cells can lead to tamoxifen resistance. The proto-oncogene B-cell lymphoma gene 6 (BCL-6) has been characterized as a regulator of B-lymphocyte growth and development. BCL-6 is also expressed in the mammary epithelium in nonpregnant animals and during early pregnancy and is expressed in 68% of histologically high-grade ductal breast carcinomas, which are clinically the most aggressive. BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We hypothesize that increased BCL-6 in breast cancer cells might block tamoxifen resistance by repressing AP-1, eventually resulting in apoptosis. We also suggest that BCL-6 expression must be analyzed in ER-positive breast cancer patients and the results must be correlated with predictive and prognostic factors and survival. Altundag K, Altundag O, Gunduz M, Arun B. Med Hypotheses. 2004;63(5):823-6.

Tamoxifen and gallstone formation in postmenopausal breast cancer patients: retrospective cohort study.

Tamoxifen is being used successfully in breast cancer patients as adjuvant hormonal therapy. The aim of this retrospective cohort study is to evaluate the impact of tamoxifen on gallstone formation in postmenopausal breast cancer patients. A total of 3165 patients who were treated for invasive breast cancer between 1990 and 1997 were reviewed. The data were collected from four university hospitals in a population-based registry. Among these patients, 2462 were excluded from the study owing to improper follow-up and other reasons. Premenopausal patients were also excluded. Of the 703 patients included in the study, 457 had received adjuvant therapy including tamoxifen, and the other 246 had not. Gallstone formation was assessed by annual abdominal ultrasonography. The mean follow-up period was 4.6 years (range 1-7 years). There were no significant differences between the groups of breast cancer patients treated with or without tamoxifen regarding the age of the patients at the time of breast cancer diagnosis, the age at menopause, the duration between the onset of menopause and the time the breast cancer was diagnosed, the presence of diabetes, and the body mass index. At the end of 5 years the incidence of gallstone formation in tamoxifen-treated patients was 37.4%, whereas it was 2.0% in patients who did not receive tamoxifen ( p < 0.0001). The incidences of gallstones being detected in 171 tamoxifen-treated patients were 0.4%, 3.7%, 24.4%, 33.1%, and 37.4% cumulatively during the first, second, third, fourth, and fifth years, respectively. Hence adjuvant tamoxifen therapy leads to gallstone formation in postmenopausal breast cancer patients and is most apparent after 3 years of treatment. Akin ML, Uluutku H, Erenoglu C, Karadag A, Gulluoglu BM, Sakar B, Celenk T. World J Surg. 2003 Apr;27(4):395-9.

Decreased secretion of Cathepsin D in breast cancer in vivo by tamoxifen: mediated by the mannose-6-phosphate/IGF-II receptor?

The lysosomal protease Cathepsin D (Cath D) is associated with increased invasiveness and metastasis in breast cancer. Both estrogen and tamoxifen have been reported to increase Cath D, which seems to contradict the efficacy of tamoxifen as an adjuvant for estrogen dependent breast cancer. Cath D is bioactive in the extracellular space but very little is known about hormonal regulation of secreted Cath D in vivo. In this study we used microdialysis to sample the extracellular fluid in estrogen receptor positive MCF-7 tumors in nude mice. We show that tamoxifen in combination with estradiol decreased secreted Cath D compared with estradiol treatment only in solid tumors in situ. Cell culture of MCF-7 cells revealed that estradiol and tamoxifen increased intracellular proteolytic activity of Cath D in a similar fashion whereas secretion of Cath D was increased by estradiol and inhibited by tamoxifen. Immunofluorescence showed that estradiol located Cath D to the cell surface, while tamoxifen accumulated Cath D to dense lysosomes in perinuclear regions. Moreover, tamoxifen increased the intracellular transporter of Cath D, the mannose 6-phosphate/IGF-II receptor (M6P/IGF2R). In contrast, estradiol decreased the levels of this receptor. Thus, secretion of Cath D is hormone dependent and may be mediated by altered expression of the M6P/IGF2R. Our results highlight the importance of measurements of proteins in all compartments where they are biological active and show that microdialysis is a viable technique for sampling of Cath D in vivo. Dabrosin C, Johansson AC, Ollinger K. Breast Cancer Res Treat. 2004 Jun;85(3):229-38.

Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential.

BACKGROUND: The metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. The role of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in the metastatic process has previously been established. However, in all published studies only a limited number of MMPs/MMP inhibitors was analyzed in a limited number of cell lines. Here, we propose a more comprehensive approach by analyzing the expression levels of several MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors (TIMP-1, TIMP-2 and RECK) in different models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues). METHODS: We analyzed the expression levels of MMP-2, MMP-9 and MMP-14 and their inhibitors (TIMP-1, TIMP-2 and RECK) by quantitative RT-PCR (qRT-PCR) in five human breast cancer cell lines presenting increased invasiveness and metastatic potential, 72 primary breast tumors and 30 adjacent normal tissues. Moreover, the role of cell-extracellular matrix elements interactions in the regulation of expression and activity of MMPs and their inhibitors was analyzed by culturing these cell lines on plastic or on artificial ECM (Matrigel). RESULTS: The results demonstrated that MMPs mRNA expression levels displayed a positive and statistically significant correlation with the transcriptional expression levels of their inhibitors both in the cell line models and in the tumor tissue samples. Furthermore, the expression of all MMP inhibitors was modulated by cell-Matrigel contact only in highly invasive and metastatic cell lines. The enzyme/inhibitor balance at the transcriptional level significantly favors the enzyme which is more evident in tumor than in adjacent non-tumor tissue samples. CONCLUSION: Our results suggest that the expression of MMPs and their inhibitors, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, the multi-factorial analysis of these molecules could provide new and independent prognostic information contributing to the determination of more adequate therapy strategies for each patient. Figueira RC, Gomes LR, Neto JS, Silva FC, Silva ID, Sogayar MC. BMC Cancer. 2009 Jan 14;9:20.

Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer.

BACKGROUND: AIB1 (SRC-3) is an estrogen receptor (ER) coactivator that, when overexpressed in cultured cells, can reduce the antagonist activity of tamoxifen-bound ERs. Signaling through the HER-2 receptor pathway activates AIB1 by phosphorylation. To determine whether high AIB1 expression alone or together with HER-2 reduces the effectiveness of tamoxifen in breast cancer patients, we quantified expression of AIB1 and HER-2 in tumors from breast cancer patients with long-term clinical follow-up who received either no adjuvant therapy or adjuvant tamoxifen therapy after breast cancer surgery. METHODS: AIB1 and HER-2 protein levels in tumors from 316 breast cancer patients were determined using western blot analysis. Molecular variables (e.g., expression of AIB1, ER, progesterone receptor, p53, Bcl-2), tumor characteristics, and patient outcome were assessed using Spearman rank correlation. Disease-free survival (DFS) curves were derived from Kaplan-Meier estimates, and the curves were compared by log-rank tests. The effect of AIB1 on DFS adjusted for other prognostic factors was assessed by multivariable analysis using the Cox proportional hazards model. All statistical tests were two-sided. RESULTS: High AIB1 expression in patients not receiving adjuvant tamoxifen therapy was associated with better prognosis and longer DFS (P =.018, log-rank test). In contrast, for patients who did receive tamoxifen therapy, high AIB1 expression was associated with worse DFS (P =.049, log-rank test), which is indicative of tamoxifen resistance. The test for interaction between AIB1 expression and tamoxifen therapy was statistically significant (P =.004). When expression of AIB1 and HER-2 were considered together, patients whose tumors expressed high levels of both AIB1 and HER-2 had worse outcomes with tamoxifen therapy than all other patients combined (P =.002, log-rank test). CONCLUSIONS: The antitumor activity of tamoxifen in patients with breast cancer may be determined, in part, by tumor levels of AIB1 and HER-2. Thus, AIB1 may be an important diagnostic and therapeutic target. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SA, Wong J, Allred DC, Clark GM, Schiff R. J Natl Cancer Inst. 2003 Mar 5;95(5):353-61.

Human X-box binding protein-1 confers both estrogen independence and antiestrogen resistance in breast cancer cell lines.

Human X-box binding protein-1 (XBP1) is an alternatively spliced transcription factor that participates in the unfolded protein response (UPR), a stress-signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen. We have previously demonstrated that XBP1 expression is increased in antiestrogen-resistant breast cancer cell lines and is coexpressed with estrogen receptor alpha (ER) in breast tumors. The purpose of this study is to investigate the role of XBP1 and the UPR in estrogen and antiestrogen responsiveness in breast cancer. Overexpression of spliced XBP1 [XBP1(S)] in ER-positive breast cancer cells leads to estrogen-independent growth and reduced sensitivity to growth inhibition induced by the antiestrogens Tamoxifen and Faslodex in a manner independent of functional p53. Data from gene expression microarray analyses imply that XBP1(S) acts through regulation of the expression of ER, the antiapoptotic gene BCL2, and several other genes associated with control of the cell cycle and apoptosis. Testing this hypothesis, we show that overexpression of XBP1(S) prevents cell cycle arrest and antiestrogen-induced cell death through the mitochondrial apoptotic pathway. XBP1 and/or the UPR may be a useful molecular target for the development of novel predictive and therapeutic strategies in breast cancer. Gomez BP, Riggins RB, Shajahan AN, Klimach U, Wang A, Crawford AC, Zhu Y, Zwart A, Wang M, Clarke R. FASEB J. 2007 Dec;21(14):4013-27. Epub 2007 Jul 27.

CYP17 polymorphism and tamoxifen-induced hepatic steatosis.

Hepatic steatosis is a frequent complication, which sometimes develops nonalcoholic steatohepatitis (NASH), in breast cancer patients treated with tamoxifen, a potent antagonist of estrogen. Recently we reported the impairment of fatty acid beta-oxidation and the enhancing fatty infiltration to hepatocytes in aromatase deficiency (ArKO) mice as the estrogen deficiency models. This experimental observation let us speculate strong link between estrogen and hepatic steatosis. In this study, we investigated whether a polymorphism in the cytochrome P450c17alpha gene (CYP17), which is associated with circulating estrogen levels, influences the development of tamoxifen-induced hepatic steatosis. This consecutive study included 180 breast cancer patients undergoing tamoxifen treatment. Genomic DNA extracted from the peripheral blood of each patient was analyzed by restriction fragment length polymorphism (defined as the A1 and A2 alleles). The extent of hepatic steatosis was assessed by computed tomography (CT) as the liver/spleen (L/S) ratio. While receiving adjuvant tamoxifen, 57 of 180 patients developed hepatic steatosis (L/S ratio <0.9) without obvious changes in body mass index (BMI). We observed a significant association between the A2/A2 genotype and the development of hepatic steatosis compared with the A1/A1 genotype [odds ratio (OR), 3.60; 95% confidence interval (C.I.)=1.42-9.10]. The A1/A2 genotype was at an intermediately increased risk of hepatic steatosis (OR, 2.24; 95% C.I.=0.99-5.08). The presence of the A2 allele possibly increased the progression of hepatic steatosis with a gene dosage effect (P=0.06). Our results suggest that functional polymorphism in CYP17 may be involved in determining susceptibility of tamoxifen-induced hepatic steatosis. Ohnishi T, Ogawa Y, Saibara T, Nishioka A, Kariya S, Fukumoto M, Onishi S, Yoshida S. Hepatol Res. 2005 Oct;33(2):178-80.

Significant effect of polymorphisms in CYP2D6 and ABCC2 on clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients.

PURPOSE: The clinical efficacy of tamoxifen is suspected to be influenced by the activity of drug-metabolizing enzymes and transporters involved in the formation, metabolism, and elimination of its active forms. We investigated relationships of polymorphisms in transporter genes and CYP2D6 to clinical outcome of patients receiving tamoxifen. PATIENTS AND METHODS: We studied 282 patients with hormone receptor-positive, invasive breast cancer receiving tamoxifen monotherapy, including 67 patients who have been previously reported. We investigated the effects of allelic variants of CYP2D6 and haplotype-tagging single nucleotide polymorphisms (tag-SNPs) of ABCB1, ABCC2, and ABCG2 on recurrence-free survival using the Kaplan-Meier method and Cox regression analysis. Plasma concentrations of tamoxifen metabolites were measured in 98 patients receiving tamoxifen 20 mg/d. RESULTS: CYP2D6 variants were significantly associated with shorter recurrence-free survival (P = .000036; hazard ratio [HR] = 9.52; 95% CI, 2.79 to 32.45 in patients with two variant alleles v patients without variant alleles). Among 51 tag-SNPs in transporter genes, a significant association was found at rs3740065 in ABCC2 (P = .00017; HR = 10.64; 95% CI, 1.44 to 78.88 in patients with AA v GG genotypes). The number of risk alleles of CYP2D6 and ABCC2 showed cumulative effects on recurrence-free survival (P = .000000055). Patients carrying four risk alleles had 45.25-fold higher risk compared with patients with <or= one risk allele. CYP2D6 variants were associated with lower plasma levels of endoxifen and 4-hydroxytamoxifen (P = .0000043 and .00052), whereas no significant difference was found among ABCC2 genotype groups. CONCLUSION: Our results suggest that polymorphisms in CYP2D6 and ABCC2 are important predictors for the prognosis of patients with breast cancer treated with tamoxifenKiyotani K, Mushiroda T, Imamura CK, Hosono N, Tsunoda T, Kubo M, Tanigawara Y, Flockhart DA, Desta Z, Skaar TC, Aki F, Hirata K, Takatsuka Y, Okazaki M, Ohsumi S, Yamakawa T, Sasa M, Nakamura Y, Zembutsu H. J Clin Oncol. 2010 Mar 10;28(8):1287-93. Epub 2010 Feb 1.

Impaired tamoxifen metabolism reduces survival in familial breast cancer patients.

PURPOSE: Tamoxifen has been the mainstay adjuvant hormonal treatment for breast cancer for many years. Conversion of tamoxifen to its active metabolite, endoxifen, is reduced by low activity of the cytochrome P450 enzyme, CYP2D6. We examined the effect of reduced CYP2D6 activity on the response to tamoxifen in patients with familial early-onset breast cancer. EXPERIMENTAL DESIGN: We conducted a case note review and genotyping for the CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*41 alleles in 115 patients (47 BRCA1, 68 BRCA2) with familial breast cancer who had been treated with 20-mg tamoxifen following surgery. RESULTS: Eight (7%) individuals had genotypes consistent with poor metabolizer status, and 4 (3.5%) individuals took CYP2D6 inhibitor drugs concomitant with their tamoxifen and were also considered poor metabolizer. Time to tumor recurrence, disease-free survival, and overall survival were reduced in the patient group with poor metabolizer CYP2D6 activity. However, a significant effect was confined to patients with BRCA2 mutations with a worse overall survival (median survival, 7 versus 28 years; P = 0.008; adjusted hazard ratio, 9.7). CONCLUSIONS: Poor metabolizer status for CYP2D6 predicts worse overall survival in patients with familial breast cancer. Therefore, CYP2D6 inhibitor drugs should not be prescribed concomitantly with tamoxifen. Prospective studies should be undertaken to establish the effect of CYP2D6 status on outcome in familial breast cancer patients treated with tamoxifen. Newman WG, Hadfield KD, Latif A, Roberts SA, Shenton A, McHague C, Lalloo F, Howell S, Evans DG. Clin Cancer Res. 2008 Sep 15;14(18):5913-8.

Pharmacogenomic variation of CYP2D6 and the choice of optimal adjuvant endocrine therapy for postmenopausal breast cancer: a modeling analysis.

BACKGROUND: Adjuvant endocrine treatment with aromatase inhibitors improves disease-free survival compared with tamoxifen in postmenopausal women with estrogen receptor-positive breast cancer. This difference could be due to differences in tamoxifen metabolism because levels of endoxifen, the active tamoxifen metabolite, vary with the number of mutant alleles, including the *4 allele, of the gene encoding cytochrome P450 2D6 (CYP2D6). METHODS: We created a Markov model to determine whether tamoxifen or aromatase inhibitor monotherapy maximized 5-year disease-free survival for patients with the wild-type CYP2D6 genotype (wt/wt). Annual risks of recurrence with aromatase inhibitors and tamoxifen in breast cancer patients who were not selected by CYP2D6 genotype were derived from the Breast International Group 1-98 trial. Genotype frequencies and the hazard ratio for cancer recurrence on tamoxifen among patients with the *4/*4 genotype relative to the wt/wt or wt/*4 genotypes (HR(*4/*4) = 1.86) were based on data from an analysis of the North Central Cancer Treatment Group trial of adjuvant tamoxifen. We explored the impact of CYP2D6(*4) heterozygosity on disease-free survival for wt/wt patients by studying a range of effect (ie, recurrence on tamoxifen) estimates, from no effect of the single mutation (Eff(wt/*4) = 0, recurrence rate in wt/*4 patients same as that in wt/wt patients) to complete effect (Eff(wt/*4) = 1 recurrence rate in wt/*4 patients same as that in *4/*4 patients). RESULTS: With HR(*4/*4) = 1.86 and Eff(wt/*4) = 0.5, the 5-year disease-free survival of tamoxifen-treated patients with no mutations (wt/wt) was 83.9%, that is, essentially the same as that (84.0%) for genotypically unselected patients who were treated with aromatase inhibitors. With greater HR(*4/*4) estimates, disease-free survival with tamoxifen exceed that with aromatase inhibitors in wt/wt patients, even at lower assumed Eff(wt/*4) ratios. CONCLUSIONS: Modeling suggests that among patients who are wild type for CYP2D6, 5-year disease-free survival outcomes are similar to or perhaps even superior with tamoxifen than with aromatase inhibitors. Endocrine therapy tailored to CYP2D6 genotype could be considered for women who are newly diagnosed with breast cancer, particularly those who have with concerns about either the relative toxicity or the increased cost of aromatase inhibitors. Punglia RS, Burstein HJ, Winer EP, Weeks JC. J Natl Cancer Inst. 2008 May 7;100(9):642-8. Epub 2008 Apr 29.

Clinical significance of CYP2D6 polymorphisms and tamoxifen in women with breast cancer.

Tamoxifen has been used as adjuvant hormonal therapy for estrogen receptor positive breast cancer for over 30 years and is also widely used for the treatment of metastatic breast cancer. Tamoxifen is metabolized to its more active form by cytochrome P450 2D6 (CYP2D6); decreases in CYP2D6 activity, either by inactivating polymorphisms or drug interactions, can reduce concentrations of tamoxifen's active metabolites. Clinical studies demonstrate that breast cancer patients treated with adjuvant tamoxifen who have decreased CYP2D6 due to genetic polymorphisms or drug interactions may have an increased risk of recurrence and reductions in disease-free survival. Pharmacogenetic testing is currently available to predict CYP2D6 phenotypes and individualize tamoxifen therapy. Gaston C, Kolesar J. Clin Adv Hematol Oncol. 2008 Nov;6(11):825-33.

Selective serotonin reuptake inhibitors and breast cancer mortality in women receiving tamoxifen: a population based cohort study.

OBJECTIVE: To characterise whether some selective serotonin reuptake inhibitor (SSRI) antidepressants reduce tamoxifen's effectiveness by inhibiting its bioactivation by cytochrome P450 2D6 (CYP2D6). DESIGN: Population based cohort study. PARTICIPANTS: Women living in Ontario aged 66 years or older treated with tamoxifen for breast cancer between 1993 and 2005 who had overlapping treatment with a single SSRI. MAIN OUTCOME MEASURES: Risk of death from breast cancer after completion of tamoxifen treatment, as a function of the proportion of time on tamoxifen during which each SSRI had been co-prescribed. RESULTS: Of 2430 women treated with tamoxifen and a single SSRI, 374 (15.4%) died of breast cancer during follow-up (mean follow-up 2.38 years, SD 2.59). After adjustment for age, duration of tamoxifen treatment, and other potential confounders, absolute increases of 25%, 50%, and 75% in the proportion of time on tamoxifen with overlapping use of paroxetine (an irreversible inhibitor of CYP2D6) were associated with 24%, 54%, and 91% increases in the risk of death from breast cancer, respectively (P<0.05 for each comparison). By contrast, no such risk was seen with other antidepressants. We estimate that use of paroxetine for 41% of tamoxifen treatment (the median overlap in our sample) would result in one additional breast cancer death within five years of cessation of tamoxifen for every 19.7 (95% confidence interval 12.5 to 46.3) patients so treated; the risk with more extensive overlap would be greater. CONCLUSION: Paroxetine use during tamoxifen treatment is associated with an increased risk of death from breast cancer, supporting the hypothesis that paroxetine can reduce or abolish the benefit of tamoxifen in women with breast cancer.

Long-term endometrial effects in postmenopausal women with early breast cancer participating in the Intergroup Exemestane Study (IES)--a randomised controlled trial of exemestane versus continued tamoxifen after 2-3 years tamoxifen.

BACKGROUND: The antiestrogen tamoxifen may have partial estrogen-like effects on the postmenopausal uterus. Aromatase inhibitors (AIs) are increasingly used after initial tamoxifen in the adjuvant treatment of postmenopausal early breast cancer due to their mechanism of action: a potential benefit being a reduction of uterine abnormalities caused by tamoxifen. PATIENTS AND METHODS: Sonographic uterine effects of the steroidal AI exemestane were studied in 219 women participating in the Intergroup Exemestane Study: a large trial in postmenopausal women with estrogen receptor-positive (or unknown) early breast cancer, disease free after 2-3 years of tamoxifen, randomly assigned to continue tamoxifen or switch to exemestane to complete 5 years adjuvant treatment. The primary end point was the proportion of patients with abnormal (> or =5 mm) endometrial thickness (ET) on transvaginal ultrasound 24 months after randomisation. RESULTS: The analysis included 183 patients. Two years after randomisation, the proportion of patients with abnormal ET was significantly lower in the exemestane compared with tamoxifen arm (36% versus 62%, respectively; P = 0.004). This difference emerged within 6 months of switching treatment (43.5% versus 65.2%, respectively; P = 0.01) and disappeared within 12 months of treatment completion (30.8% versus 34.7%, respectively; P = 0.67). CONCLUSION: Switching from tamoxifen to exemestane significantly reverses endometrial thickening associated with continued tamoxifen. Bertelli G, Hall E, Ireland E, Snowdon CF, Jassem J, Drosik K, Karnicka-Mlodkowska H, Coombes RC, Bliss JM. Ann Oncol. 2010 Mar;21(3):498-505. Epub 2009 Aug 28.

Breast cancer recurrence risk related to concurrent use of SSRI antidepressants and tamoxifen.

BACKGROUND: Up to one-quarter of breast cancer patients suffer clinically significant depression in the year after diagnosis, which may respond to intervention. About half may be prescribed a psychotropic medication, such as a selective serotonin reuptake inhibitor (SSRI), while completing breast cancer therapy. Cytochrome P-450 2D6 (CYP2D6) metabolizes SSRIs and also metabolizes tamoxifen to more active forms. Therefore, concurrent use of SSRIs may reduce tamoxifen's effectiveness at preventing breast cancer recurrence. The SSRI citalopram has limited potency to inhibit CYP2D6 activity, so has been recommended for breast cancer patients taking tamoxifen. This study provides epidemiologic evidence to support this recommendation. MATERIAL AND METHODS: We conducted a case-control study of breast cancer recurrence nested in the population of female residents of Denmark who were diagnosed with non-metastatic estrogen-receptor positive breast cancers between 1994 and 2001 and who took tamoxifen for at least one year. We ascertained complete prescription histories by linking cases' and controls' civil registration numbers to the Danish national prescription registry. We estimated the association between SSRI use while taking tamoxifen and risk of recurrent breast cancer. RESULTS: About the same proportion of recurrent cases (37 of 366) and matched controls (35 of 366) received at least one prescription for citalopram or its s-stereoisomer while taking tamoxifen (adjusted odds ratio = 1.1, 95% confidence interval = 0.7, 1.7). Breast cancer patients taking other SSRIs were also at no increased risk of recurrence (adjusted odds ratio = 0.9, 95% confidence interval = 0.5, 1.8). DISCUSSION: Breast cancer patients with indications for an SSRI may be prescribed citalopram - and possibly other SSRI - without adversely affecting the outcome of adjuvant therapy with tamoxifen. Lash TL, Cronin-Fenton D, Ahern TP, Rosenberg CL, Lunetta KL, Silliman RA, Hamilton-Dutoit S, Garne JP, Ewertz M, Sørensen HT, Pedersen L. Acta Oncol. 2010 Apr;49(3):305-12.

BCL2 and CASP8 regulation by NF-kappaB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells.

Resistance to endocrine therapies remains a major problem in the management of estrogen receptor-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC(50)=500 nM in tamoxifen-resistant cells), synergistically restores sensitivity to 4-hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF-7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF-kappaB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase-dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)-specific inhibitor that blocks enzyme-substrate binding. Thus, increased activation of NF-kappaB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers. Nehra R, Riggins RB, Shajahan AN, Zwart A, Crawford AC, Clarke R. FASEB J. 2010 Jun;24(6):2040-55. Epub 2010 Feb 12.

Flaxseed alone or in combination with tamoxifen inhibits MCF-7 breast tumor growth in ovariectomized athymic mice with high circulating levels of estrogen.

Flaxseed (FS) is rich in mammalian lignan precursors and alpha-linolenic acid, which have been suggested as having anticancer effects. Previous studies have shown that 10% FS inhibits the growth of human estrogen-dependent breast cancer (MCF-7) in athymic mice, and it enhances the inhibitory effect of tamoxifen (TAM). This study determined whether the effect of FS, alone or in combination with TAM, is dose dependent, and it explored the potential mechanism of action. Ovariectomized athymic mice with estradiol (E2) supplementation (1.7 mg/pellet, 60-day release) and established MCF-7 tumors were treated with basal diet control (0FS), 5% FS (5FS), 10% FS (10FS), and TAM (TAM/ 0FS; 5 mg/pellet, 60-day release), alone or in combination (TAM/ 5FS and TAM/10FS) for 8 weeks. Compared with control, 5FS and 10FS significantly inhibited tumor growth by 26% and 38%, respectively. TAM/0FS had an effect similar to the 10FS. TAM/ 5FS and TAM/10FS, respectively, induced significant 48% and 43% reductions in tumor size compared with 0FS, and 18% and 10% reductions compared with TAM/0FS. The relative uterine weight was significantly lower in all TAM groups compared with the control. The reduction of tumor growth resulted from decreased cell proliferation and increased cell apoptosis. TAM/ 5FS caused a significantly higher expression of estrogen receptor-alpha (ERalpha) compared with 5FS and TAM/0FS, whereas TAM/10FS had a higher ERalpha than 10FS and TAM/0FS. Compared with the control, progesterone receptor (PgR) expression was significantly reduced in all treatment groups, but insulin-like growth factor-1 (IGF-1) expression was reduced only by 10FS, TAM/5FS and TAM/10FS. Tumor cell proliferation was significantly positively associated with expression of PgR and IGF-1 and negatively associated with apoptosis and ERalpha. Apoptosis was only associated with ERalpha. In conclusion, FS inhibited MCF-7 tumor growth in a dose-dependent manner and enhanced the inhibitory effect of TAM due to the modulation of ER and growth factor signal transduction pathways. Exp Biol Med (Maywood). 2007 Sep;232(8):1071-80. (Chen J, et al 2007)

Dietary flaxseed enhances the inhibitory effect of tamoxifen on the growth of estrogen-dependent human breast cancer (mcf-7) in nude mice.

PURPOSE: This study determined the effect of 10% dietary flaxseed (FS) and tamoxifen (TAM), alone and in combination, on the growth of estrogen-dependent human breast cancer (MCF-7) in athymic mice with or without 17beta-estradiol (E2) supplementation. EXPERIMENTAL DESIGN: Ovariectomized mice received injection with MCF-7 cells, were implanted with an E2 pellet (1.7 mg), and fed the basal diet (BD). When tumor reached approximately 40 mm2, the E2 implant was removed, and mice were randomized to the following groups and maintained at either low (E2 pellet removed) or high E2 level (new E2 pellet implanted) for 6 weeks: (a) positive control with new E2 pellet, fed BD, (b) negative control with no E2 implant, fed BD, (c) TAM group with TAM pellet (5 mg) implant, fed BD, (d) FS group fed 10% FS, (e) FS+TAM group with TAM implant, fed 10% FS. Tumor growth was monitored weekly. RESULTS: At low E2 level, FS regressed the pretreatment tumor size by 74%. TAM regressed tumor initially but later induced an increase so that the tumor size was finally similar to the pretreatment size. A tumor regression >53% was induced by FS+TAM than by TAM alone. At high E2 level, FS, TAM, and FS+TAM inhibited the tumor growth by 22, 41, and 50%, respectively, compared with the positive control. Decreased tumor size was attributable to reduced tumor cell proliferation and increased apoptosis. CONCLUSIONS: FS inhibited the growth of human estrogen-dependent breast cancer and strengthened the tumor-inhibitory effect of TAM at both low and high E2 levels. Clin Cancer Res. 2004 Nov 15;10(22):7703-11. (Chen J, et al 2004)

Selenium disrupts estrogen receptor (alpha) signaling and potentiates tamoxifen antagonism in endometrial cancer cells and tamoxifen-resistant breast cancer cells.

Tamoxifen, a selective estrogen receptor (ER) modulator, is the most widely prescribed hormonal therapy treatment for breast cancer. Despite the benefits of tamoxifen therapy, almost all tamoxifen-responsive breast cancer patients develop resistance to therapy. In addition, tamoxifen displays estrogen-like effects in the endometrium increasing the incidence of endometrial cancer. New therapeutic strategies are needed to circumvent tamoxifen resistance in breast cancer as well as tamoxifen toxicity in endometrium. Organic selenium compounds are highly effective chemopreventive agents with well-documented benefits in reducing total cancer incidence and mortality rates for a number of cancers. The present study shows that the organic selenium compound methylseleninic acid (MSA, 2.5 micromol/L) can potentiate growth inhibition of 4-hydroxytamoxifen (10(-7) mol/L) in tamoxifen-sensitive MCF-7 and T47D breast cancer cell lines. Remarkably, in tamoxifen-resistant MCF-7-LCC2 and MCF7-H2Delta16 breast cancer cell lines and endometrial-derived HEC1A and Ishikawa cells, coincubation of 4-hydroxytamoxifen with MSA resulted in a marked growth inhibition that was substantially greater than MSA alone. Growth inhibition by MSA and MSA + 4-hydroxytamoxifen in all cell lines was preceded by a specific decrease in ER(alpha) mRNA and protein without an effect on ER(beta) levels. Estradiol and 4-hydroxytamoxifen induction of endogenous ER-dependent gene expression (pS2 and c-myc) as well as ER-dependent reporter gene expression (ERE(2)e1b-luciferase) was also attenuated by MSA in all cell lines before effect on growth inhibition. Taken together, these data strongly suggest that specific decrease in ER(alpha) levels by MSA is required for both MSA potentiation of the growth inhibitory effects of 4-hydroxytamoxifen and resensitization of tamoxifen-resistant cell lines. Mol Cancer Ther. 2005 Aug;4(8):1239-49. (Shah YM, et al 2005)

Combination of methylselenocysteine with tamoxifen inhibits MCF-7 breast cancer xenografts in nude mice through elevated apoptosis and reduced angiogenesis.

To investigate the therapeutic effect of methylselenocysteine (MSC) combined with tamoxifen in MCF-7 breast cancer xenograft and the underlying mechanisms. MCF-7 breast cancer xenograft was established in ovariectomized female athymic nude mice and treated with tamoxifen and/or MSC. Tumor size was measured twice a week. Immunohistochemistry and TUNEL assays were used to measure ERalpha expression, ERalpha target genes (progesterone receptor (PR) and cyclin D1 expression), Ki-67 index, apoptosis and microvessel density. Combined treatment with tamoxifen and MSC synergistically inhibited tumor growth compared to MSC alone and tamoxifen alone. MSC alone or MSC + tamoxifen significantly reduced ERalpha, PR and cyclin D1, Ki67 index and microvessel density while increasing apoptosis in tumor tissues. These findings demonstrate synergistic growth inhibition of ERalpha positive breast cancer xenografts by combination of tamoxifen with organic selenium compounds. Organic selenium may provide added benefit when combined with tamoxifen in adjuvant therapy or prevention. Breast Cancer Res Treat. 2008 Oct 15. [Epub ahead of print] (Li Z, et al 2008)

Methylseleninic acid synergizes with tamoxifen to induce caspase-mediated apoptosis in breast cancer cells.

Tamoxifen has efficacy as a breast cancer therapy and chemoprevention agent. However, toxicity and resistance to tamoxifen limit its clinical application. There is an urgent need to develop compounds that may be combined with tamoxifen to improve efficacy and overcome toxicity and resistance. We showed previously that the organoselenium compound methylseleninic acid (MSA) increased the growth-inhibitory effect of tamoxifen and reversed tamoxifen resistance in breast cancer cells. In this study, we examined the mechanism for induction of apoptosis by MSA combined with tamoxifen in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. 4-hydroxytamoxifen (TAM; 10(-7) mol/L) alone resulted in cell cycle arrest but no apoptosis, whereas MSA alone (10 micromol/L) induced apoptosis in tamoxifen-sensitive cells. Combination of MSA with TAM resulted in a synergistic apoptosis in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells compared with either agent alone. MSA and MSA combined with TAM induced apoptosis through the intrinsic, mitochondrial apoptotic pathway. MSA induced a sequential activation of caspase-9 and then caspase-8. These results indicate that the growth inhibition synergy and reversal of tamoxifen resistance by combination of selenium with tamoxifen occurs via a tamoxifen-induced cell cycle arrest, allowing more cells to enter the intrinsic apoptotic pathway elicited by selenium. Mol Cancer Ther. 2008 Sep;7(9):3056-63. (Li Z, Carrier L, Rowan BG.2008)

-(-)Epigallocatechin-3-gallate downregulates EGFR, MMP-2, MMP-9 EMMPRIN and inhibits the invasion of MCF-7 tamoxifen resistant cells.

The activation of Epidermal Growth Factor Receptor (EGFR) signaling pathway is one of the key mechanisms underlying the development of resistance to tamoxifen in breast cancer patients. As -(-) Epigallocatechine-3-gallate (EGCG), the most active catechin present in green tea, has been shown to downregulate EGFR, we studied the effects of 10-100 microg/ml EGCG treatment on growth and invasion in a breast carcinoma cell line resistant to tamoxifen (MCF-7Tam) and parental MCF-7. A dose-dependent downregulation of EGFR mRNA expression and protein level occurred after 50 microg/ml EGCG treatment of MCF-7Tam cells. EGFR molecules on the plasma membrane surface of MCF-7Tam cells significantly decreased. EGFR phoshoporylation (Tyr992, Tyr1045 and Tyr1068) was higher in MCF-7Tam than MCF-7 and it was reduced by EGCG treatment. ERK and phosphoERK p42/44 were also dowregulated by EGCG treatment and in vitro cell growth and invasion decreased. Matrix Metalloproteinases (MMPs) -2 and -9, which are implicated in cell invasion and metastasis and Extracellular Matrix Metalloproteinase Inducer (EMMPRIN), a glycoprotein able to activate MMPs, were significantly reduced after 50 microg/ml EGCG treatment. Coherently, Tissue Inhibitors of Metallo Proteinases (TIMPs) -1 and -2, which downregulate MMPs, increased after EGCG treatment. Altogether the present data demonstrated that EGCG could attenuate the tamoxifen-resistant phenotype of MCF-7Tam cells. EGCG could contrast MCF-7Tam cell growth and in vitro invasion through downregulation of EGFR and other molecules implicated in aggressive biologic behavior. The present data support the hypothesis that EGCG is an interesting molecule to be investigated in tamoxifen-resistant breast carcinoma. Farabegoli F, Papi A, Orlandi M. Biosci Rep. 2010 May 7. [Epub ahead of print]

The sensitization of glioma cells to cisplatin and tamoxifen by the use of catechin.

Telomerase expression strongly correlates with the grade of malignancy in glioma with inhibition illustrating a definite increase in chemosensitivity. This study was designed to investigate the effects of a green tea derivative, epigallocatechin-3-gallate (EGCG); together with either cisplatin or tamoxifen in glioma, and to investigate whether these effects are mediated through telomerase suppression. EGCG showed a significant cytotoxic effect on 1321N1 cells after 24 h and on U87-MG cells after 72 h (P < 0.001) without significantly affecting the normal astrocytes. Treatment with EGCG inhibited telomerase expression significantly (P < 0.01) and enhanced the effect of cisplatin and tamoxifen in both 1321N1 (P < 0.01) and U87-MG (P < 0.001) cells. EGCG, as a natural product has enormous potential to be an anti-cancer agent capable of enhancing tumour cell sensitivity to therapy. Shervington A, Pawar V, Menon S, Thakkar D, Patel R. Mol Biol Rep. 2009 May;36(5):1181-6. Epub 2008 Jun 26.

(-)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells.

BACKGROUND: (-)-Epigallocatechin-3-gallate (EGCG) is the most active catechin present in green tea, demonstrated to have chemopreventive action and to kill cancer cells selectively. As a previous study found that catechins could compete with 17-beta-estradiol for binding to estrogen receptor alpha (ERalpha), we asked whether EGCG could regulate ERalpha action. METHODS: We used MCF-7, a breast carcinoma cell line having a high level of ERalpha expression. The cells were treated with various EGCG concentrations and cell viability was evaluated by MTT assay. ERalpha and pS2 expression were analyzed by RT-PCR after RNA extraction. To better define EGCG action in relation to ERalpha, we studied EGCG cytotoxicity on MCF-7 resistant to tamoxifen (MCF-7tam), MCF-7 treated with 10(-7)M ICI 182,780 for 8 days and on MDA-MB-231, a cell line that lacked ERalpha by flow cytometry (FCM). RESULTS: Both ERalpha and pS2 mRNA were expressed in samples treated with low EGCG concentration (30 microg/ml). At this concentration, no cell change was detectable. In contrast, pS2 expression was lost in samples treated with 100 microg/ml EGCG for 24h, indicating ERalpha alteration. EGCG cytotoxicity was lower when ERalpha was not present (MDA-MB-231) or inactivated (by tamoxifen or ICI 182,780). CONCLUSIONS: Functionally active ERalpha may have a role in EGCG cytotoxicity, increasing the sensitivity to the drug. As higher EGCG concentrations also killed cells resistant to tamoxifen or treated by 10(-7)M ICI 182,780, EGCG ought to be better investigated in breast carcinoma cells treated with drugs targeted to steroid receptors, as a potential complement of therapy. Farabegoli F, Barbi C, Lambertini E, Piva R. Cancer Detect Prev. 2007;31(6):499-504.

The combination of green tea and tamoxifen is effective against breast cancer.

Epidemiologic data have suggested that green tea may prevent breast cancer. Studies in our laboratory have provided evidence that green tea extract inhibits breast cancer growth by a direct anti-proliferative effect on the tumor cells, as well as by indirect suppressive effects on the tumor-associated endothelial cells. In this study, we asked whether concurrent administration of green tea may add to the anti-tumor effects of standard breast cancer therapy. We observed that green tea increased the inhibitory effect of tamoxifen on the proliferation of the ER (estrogen receptor)-positive MCF-7, ZR75, T47D human breast cancer cells in vitro. This combination regimen was also more potent than either agent alone at increasing cell apoptosis. In animal experiments, mice treated with both green tea and tamoxifen had the smallest MCF-7 xenograft tumor size, and the highest levels of apoptosis in tumor tissue, as compared with either agent administered alone. Moreover, the suppression of angiogenesis in vivo correlated with larger areas of necrosis and lower tumor blood vessel density in treated xenografts. Green tea decreased levels of ER-alpha in tumors both in vitro and in vivo. We also observed that green tea blocked ER-dependent transcription, as well as estradiol-induced phosphorylation and nuclear localization of mitogen-activated protein kinase. To our knowledge, this study is the first to show the interaction of green tea with the ER pathway, as well as provide mechanistic evidence that the combination of green tea and tamoxifen is more potent than either agent alone in suppressing breast cancer growth. These results may lead to future improvements in breast cancer treatment and prevention. Sartippour MR, Pietras R, Marquez-Garban DC, Chen HW, Heber D, Henning SM, Sartippour G, Zhang L, Lu M, Weinberg O, Rao JY, Brooks MN. Carcinogenesis. 2006 Dec;27(12):2424-33. Epub 2006 Jun 19.

A new role for tamoxifen in oestrogen receptor-negative breast cancer when it is combined with epigallocatechin gallate.

We have previously shown that tamoxifen+epigallocatechin gallate (EGCG) is synergistically cytotoxic towards oestrogen receptor (ER)-negative breast cancer cells. To determine if this response would correlate with significant tumour suppression in vivo, athymic nude female mice were implanted with MDA-MB-231 cells and treated with tamoxifen, EGCG, EGCG+tamoxifen, or vehicle control for 10 weeks. Tumour volume in EGCG- (25 mg kg(-1))+tamoxifen (75 microg kg(-1))-treated mice decreased by 71% as compared with vehicle control (P<0.05), whereas tumour weight was decreased by 80% compared with control (P<0.01). Epigallocatechin gallate treatment did not alter ER protein expression in MDA-MB-231 cells and thus was not a mechanism for the observed tumour suppression. However, western blotting of tumour extracts demonstrated that epidermal growth factor receptor (EGFR; 85% lower than control), pEGFR (78% lower than control), mammalian target of rapamycin (mTOR; 78% lower than control), and CYP1B1 (75% lower than control) were significantly lower after the combination treatment as compared with all other treatments. Nuclear factor-kappaB (NF-kappaB), b-Raf, p-MEK, S6K, 4EBP1, Akt, vascular EGFR-1 (VEGFR-1) and VEGF expressions were decreased in control but not in the individual treatments, whereas MEK, phospholipase D 1/2, TGF alpha, and ERK expressions were not changed after any treatment. The results demonstrate that tamoxifen at realistic doses (75 mug kg(-1)) can suppress the growth of ER-negative breast cancer when combined with EGCG. In addition, the dominant mechanism for tumour suppression is the concomitant decrease in tumour protein expressions of mTOR and the EGFR. Scandlyn MJ, Stuart EC, Somers-Edgar TJ, Menzies AR, Rosengren RJ. Br J Cancer. 2008 Oct 7;99(7):1056-63. Epub 2008 Sep 16.

Hepatoprotective effect of green tea (Camellia sinensis) extract against tamoxifen-induced liver injury in rats.

Tamoxifen citrate (TAM), is widely used for treatment of breast cancer. It showed a degree of hepatic carcinogenesis. The purpose of this study was to elucidate the antioxidant capacity of green tea (Camellia sinensis) extract (GTE) against TAM-induced liver injury. A model of liver injury in female rats was done by intraperitoneal injection of TAM in a dose of 45mg Kg(-1) day(-1), i.p. for 7 successive days. GTE in the concentration of 1.5 %, was orally administered 4 days prior and 14 days after TAM-intoxication as a sole source of drinking water. The antioxidant flavonoid; epicatechin (a component of green tea) was not detectable in liver and blood of rats in either normal control or TAM-intoxicated group, however, TAM intoxication resulted in a significant decrease of its level in liver homogenate of tamoxifenintoxicated rats. The model of TAM-intoxication elicited significant declines in the antioxidant enzymes (glutathione-S-transferase,glutathione peroxidase, superoxide dismutase and catalase) and reduced glutathione concomitant with significant elevations in TBARS (thiobarbituric acid reactive substance) and liver transaminases; sGPT (serum glutamate pyruvate transaminase) and sGOT (serum glutamate oxaloacetate transaminase) levels. The oral administration of 1.5 % GTE to TAM-intoxicated rats, produced significant increments in the antioxidant enzymes and reduced glutathione concomitant with significant decrements in TBARS and liver transaminases levels. The data obtained from this study speculated that 1.5 % GTE has the capacity to scavenge free radical and can protect against oxidative stress induced by TAM intoxication. Supplementation of GTE could be useful in alleviating tamoxifen-induced liver injury in rats. El-Beshbishy HA. Biochem Mol Biol. 2005 Sep 30;38(5):563-70.

The combination of green tea and tamoxifen is effective against breast cancer.

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Decreased BRCA1 confers tamoxifen resistance in breast cancer cells by altering estrogen receptor-coregulator interactions.

Genetic screen identifies insulin-like growth factor binding protein 5 as a modulator of tamoxifen resistance in breast cancer.

Estrogen receptor-alpha phosphorylation at serine 305, nuclear p21-activated kinase 1 expression, and response to tamoxifen in postmenopausal breast cancer.

Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen.

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells.

Association between CYP2D6 polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen.

Genetic variants of CYP3A5, CYP2D6, SULT1A1, UGT2B15 and tamoxifen response in postmenopausal patients with breast cancer.

Pharmacogenetics of tamoxifen: who should undergo CYP2D6 genetic testing?

Pharmacogenomics of tamoxifen therapy.

Clinical relevance of circulating CK-19 mRNA-positive cells detected during the adjuvant tamoxifen treatment in patients with early breast cancer.

Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation.

Factor V Leiden Mutation and Thromboembolism Risk in Women Receiving Adjuvant Tamoxifen for Breast Cancer

Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.

Enhancement of vascular endothelial growth factor-mediated angiogenesis in tamoxifen-resistant breast cancer cells: role of Pin1 overexpression.

Possible interaction between activator protein-1 and proto-oncogene B-cell lymphoma gene 6 in breast cancer patients resistant to tamoxifen.

Tamoxifen and gallstone formation in postmenopausal breast cancer patients: retrospective cohort study.

Decreased secretion of Cathepsin D in breast cancer in vivo by tamoxifen: mediated by the mannose-6-phosphate/IGF-II receptor?

Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential.

Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer.

Human X-box binding protein-1 confers both estrogen independence and antiestrogen resistance in breast cancer cell lines.

CYP17 polymorphism and tamoxifen-induced hepatic steatosis.

Significant effect of polymorphisms in CYP2D6 and ABCC2 on clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients.

Impaired tamoxifen metabolism reduces survival in familial breast cancer patients.

Pharmacogenomic variation of CYP2D6 and the choice of optimal adjuvant endocrine therapy for postmenopausal breast cancer: a modeling analysis.

Clinical significance of CYP2D6 polymorphisms and tamoxifen in women with breast cancer.

Selective serotonin reuptake inhibitors and breast cancer mortality in women receiving tamoxifen: a population based cohort study.

Long-term endometrial effects in postmenopausal women with early breast cancer participating in the Intergroup Exemestane Study (IES)--a randomised controlled trial of exemestane versus continued tamoxifen after 2-3 years tamoxifen.

Breast cancer recurrence risk related to concurrent use of SSRI antidepressants and tamoxifen.

BCL2 and CASP8 regulation by NF-kappaB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells.

Flaxseed alone or in combination with tamoxifen inhibits MCF-7 breast tumor growth in ovariectomized athymic mice with high circulating levels of estrogen.

Selenium disrupts estrogen receptor (alpha) signaling and potentiates tamoxifen antagonism in endometrial cancer cells and tamoxifen-resistant breast cancer cells.

Combination of methylselenocysteine with tamoxifen inhibits MCF-7 breast cancer xenografts in nude mice through elevated apoptosis and reduced angiogenesis.

Methylseleninic acid synergizes with tamoxifen to induce caspase-mediated apoptosis in breast cancer cells.

-(-)Epigallocatechin-3-gallate downregulates EGFR, MMP-2, MMP-9 EMMPRIN and inhibits the invasion of MCF-7 tamoxifen resistant cells.

The sensitization of glioma cells to cisplatin and tamoxifen by the use of catechin.

(-)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells.

The combination of green tea and tamoxifen is effective against breast cancer.

A new role for tamoxifen in oestrogen receptor-negative breast cancer when it is combined with epigallocatechin gallate.

Hepatoprotective effect of green tea (Camellia sinensis) extract against tamoxifen-induced liver injury in rats.

The combination of green tea and tamoxifen is effective against breast cancer.