Amanda Beyer

Androgen Receptor Overexpression Induces Tamoxifen Resistance in Human Breast Cancer Cells

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) α mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERα-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex™ (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERα that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen’s agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor–positive human breast cancer cells

It has long been appreciated that estrogenic signaling contributes to breast cancer progression. c-Src is also required for a number of processes involved in tumor progression and metastasis. We have previously identified the K303R mutant estrogen receptor α (ERα) that confers hypersensitivity to low levels of estrogen. Because ERα and c-Src have been shown to interact in a number of different systems, we wanted to evaluate the role of c-Src kinase in estrogen-stimulated growth and survival of ERα-positive breast cancer cells. MCF-7 cells stably expressing the mutant receptor showed increased c-Src kinase activity and c-Src tyrosine phosphorylation when compared with wild-type ERα-expressing cells. A c-Src inhibitor, AZD0530, was used to analyze the biological effects of pharmacologically inhibiting c-Src kinase activity. MCF-7 cells showed an anchorage-dependent growth IC50 of 0.47 μmol/L, which was increased 4-fold in the presence of estrogen. In contrast, cells stably expressing the mutant ERα had an elevated IC50 that was only increased 1.4-fold by estrogen stimulation. The c-Src inhibitor effectively inhibited the anchorage-independent growth of both of these cells, and estrogen was able to reverse these effects. When cells were treated with suboptimal concentrations of c-Src inhibitor and tamoxifen, synergistic inhibition was observed, suggesting a cooperative interaction between c-Src and ERα. These data clearly show an important role for ERα and estrogen signaling in c-Src–mediated breast cancer cell growth and survival. Here, we show that c-Src inhibition is blocked by estrogen signaling; thus, the therapeutic use of c-Src inhibitors may require inhibition of ERα in estrogen-dependent breast cancer. [Mol Cancer Ther 2006;5(12):3023–31]

Loss of Rho GDIα and Resistance to Tamoxifen via Effects on Estrogen Receptor α

BACKGROUND Estrogen receptor (ER) α is a successful therapeutic target in breast cancer, but patients eventually develop resistance to antiestrogens such as tamoxifen. METHODS To identify genes whose expression was associated with the development of tamoxifen resistance and metastasis, we used microarrays to compare gene expression in four primary tumors from tamoxifen-treated patients whose breast cancers did not recur vs five metastatic tumors from patients whose cancers progressed during adjuvant tamoxifen treatment. Because Rho guanine dissociation inhibitor (GDI) α was underexpressed in the tamoxifen-resistant group, we stably transfected ERα-positive MCF-7 breast cancer cells with a plasmid encoding a short hairpin (sh) RNA to silence Rho GDIα expression. We used immunoblots and transcription assays to examine the role of Rho GDIα in ER-related signaling and growth of cells in vitro and as xenografts in treated nude mice (n = 8-9 per group) to examine the effects of Rho GDIα blockade on hormone responsiveness and metastatic behavior. The time to tumor tripling as the time in weeks from randomization to a threefold increase in total tumor volume over baseline was examined in treated mice. The associations of Rho GDIα and MTA2 levels with tamoxifen resistance were examined in microarray data from patients. All statistical tests were two-sided. RESULTS Rho GDIα was expressed at lower levels in ERα-positive tumors that recurred during tamoxifen treatment than in ERα-positive tamoxifen-sensitive primary tumors. MCF-7 breast cancer cells in which Rho GDIα expression had been silenced were tamoxifen-resistant, had increased Rho GTPase and p21-activated kinase 1 activity, increased phosphorylation of ERα at serine 305, and enhanced tamoxifen-induced ERα transcriptional activity compared with control cells. MCF-7 cells in which Rho GDIα expression was silenced metastasized with high frequency when grown as tumor xenografts. When mice were treated with estrogen or estrogen withdrawal, tripling times for xenografts from cells with Rho GDIα silencing were similar to those from vector-containing control cells; however, tripling times were statistically significantly faster than control when mice were treated with tamoxifen (median tripling time for tumors with Rho GDIα small interfering RNA = 2.34 weeks; for control tumors = not reached, hazard ratio = 4.13, 95% confidence interval = 1.07 to 15.96, P = .040 [adjusted for multiple comparisons, P = .119]). Levels of the metastasis-associated protein MTA2 were also increased upon Rho GDIα silencing, and combined Rho GDIα and MTA2 levels were associated with recurrence in 250 tamoxifen-treated patients. CONCLUSION Loss of Rho GDIα enhances metastasis and resistance to tamoxifen via effects on both ERα and MTA2 in models of ERα-positive breast cancer and in tumors of tamoxifen-treated patients.

Dicer-Mediated Upregulation of BCRP Confers Tamoxifen Resistance in Human Breast Cancer Cells

Purpose: Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer. Experimental Design: We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux. Results: In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux. Conclusion: Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity. Clin Cancer Res; 17(20); 6510–21. ©2011 AACR.

Phosphorylation of the mutant K303R estrogen receptor |[alpha]| at serine 305 affects aromatase inhibitor sensitivity

We earlier identified a lysine to arginine transition at residue 303 (K303R) in estrogen receptor alpha (ERα) in invasive breast cancers, which confers resistance to the aromatase inhibitor (AI) anastrozole (Ana) when expressed in MCF-7 breast cancer cells. Here, we show that AI resistance arises through an enhanced cross talk of the insulin-like growth factor receptor-1 (IGF-1R)/insulin receptor substrate (IRS)-1/Akt pathway with ERα, and the serine (S) residue 305 adjacent to the K303R mutation has a key function in mediating this cross talk. The ERα S305 residue is an important site that modifies response to tamoxifen; thus, we questioned whether this site could also influence AI response. We generated stable transfectants-expressing wild-type, K303R ERα or a double K303R/S305A mutant receptor, and found that the AI-resistant phenotype associated with expression of the K303R mutation was dependent on activation of S305 within the receptor. Ana significantly reduced growth in K303R/S305A-expressing cells. Preventing S305 phosphorylation with a blocking peptide inhibited IGF-1R/IRS-1/Akt activation and also restored AI sensitivity. Our data suggest that the K303R mutation and the S305 ERα residue may be a novel determinant of AI response in breast cancer, and blockade of S305 phosphorylation represents a new therapeutic strategy for treating tumors resistant to hormone therapy.

Association between the Estrogen Receptor α A908G Mutation and Outcomes in Invasive Breast Cancer

Purpose: Estrogen receptor α (ERα) predicts the natural history of breast cancer without intervening therapy. Here, we have optimized the detection of a somatic mutation, an A908G transition of ERα, and examined its association with clinical and biological features of invasive breast cancer. Experimental Design: We compared two methods of sequencing to detect the A908G ERα mutation. We then used primer extension sequencing with genomic DNA isolated from invasive breast tumors to determine whether the mutation was associated with clinical outcome in 267 axillary node–negative and axillary node–positive breast tumors. The presence of the mutation and clinical variables were analyzed for association with recurrence-free survival and overall survival by Cox proportional hazards regression models. Results: We determined that dye-labeled terminator sequencing was not adequate for detection of the A908G ERα mutation. The mutation was detected at a high frequency (50%) in invasive breast tumors using primer extension sequencing, and was found to be associated with clinical measures of poor outcome, including larger tumor size and axillary lymph node positivity. Although the mutation was associated with recurrence-free survival in univariate analysis, it was not an independent predictor of outcomes in multivariate analysis. Conclusions: Consistent with our previous finding of this somatic ERα mutation in breast ductal hyperplasias, we now present evidence that the A908G mutation is present in invasive breast tumors using an optimized sequencing method. We find that the mutation is significantly associated with aggressive biological tumor features, and with an unfavorable prognosis, but was not an independent prognostic marker in untreated patients.

Androgen receptor promotes tamoxifen agonist activity by activation of EGFR in ERα-positive breast cancer

Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) α-positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI) α, a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDIα and seek to determine AR’s contribution to resistance. We engineered ERα-positive cell lines with stable knockdown (KD) of Rho GDIα (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Tam-resistant tumors and cell lines with low Rho GDIα levels exhibited upregulated AR expression. Microarray of Rho GDIα KD cells indicated that activation of EGFR and ERα was associated with Tam treatment. When AR levels were elevated, interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ERα. Tam exhibited agonist activity in AR overexpressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ERα-positive cells with low expression of Rho GDIα.

Targeting thyroid hormone receptor beta in triple-negative breast cancer

The purpose of this study was to discover novel nuclear receptor targets in triple-negative breast cancer. Expression microarray, Western blot, qRT-PCR analyses, MTT growth assay, soft agar anchorage-independent growth assay, TRE reporter transactivation assay, and statistical analysis were performed in this study. We performed microarray analysis using 227 triple-negative breast tumors, and clustered the tumors into five groups according to their nuclear receptor expression. Thyroid hormone receptor beta (TRβ) was one of the most differentially expressed nuclear receptors in group 5 compared to other groups. TRβ low expressing patients were associated with poor outcome. We evaluated the role of TRβ in triple-negative breast cancer cell lines representing group 5 tumors. Knockdown of TRβ increased soft agar colony and reduced sensitivity to docetaxel and doxorubicin treatment. Docetaxel or doxorubicin long-term cultured cell lines also expressed decreased TRβ protein. Microarray analysis revealed cAMP/PKA signaling was the only KEGG pathways upregulated in TRβ knockdown cells. Inhibitors of cAMP or PKA, in combination with doxorubicin further enhanced cell apoptosis and restored sensitivity to chemotherapy. TRβ-specific agonists enhanced TRβ expression, and further sensitized cells to both docetaxel and doxorubicin. Sensitization was mediated by increased apoptosis with elevated cleaved PARP and caspase 3. TRβ represents a novel nuclear receptor target in triple-negative breast cancer; low TRβ levels were associated with enhanced resistance to both docetaxel and doxorubicin treatment. TRβ-specific agonists enhance chemosensitivity to these two agents. Mechanistically enhanced cAMP/PKA signaling was associated with TRβ’s effects on response to chemotherapy.

Abstract 1468: MTA2 enhances breast cancer metastasis via regulation of the cytoskeleton and rho signaling pathways

Metastasis tumor associated (MTA) 2 is a component of the nucleosome remodeling and histone deacetylation complex and is implicated in regulating gene expression through its associated histone deacetylase activity. We have previously found that MTA2 enhanced the anchorage independent growth phenotype of estrogen receptor alpha (ER)-positive cells, implicating a potential role in metastasis. As MTA2-overexpression in these cells also resulted in an estrogen independent phenotype, we hypothesized that MTA2-overexpression may similarly enhance the aggressiveness of ER-negative cells. We found that MTA2-overexpressing MDA-MB-231 cells were more invasive through Matrigel, had enhanced soft-agar colony formation activity compared with vector controls, and demonstrated increased migration in wound healing assays. Furthermore, we found that MTA2 overexpression resulted in rapid systemic metastases when grown as orthotopic xenografts, while vector control cells failed to form metastases, in the same time period. Microarray analysis revealed enrichment for genes regulating the cytoskeleton and cell-environment interactions, which was validated using a previously published microarray dataset. We found that the negative regulator of the Rho pathway, Rho GDP dissociation inhibitor alpha (RhoGDI-alpha), was also decreased in expression at the protein level in the MTA2-overexpressing MDA-MB-231 cells. We found that blocking signaling through the Rho pathway by either using a Rho Kinase inhibitor or by overexpressing RhoGDI-alpha significantly reduced the soft-agar colony forming ability of MTA2-overexpressing cells. We examined the effect of MTA2 and RhoGDI-alpha expression in a retrospective breast cancer microarray dataset and found that patients classified as MTA2-high and RhoGDI-alpha-low had significantly earlier recurrence compared with all other patients. These data indicate that MTA2-overexpression results in an increase in the metastatic potential of breast cancer cells. These results have potential clinical implications as patients who have the MTA2-high/RhoGDI-alpha-low phenotype are at increased risk for early recurrence. In conclusion, our data show that MTA2 and RhoGDI-alpha enhances breast cancer metastasis possibly by promoting invasiveness and anchorage independent growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1468. doi:10.1158/1538-7445.AM2011-1468

Abstract 2280: Increased activity of the Rho family of proteins results in a tamoxifen-resistant phenotype in ERα-positive breast cancer cells

Background: We discovered that shRNA knockdown of Rho GDIα can render estrogen receptor (ER)-positive breast cancer cells more aggressive by increasing their metastatic ability and altering their tamoxifen (tam) sensitivity. Rho GDIα is a negative regulator of the Rho family of proteins (Rho, Rac, and Cdc42), which play an important role in the regulation of the actin cytoskeleton. While the Rho GDIα pathway is known to influence metastasis in breast cancer, it is unclear how this pathway causes ERα-positive breast cancer cells to become tam-resistant. Since loss of Rho GDIα causes an increased activity of the Rho proteins it is possible that increased activity of downstream effectors may lead to tam resistance through crosstalk with the ERα signaling pathway. We have shown that increased phosphorylation of the S305 site in ERα leads to tam resistance and this site is a target for PAK1 phosphorylation which lies downstream of several growth factor signaling pathways. Results: Stable knockdown of Rho GDIα in ER-positive breast cancer cells, using shRNA, resulted in tam resistance in vivo. Tam stimulated tumor growth in athymic nude mice injected with Rho GDIα knockdown cells. These mice exhibted metastatic lung lesions when treated with either estrogen or tam but not in untreated mice. Tam was unable to reduce in vitro growth of Rho GDIα knockdown cells in soft agar assays. Rhotekin pulldown assays revealed higher activity of Rho proteins in Rho GDIα knockdown cells compared to vector control cells. PAK1 kinase assays revealed that PAK1, a downstream effector of the Rho pathway, had increased activity in Rho GDIα knockdown cells. This increased activity led to increased phosphorylation of ERα on S305. As expected, acetylation of ERα was decreased and this decrease in acetylation was dependent on phosphorylation of S305. ERE-luciferase assays showed that Rho GDIα knockdown cells had higher levels of ERE luciferase activity in these cells, tam significantly stimulated ERE-luciferase activity, suggesting agonist activity as a mechanism of resistance. Further possible effectors of Rho GDIα are being investigated as possible candidates playing a role in the crosstalk between these two pathways, as well as growth factor signaling pathways which may signal to ER and play a role in the tam-resistant phenotype. Interestingly, cells which became tam resistant due to long term culture in the presence of tam showed a decrease in endogenous Rho GDIα levels. Discussion: Loss of Rho GDIα in ERα-positive breast cancer cells conferred a tam-resistant phenotype in vitro and in vivo. Cells with decreased Rho GDIα levels had increased phosphorylation of ERα, suggesting crosstalk between the Rho family of signaling pathways and the ERα signaling pathways. Disrupting this crosstalk may be a mechanism of preventing or reversing tam resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2280. doi:10.1158/1538-7445.AM2011-2280