AR-positive Breast Cancer Research Articles

Androgen Triggers the Pro-Migratory CXCL12/CXCR4 Axis in AR-Positive Breast Cancer Cell Lines: Underlying Mechanism and Possible Implications for the Use of Aromatase Inhibitors in Breast Cancer

Background/Aims: Reports regarding the role of androgen in breast cancer (BC) are conflicting. Some studies suggest that androgen could lead to undesirable responses in the presence of certain BC tumor characteristics. We have shown that androgen induces C-X-C motif chemokine 12 (CXCL12) in BC cell lines. Our aim was to identify the mechanisms regulating the phenotypic effects of androgen-induced CXCL12 on Androgen Receptor (AR) positive BC cell lines. Methods: We analyzed the expression of CXCL12 and its receptors with qPCR and ELISA and the role of Nuclear Receptor Coactivator 1 (NCOA1) in this effect. AR effects on the CXCL12 promoter was studied via Chromatin-immunoprecipitation. We also analyzed publically available data from The Cancer Genome Atlas to verify AR-CXCL12 interactions and to identify the effect or Aromatase Inhibitors (AI) therapy on CXCL12 expression and disease progression in AR positive cases. Results: CXCL12 induction occurs only in AR-positive BC cell lines, possibly via an Androgen Response Element, upstream of the CXCL12 promoter. The steroid receptor co-regulator NCOA1 is critical for this effect. Androgen only induced the motility of p53-mutant BC cells T47D cells via upregulation of CXCR4 expression while they had no effect on wild-type p53 MCF-7 cells. Loss of CXCR4 expression and depletion of CXCL12 abolished the effect of androgen in T47D cells while inhibition of p53 expression in MCF-7 cells made them responsive to androgen and increased their motility in the presence to androgen. Patients with estrogen receptor positive (ER+)/AR+ BC treated with AIs were at increased risk of disease progression compared to ER+/AR+ non-AI treated and ER+/AR- AI treated cases. Conclusion: AIs may lead to unfavorable responses in some ER/AR positive BC cases, especially in patients with AR+, p53 mutant tumors.

Abstract P3-14-04: Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo

Abstract VT-464 is a lyase-selective inhibitor of the dual-activity CYP17A1 enzyme that is required for the synthesis of androgens and estrogens in the gonads, adrenals, and tumors. In addition to its role as a CYP17A1 lyase inhibitor, we previously showed that VT-464 also functions as a direct and effective AR antagonist in prostate cancer models. In our current study, we evaluated the therapeutic potential of VT-464 in breast cancer by analyzing its effectiveness in several AR-positive breast cancer cell lines, including those that are ER-positive and ER-negative. Through in vitro assays and xenograft analysis, we compared the activity of VT-464 to enzalutamide, a second generation AR antagonist that is approved for the treatment of castration resistant prostate cancer and is in multiple Phase 2 breast cancer studies. Our results showed that VT-464 was highly effective in preventing proliferation of both ER-positive and ER-negative breast cancer cell lines in vitro. Importantly, significant inhibition was also observed in soft agar assays that assesses anchorage-independent growth. In mechanistic studies, VT-464 and enzalutamide both inhibited induction of AR target genes and recruitment of AR to target promoters, verifying direct AR antagonistic activity observed previously in prostate cancer cells. Furthermore, we evaluated the ability of VT-464 and enzalutamide to inhibit tumor formation in a tamoxifen-resistant model of breast cancer. Oral administration of either enzalutamide or VT-464 significantly decreased tumor growth in mice, with VT-464 achieving greater growth inhibition than enzalutamide. Together, these data provide further rationale for the future study of the AR as a viable therapeutic target in breast cancer, and importantly, suggest that AR inhibition can impact tamoxifen-resistant tumor growth. The dual effects of CYP17A1 lyase inhibition and AR antagonism that are achieved with VT-464 further supports its development as an effective oral therapy option for AR-positive breast cancer. A phase 1 / 2 clinical study of oral VT-464 in women with AR+ triple-negative breast cancer or ER+ cancer resistant to aromatase inhibitors will commence in 2015. Citation Format: Ellison SJ, Norris JD, Wardell S, Eisner JR, Hoekstra WJ, Stagg DB, Alley HM, Moore WR, McDonnell DP. Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-04.

Abiraterone acetate, exemestane or the combination in postmenopausal patients with estrogen receptor-positive metastatic breast cancer

Androgen receptor (AR) signaling and incomplete inhibition of estrogen signaling may contribute to metastatic breast cancer (MBC) resistance to a nonsteroidal aromatase inhibitor (NSAI; letrozole or anastrozole). We assessed whether combined inhibition of androgen biosynthesis with abiraterone acetate plus prednisone and estradiol synthesis with exemestane (E) may be of clinical benefit to postmenopausal patients with NSAI-pretreated estrogen receptor-positive (ER+) MBC. Patients (N = 297) were stratified by the number of prior therapies for metastatic disease (0-1 versus 2) and by prior NSAI use (adjuvant versus metastatic), and randomized (1 : 1 : 1) to receive oral once daily 1000 mg abiraterone acetate plus 5 mg prednisone (AA) versus AA with 25 mg E (AAE) versus 25 mg E alone (E). Each treatment arm was well balanced with regard to the proportion of patients with AR-positive breast cancer. The primary end point was progression-free survival (PFS). Secondary end points included overall survival, clinical benefit rate, duration of response, and overall response rate. There was no significant difference in PFS with AA versus E (3.7 versus 3.7 months; hazard ratio [HR] = 1.1; 95% confidence interval [CI] 0.82-1.60; P = 0.437) or AAE versus E (4.5 versus 3.7 months; HR = 0.96; 95% CI 0.70-1.32; P = 0.794). Increased serum progesterone concentrations were observed in both arms receiving AA, but not with E. Grade 3 or 4 treatment-emergent adverse events associated with AA, including hypokalemia and hypertension, were less common in patients in the E (2.0% and 2.9%, respectively) and AA arms (3.4% and 1.1%, respectively) than in the AAE arm (5.8% for both). Adding AA to E in NSAI-pretreated ER+ MBC patients did not improve PFS compared with treatment with E. An AA-induced progesterone increase may have contributed to this lack of clinical activity. NCT01381874.

Activation of AR Sensitizes Breast Carcinomas to NVP-BEZ235's Therapeutic Effect Mediated by PTEN and KLLN Upregulation

NVP-BEZ235 is a newly developed dual PI3K/mTOR inhibitor, being tested in multiple clinical trials, including breast cancer. NVP-BEZ235 selectively induces cell growth inhibition in a subset, but not all, breast cancer cell lines. However, it remains a challenge to distinguish between sensitive and resistant tumors, particularly in the pretreatment setting. Here, we used ten breast cancer cell lines to compare NVP-BEZ235 sensitivity and in the context of androgen receptor (AR) activation during NVP-BEZ235 treatment. We also used female SCID mice bearing breast tumor xenografts to investigate the beneficial effect of dihydrotestosterone/NVP-BEZ235 combination treatment compared with each alone. We found that AR-positive breast cancer cell lines are much more sensitive to NVP-BEZ235 compared with AR-negative cells, regardless of PTEN or PI3KCA status. Reintroducing AR expression in NVP-BEZ235 nonresponsive AR-negative cells restored the response. DHT/NVP-BEZ235 combination not only resulted in a more significant growth inhibition than either drug alone, but also achieved tumor regression and complete responses for AR(+)/ER(+) tumors. This beneficial effect was mediated by dihydrotestosterone (DHT)-induced PTEN and KLLN expression. Furthermore, DHT could also reverse NVP-BEZ235-induced side effects such as skin rash and weight loss. Our data suggest that AR expression may be an independent predictive biomarker for response to NVP-BEZ235. AR induction could add benefit during NVP-BEZ235 treatment in patients, especially with AR(+)/ER(+) breast carcinomas.

Highlights of This Issue

Despite the clinical success of selective BRAF-targeted agents in melanoma, strategies to counteract the invariable development of resistance are not yet established. Acquaviva and colleagues show that HSP90 inhibition using the investigational drug ganetespib resulted in superior cytotoxic activity and antitumor efficacy over targeted BRAF or MEK inhibitors in preclinical models of mutant BRAF-driven melanoma. Importantly, ganetespib treatment readily overcame mechanisms of intrinsic and acquired resistance to vemurafenib, and dual targeting of BRAFV600E or MEK with ganetespib afforded combinatorial benefit in vitro and in vivo. Thus, HSP90 blockade may represent an alternative, and potentially complementary strategy, for therapeutic intervention in mutant-BRAF disease.An oncogenic fusion protein, EML4-ALK is involved in the pathogenesis of a subset of non–small cell lung cancer (NSCLC). The ALK/MET inhibitor crizotinib has already shown efficacy in ALK-driven NSCLC patients, but the treatment is not curative with rapid acquisition of resistance. In this report, Mori and colleagues have demonstrated that ASP3026, a novel small-molecule ALK inhibitor, induced tumor regression and prolonged survival in mice with ALK-driven NSCLC tumors, for which crizotinib showed limited efficacy, including the L1196M ‘gatekeeper’ mutant. These findings highlight the therapeutic potential of ASP3026 against cancer in patients with ALK abnormality.Although inhibition of IGF-1R was expected to eliminate a key resistance mechanism to anticancer therapies, clinical results to date have been disappointing. Fitzgerald and colleagues show that adaptive ErbB3 signaling is a key factor limiting the utility of anti-IGF-1R agents. The authors describe a novel bispecific tetravalent antibody, MM-141, that co-blocks IGF and heregulin-induced signaling and induces degradation of receptor complexes containing IGF-1R and ErbB3, including their heterodimers with insulin receptor and ErbB2. MM-141 shows superior cellular and xenograft growth inhibition to antibody monotherapies and potentiates the effect of mTOR inhibitors and taxanes, supporting its ongoing clinical development.It is challenging to predict patients' responses to cancer treatment and mTOR inhibitors are no exception. Wang and colleagues used androgen receptor (AR) expression as a marker to evaluate breast cancers' sensitivity to NVP-BEZ235, an experimental mTOR inhibitor, and investigated the beneficial effect of combining NVP-BEZ235 with dihydrotestosterone (DHT), an unaromatizable androgen. AR-positive breast cancer cell lines are more sensitive to NVP-BEZ235 compared with AR-negative cells. DHT/NVP-BEZ235 combination achieved therapeutic effects superior to either drug alone and reversed therapy-induced side effects. It suggests that AR expression may be used to predict NVP-BEZ235 response and AR induction could benefit NVP-BEZ235 treatment for breast carcinomas.

Phase II Trial of Bicalutamide in Patients with Androgen Receptor–Positive, Estrogen Receptor–Negative Metastatic Breast Cancer

Patients with hormone receptor-negative breast cancer generally do not benefit from endocrine-targeted therapies. However, a subset with androgen receptor (AR) expression is predicted to respond to antiandrogen therapies. This phase II study explored bicalutamide in AR-positive, estrogen receptor (ER), and progesterone receptor (PgR)-negative metastatic breast cancer. Tumors from patients with ER/PgR-negative advanced breast cancer were tested centrally for AR [immunohistochemistry (IHC) > 10% nuclear staining considered positive]. If either the primary or a metastatic site was positive, patients were eligible to receive the AR antagonist bicalutamide at a dose of 150 mg daily. Clinical benefit rate (CBR), the primary endpoint, was defined as the total number of patients who show a complete response (CR), partial response (PR), or stable disease (SD) > 6 months; secondary endpoints included progression-free survival (PFS) and toxicity. Correlative studies included measurement of circulating endocrine markers and IHC surrogates for basal-like breast cancer. Of 424 patients with ER/PgR-negative breast cancer, 12% tested AR-positive. The 6-month CBR was 19% [95% confidence interval (CI), 7%-39%] for bicalutamide. The median PFS was 12 weeks (95% CI, 11-22 weeks). Bicalutamide was well-tolerated with no grade 4/5 treatment-related adverse events observed. AR was expressed in 12% of patients with ER/PgR-negative breast cancer screened for this trial. The CBR of 19% observed with bicalutamide shows proof of principle for the efficacy of minimally toxic androgen blockade in a select group of patients with ER/PgR-negative, AR-positive breast cancer.

Abstract 4170: MicroRNA-135b influences androgen and estrogen receptor protein levels and regulates proliferation of hormone-receptor positive prostate and breast cancer cells.

Abstract MicroRNAs (miRNAs) regulate genes and impact on a wide range of cellular signaling pathways and biological processes in both physiological and pathological states. To understand their role in prostate and breast cancer, we have carried out systematic functional high-throughput miRNA-overexpression studies in cell lines, and assessed the effect of miRNAs on androgen receptor (AR) and estrogen receptor (ER) protein levels and on cell proliferation. We have observed that overexpression of miR-135b affected the growth of cells where the endogenous expression of miR-135b was low. Furthermore, the cells with low miR-135b expression mostly had a higher expression of AR and ER in prostate respective breast cancer cells. We identified that miR-135b is able to target AR and regulate the AR protein level in prostate cancer cells. Our aim was thus to further explore the function and significance of miR-135b in prostate and breast cancer. We found that miR-135b inhibited the growth of AR-positive prostate cancer (LNCaP, 22Rv1 and VCaP) and ER-positive breast cancer (MCF-7 and BT-474) cells, whereas it did not have any effect on the AR- and ER-negative cells. Here, we demonstrate that miR-135b decreases the ER protein level in breast cancer cells and that miR-135b expression inversely correlates with ER protein and mRNA as well as AR mRNA levels in 101 clinical breast cancer samples and with AR protein level in 47 clinical prostate cancer samples. In order to characterize novel target genes and to understand the mechanisms for the observed effect, we conducted gene expression analyses and identified 65 genes that were deregulated by miR-135b at three different time points (12 h, 24 h and 36 h). We discovered that for almost 50% of the genes, the expression significantly (p≤0.05) positively or negatively correlated with the expression of miR-135b in clinical breast cancer samples, suggesting that they may be relevant. For 14 genes we observed a significant inverse correlation with miR-135b in the clinical material. Hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) showed an inverse correlation and is predicted to be targeted by miR-135b by TargetScan 6.2. Our results indicate that miR-135b influences the protein level of HIF1AN. This could imply that miR-135b is involved in the regulation of oxygen homeostasis, and subsequently angiogenesis and glycolytic metabolism, in breast and prostate cancer tumors by negatively regulating the inhibitor of HIF1α that plays an important role in cancer. Citation Format: Anna Aakula, Suvi-Katri Leivonen, Pekka Kohonen, Petteri Hintsanen, Tero Aittokallio, Yvonne Ceder, Anne-Lise Børresen-Dale, Merja Perälä, Päivi Östling, Olli Kallioniemi. MicroRNA-135b influences androgen and estrogen receptor protein levels and regulates proliferation of hormone-receptor positive prostate and breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2013-4170

Evaluation of the prognostic significance of androgen receptor expression in patients with breast cancer

To evaluate the prognostic significance of androgen receptor (AR) expression in patients with breast cancer. The clinicopathological data of 184 consecutive female patients with primary breast cancer treated from January 2003 to June 2003 were analyzed retrospectively. The expression of estrogen receptor (ER), progesterone receptor (PR), AR and c-erbB2 protein were assessed in breast cancer tissue before chemotherapy by using immunohistochemistry methods. The correlations between AR expression and lymph node status, c-erbB2 expression in patients who were classified as two subsets according to the lymph node status and c-erbB2 over-expression and the prognostic significance of AR expression were analyzed. All the 184 patients were followed up for more than 5 years. The lymph node involvement, 5-year metastasis and 5-year survival rate were 45.1%, 16.9% and 86.4%, respectively. The 184 patients were classified as two subsets of node-negative and node-positive group. The Chi-square test showed an lowly inverse correlation between AR and lymph node status (P<0.05, rs=-0.236), AR expression rate was lower in patients with positive nodes. The Kaplan-Meier test showed that AR expression had no influence on prognosis in lymph node-negative patients. In patients with positive nodes, the ones with AR-positive tumors had significantly better survival rate (P<0.05), and the 5-year survival rate and the median survival time of patients with AR-positive tumors were significantly better than those of patients with AR-negative tumors (89.3% vs. 67.3% and 56 months vs. 37 months). When the patients were classified as two subsets: c-erbB2-negative and c-erbB2-positive, it indicated that AR expression had on impact on prognosis in c-erbB2-negative patients; and in c-erbB2-positive patients, the survival status was significantly better in patients with AR-positive breast cancer, the 5-year survival rate and the median survival time of patients with AR-positive tumors were significantly better than those of patients with AR-negative tumors (80.0% vs. 57.1% and 57 months vs. 37 months). The patients with positive lymph nodes and positive c-erbB2 expression have a poorer prognosis in the female breast cancer patients, and the expression of AR has a prognostic significance in those patients. The patients with AR-positive tumors have a better prognosis than these with AR-negative tumors.

A New Hormonal Therapy for Estrogen Receptor–Negative Breast Cancer

We postulate that the androgen dehydroepiandrosterone sulfate (DHEAS) may represent an innovative hormonal treatment for estrogen (ER), progesterone (PR) receptor-negative, but androgen receptor (AR)-positive breast cancers by inhibiting breast cancer cell growth through AR stimulation. Three ER,PR-negative breast cancer cell lines (HCC 1137, 1954, and 38), were treated with DHEAS. DHEAS-induced growth was measured by a methylthiotetrazole (MTT) proliferation assay and apoptosis by TUNEL fluorescence. Androgen receptor gene expression levels were determined using quantitative real-time polymerase chain reaction (q-RT-PCR). HCC cell lines 1954 and 1937 were positive for AR expression; HCC 38 was weakly positive. MTT analysis showed DHEAS-induced decreases in cell proliferation of 47% in HCC 1937, 27% in HCC 1954, and 0.4% in HCC 38. Ten days of culturing HCC 1954 cells after the removal of DHEAS resulted in a 3.5-fold increase in growth. Continuous treatment for the same duration induced a 2.8-fold decrease in growth. Parallel experiments showed no significant changes in HCC 38 cultures. TUNEL assays showed DHEAS-induced apoptosis fold increases of 2.8 in HCC 1937, 1.9 in HCC 1954, and no significant difference in HCC 38 cultures. Q-RT-PCR of HCC 1954 cells showed a 6-fold DHEAS-induced decrease in AR gene expression at 4 h. Co-treatment with Casodex nullified this effect. DHEAS inhibited growth of ER,PR-negative, AR-positive breast cancer cells. DHEAS was cytotoxic to these breast cancer cells via the apoptosis pathway. DHEAS may be an effective treatment for a population previously excluded from hormone therapy.