The Akt/mTOR Pathway and Estrogen Receptor Phosphorylations : a crosstalk with potential to predict tamoxifen resistance in breast cancer

Estrogen receptor α content is the primary breast cancer biomarker distinguishing the patients responsive from the non-responsive to endocrine treatments. Tamoxifen is an estrogen competitor with large potential to treat breast cancer patients and prolongs time to recurrence. Despite the estrogen receptor positivity and tamoxifen treatment, many women face recurrence of the disease. An important mechanism of resistance to endocrine treatments is upregulated growth factor signaling, and the subsequent effect on the estrogen receptor, rendering an active receptor that stimulates cell proliferation or reduced estrogen-receptor dependence. This thesis concerns the investigation of biomarkers, as a complement to the existing markers, for determining optimal treatment for patients with primary invasive breast cancer. Randomized patient tumor materials were used in order to measure variations in gene copies, proteins, and protein phosphorylations and to further relate these variations to time-to-recurrence. Endocrine untreated groups within the patient tumor sets gave us the opportunity to study the prognostic potential of selected markers and to compare tamoxifen-treated patients with endocrine untreated, thus obtaining a treatment-predictive value of each marker or marker combination. In endocrine-dependent cancer the 11q13 chromosomal region is frequently amplified, harboring the genes encoding the cell cycle stimulator cyclin D1 and the estrogen receptor phosphorylating kinase Pak1, respectively. Amplification of the genes was associated with reduced time-torecurrence, indicating a prognostic value, whereas PAK1 gene amplification predicted reduced response to tamoxifen treatment. Moreover, the protein expression of Pak1 tended to predict treatment response, which led to the investigation of this protein in a larger cohort. Together with one of its targets, the estrogen receptor phosphorylation at serine 305, Pak1 predicted reduced response to tamoxifen treatment when detected in the nucleus of tumor cells, suggesting activation of this pathway as a mechanism for tamoxifen-treatment resistance. The estrogen receptor is phosphorylated by several growth factor stimulated kinases. The role of serine-167 phosphorylation has been debated, with inconsistent results. To study the biomarker value of this site the upstream activity of Akt, mTOR, and the S6 kinases were analyzed individually and in combinations. As a prognostic factor, serine 167 indicated an improved breast cancer survival, and as a treatment predictive factor we could not detect a significant value of serine 167 as a single marker. However, in combination with serine 305, and Akt/mTOR-pathway activation, the response to tamoxifen treatment was reduced. The mTOR effector protein S6K1 was found to be associated with HER2 positivity and a worse prognosis. In the group of patients with S6K1 accumulation in the tumor cell nuclei, treatment did not prolong time-to-recurrence, similarly as observed with expression of active S6 kinases. In vitro, a simultaneous knockdown of the S6 kinases in estrogen receptor-positive breast cancer cells resulted in G1 arrest, and tamoxifen-induced G1 arrest was in part S6 kinase dependent. The results presented herein suggest biomarkers that would improve treatment decisions in the clinic, specifically for estrogen receptor-positive breast cancer and tamoxifen treatment but in a broader perspective, also for other endocrine treatments and targeted treatments

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 (ER alpha) and influences the response of breast cancer cells to tamoxifen. We investigated the influence of Pak1 and pER alpha(ser305) on breast cancer prognosis and results of tamoxifen therapy. Experimental Design: We examined Pak1 and pER alpha(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 pER alpha(ser305) correlated to small tumors and ER positivity. Nuclear expression of Pak1 and pER alpha(ser305) predicted reduced response to tamoxifen in patients with ER alpha-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 less than 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 pER alpha(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.Original Publication:Josefine Bostner, Lambert Skoog, Tommy Fornander, Bo Nordenskjöld and Olle Stål, Estrogen Receptor-alpha Phosphorylation at Serine 305, Nuclear p21-Activated Kinase 1 Expression, and Response to Tamoxifen in Postmenopausal Breast Cancer, 2010, Clinical Cancer Research, (16), 5, 1624-1633.http://dx.doi.org/10.1158/1078-0432.CCR-09-1733Copyright: American Association for Cancer Research, Inc.http://www.aacr.org

Experimental Medicine

– a crosstalk with potential to predict tamoxifen resistance in breast cance

Raptor localization predicts prognosis and tamoxifen response in estrogen receptor-positive breast cancer

Deregulated PI3K/mTOR signals can promote the growth of breast cancer and contribute to endocrine treatment resistance. This report aims to investigate raptor and its intracellular localization to further understand its role in ER-positive breast cancer. Raptor protein expression was evaluated by immunohistochemistry in 756 primary breast tumors from postmenopausal patients randomized to tamoxifen or no tamoxifen. In vitro, the MCF7 breast cancer cell line and tamoxifen-resistant MCF7 cells were studied to track the raptor signaling changes upon resistance, and raptor localization in ER alpha-positive cell lines was compared with that in ER alpha-negative cell lines. Raptor protein expression in the nucleus was high in ER/PgR-positive and HER2-negative tumors with low grade, features associated with the luminal A subtype. Presence of raptor in the nucleus was connected with ER alpha signaling, here shown by a coupled increase of ER alpha phosphorylation at S167 and S305 with accumulation of nuclear raptor. In addition, the expression of ER alpha-activated gene products correlated with nuclear raptor. Similarly, in vitro we observed raptor in the nucleus of ER alpha-positive, but not of ER-negative cells. Interestingly, raptor localized to the nucleus could still be seen in tamoxifen-resistant MCF7 cells. The clinical benefit from tamoxifen was inversely associated with an increase of nuclear raptor. High cytoplasmic raptor expression indicated worse prognosis on long-term follow-up. We present a connection between raptor localization to the nucleus and ER alpha-positive breast cancer, suggesting raptor as a player in stimulating the growth of the luminal A subtype and a possible target along with endocrine treatment.Funding Agencies|Swedish Cancer Society; Region of Ostergotland; Cancer Society in Stockholm; King Gustav V Jubilee Clinical Research Foundation; National Cancer Institute (NCI); American Cancer Society; Atol Charitable Trust</p

High-Resolution Genomic Analysis of the 11q13 Amplicon in Breast Cancers Identifies Synergy with 8p12 Amplification, Involving the mTOR Targets S6K2 and 4EBP1

The chromosomal region 11q13 is amplified in 15-20% of breast cancers; an event not only associated with estrogen receptor (ER) expression but also implicated in resistance to endocrine therapy. Coamplifications of the 11q13 and 8p12 regions are common, suggesting synergy between the amplicons. The aim was to identify candidate oncogenes in the 11q13 region based on recurrent amplification patterns and correlations to mRNA expression levels. Furthermore, the 11q13/8p12 coamplification and its prognostic value, was evaluated at the DNA and the mRNA levels. Affymetrix 250K NspI arrays were used for whole-genome screening of DNA copy number changes in 29 breast tumors. To identify amplicon cores at 11q13 and 8p12, genomic identification of significant targets in cancer (GISTIC) was applied. The mRNA expression levels of candidate oncogenes in the amplicons [ RAD9A, RPS6KB2 (S6K2), CCND1, FGF19, FGF4, FGF3, PAK1, GAB2 (11q13); EIF4EBP1 (4EBP1), PPAPDC1B, and FGFR1 (8p12)] were evaluated using real-time PCR. Resulting data revealed three main amplification cores at 11q13. ER expression was associated with the central 11q13 amplification core, encompassing CCND1, whereas 8p12 amplification/gene expression correlated to S6K2 in a proximal 11q13 core. Amplification of 8p12 and high expression of 4EBP1 or FGFR1 was associated with a poor outcome in the group. In conclusion, single nucleotide polymorphism arrays have enabled mapping of the 11q13 amplicon in breast tumors with high resolution. A proximal 11q13 core including S6K2 was identified as involved in the coamplification/coexpression with 8p12, suggesting synergy between the mTOR targets S6K2 and 4EBP1 in breast cancer development and progression.Funding Agencies|Swedish Cancer Foundation||Swedish Research Council||</p

Revealing Different Roles of the mTOR-Targets S6K1 and S6K2 in Breast Cancer by Expression Profiling and Structural Analysis

Background The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer. Materials and methods Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1. Results Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.At the time for thesis presentation publication was in status: ManuscriptFunding Agencies|Swedish Research Council [2012-5136, 2007-3475]; Swedish Cancer Foundation; LiU Cancer; LiU Cancer Foundation</p

Open Access

The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer: a retrospective study including patients from the randomised Stockholm tamoxifen trial