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Supplementary Methods from Loss of Phosphatase and Tensin Homologue Deleted on Chromosome 10 Engages ErbB3 and Insulin-Like Growth Factor-I Receptor Signaling to Promote Antiestrogen Resistance in Breast Cancer

Knockdown of the tumor suppressor phosphatase Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) with shRNA in three estrogen receptor (ER)-positive breast cancer cell lines resulted in increased phosphatidylinositol-3 kinase (PI3K) and AKT activities, resistance to tamoxifen and fulvestrant, and hormone-independent growth. PTEN knockdown induced the up-regulation of ER transcriptional activity in MCF-7 cells but decreased ER protein levels and transcriptional activity in T47D and MDA-361 cells. Tamoxifen and fulvestrant treatment inhibited estradiol-induced ER transcriptional activity in all shPTEN cell lines but did not abrogate the increased cell proliferation induced by PTEN knockdown. PTEN knockdown increased basal and ligand-induced activation of the insulin-like growth factor-I (IGF-I) and ErbB3 receptor tyrosine kinases, and prolonged the association of the p85 PI3K subunit with the IGF-I receptor (IGF-IR) effector insulin receptor substrate-1 and with ErbB3, implicating PTEN in the modulation of signaling upstream of PI3K. Consistent with these data, PTEN levels inversely correlated with levels of tyrosine-phosphorylated IGF-IR in tissue lysate arrays of primary breast cancers. Inhibition of IGF-IR and/or ErbB2-mediated activation of ErbB3 with tyrosine kinase inhibitors restored hormone dependence and the growth inhibitory effect of tamoxifen and fulvestrant on shPTEN cells, suggesting that cotargeting both ER and receptor tyrosine kinase pathways holds promise for the treatment of patients with ER+, PTEN-deficient breast cancers. [Cancer Res 2009;69(10):4192–201]

Supplementary Figures 1-8 from Loss of Phosphatase and Tensin Homologue Deleted on Chromosome 10 Engages ErbB3 and Insulin-Like Growth Factor-I Receptor Signaling to Promote Antiestrogen Resistance in Breast Cancer

PurposeThe protein tyrosine phosphatase PTPN2 dephosphorylates several tyrosine kinases in cancer-related signalling pathways and is thought to be a tumour suppressor. As PTPN2 is not frequently studied in breast cancer, we aimed to explore the role of PTPN2 and the effects of its loss in breast cancer.MethodsProtein expression and gene copy number of PTPN2 were analysed in a cohort of pre-menopausal breast cancer patients with immunohistochemistry and droplet digital PCR, respectively. PTPN2 was knocked down in three cell lines, representing different breast cancer subtypes, with siRNA transfection. Several proteins related to PTPN2 were analysed with Western blot.ResultsLow PTPN2 protein expression was found in 50.2% of the tumours (110/219), gene copy loss in 15.4% (33/214). Low protein expression was associated with a higher relapse rate in patients with Luminal A and HER2-positive tumours, but not triple-negative tumours. In vitro studies further suggested a subtype-specific role of PTPN2. Knockdown of PTPN2 had no effect on the triple-negative cell line, whilst knockdown in MCF7 inhibited phosphorylation of Met and promoted that of Akt. Knockdown in SKBR3 led to increased Met phosphorylation and decreased Erk phosphorylation as well as EGF-mediated STAT3 activation.ConclusionWe confirm previous studies showing that the PTPN2 protein is lost in half of the breast cancer cases and gene deletion occurs in 15-18% of the cases. Furthermore, the results suggest that the role of PTPN2 is subtype-related and should be further investigated to assess how this could affect breast cancer prognosis and treatment response. Funding Agencies|Swedish Cancer Society; ALF Grants, Region Ostergotland; LiU Cancer Foundation; Cancer Research Foundations of Radiumhemmet; Onkologiska Klinikernas i Linkoping Forskningsfond

BackgroundBreast cancer tumors are heterogenous, including their metastatic potential and time to distant metastasis. Patients with estrogen receptor (ER)-positive tumors have a continuous long-term risk of fatal disease decades after diagnosis. Recently, ER-positive breast cancer patients classified as having ultralow risk tumors by the 70-gene expression signature (MammaPrint) were associated with a minimal risk of fatal disease. However, the underlying tumor characteristics of ultralow risk tumors are unknown.MethodsSecondary analysis of the Stockholm tamoxifen randomized trial (STO-3, 1976-1990) enrolling postmenopausal lymph node-negative breast cancer patients. Immunohistochemistry of the clinically used breast cancer markers (n=727 patients) and gene expression profiling by Agilent microarrays (n=652 patients) were performed in 2014. Ultralow risk tumors, identified by the 70-gene signature, were compared to other ER-positive tumors (of low/high risk) and Luminal A and B PAM50 subtype tumors (ER-positive, low/high risk) by the clinical markers and multi-gene modules, representative of specific biological processes and pathways, using Fisher’s exact test. Furthermore, differential gene expression analysis was performed contrasting ultralow risk tumors to other ER-positive tumors (of low/high risk) using t-statistics and false discovery rate (FDR).ResultsUltralow risk tumors were significantly (P

SUMMARY Overactivation of the PI3K/AKT signaling pathway is frequently reported in breast cancer, consequently inhibitors targeting this pathway are clinically useful. AKT constitutes a hub in the regulation of several cancer hallmarks, such as proliferation, survival and migration. Three AKT isoforms, named AKT1, AKT2 and AKT3, are identified in humans. AKT alterations, mainly upregulation of phosphorylated AKT in tumors may have prognostic and predictive value. Moreover, the AKT isoforms may possess partly divergent cellular functions and be upregulated in certain breast cancer subtypes, suggesting the importance of isoform-specific analyses. In conclusion, AKT isoform-specific detection and targeting in different tumor subtypes will hopefully result into a further developed personalized medicine.

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

Knockdown of the tumor suppressor phosphatase Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) with shRNA in three estrogen receptor (ER)-positive breast cancer cell lines resulted in increased phosphatidylinositol-3 kinase (PI3K) and AKT activities, resistance to tamoxifen and fulvestrant, and hormone-independent growth. PTEN knockdown induced the up-regulation of ER transcriptional activity in MCF-7 cells but decreased ER protein levels and transcriptional activity in T47D and MDA-361 cells. Tamoxifen and fulvestrant treatment inhibited estradiol-induced ER transcriptional activity in all shPTEN cell lines but did not abrogate the increased cell proliferation induced by PTEN knockdown. PTEN knockdown increased basal and ligand-induced activation of the insulin-like growth factor-I (IGF-I) and ErbB3 receptor tyrosine kinases, and prolonged the association of the p85 PI3K subunit with the IGF-I receptor (IGF-IR) effector insulin receptor substrate-1 and with ErbB3, implicating PTEN in the modulation of signaling upstream of PI3K. Consistent with these data, PTEN levels inversely correlated with levels of tyrosine-phosphorylated IGF-IR in tissue lysate arrays of primary breast cancers. Inhibition of IGF-IR and/or ErbB2-mediated activation of ErbB3 with tyrosine kinase inhibitors restored hormone dependence and the growth inhibitory effect of tamoxifen and fulvestrant on shPTEN cells, suggesting that cotargeting both ER and receptor tyrosine kinase pathways holds promise for the treatment of patients with ER+, PTEN-deficient breast cancers. [Cancer Res 2009;69(10):4192–201]

// Cynthia Veenstra 1 , Gizeh Perez-Tenorio 1 , Anna Stelling 1 , Elin Karlsson 1 , Sanam Mirwani Mirwani 1 , Bo Nordenskoljd 1 , Tommy Fornander 2 , Olle Stal 1 1 Department of Clinical and Experimental Medicine and Department of Oncology, Linkoping University, Linkoping, Sweden 2 Department of Oncology-Pathology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Correspondence to: Cynthia Veenstra, email: cynthia.veenstra@liu.se Keywords: radiation, copy number variation, droplet digital PCR, triple-negative breast cancer, radiotherapy Received: February 24, 2016 Accepted: April 26, 2016 Published: May 10, 2016 ABSTRACT Few biomarkers exist to predict radiotherapy response in breast cancer. In vitro studies suggest a role for Met and its ligand HGF. To study this suggested role, MET and HGF gene copy numbers were determined by droplet digital PCR in tumours from 205 pre-menopausal and 184 post-menopausal patients, both cohorts randomised to receive either chemo- or radiotherapy. MET amplification was found in 8% of the patients in both cohorts and HGF amplification in 7% and 6% of the patients in the pre- and post-menopausal cohort, respectively. Met, phosphorylated Met (pMet), and HGF protein expression was determined by immunohistochemistry in the pre-menopausal cohort. Met, pMet, and HGF was expressed in 33%, 53%, and 49% of the tumours, respectively. MET amplification was associated with increased risk of distant recurrence for patients receiving chemotherapy. For the pre-menopausal patients, expression of cytoplasmic pMet and HGF significantly predicted benefit from radiotherapy in terms of loco-regional recurrence. Similar trends were seen for MET and HGF copy gain. In the post-menopausal cohort, no significant association of benefit from radiotherapy with neither genes nor proteins was found. The present results do not support that inhibition of Met prior to radiotherapy would be favourable for pre-menopausal breast cancer, as previously suggested.

// Anna-Maria Husa 1, 2 , Željana Magic 1 , Malin Larsson 3 , Tommy Fornander 4 , Gizeh Perez-Tenorio 1 1 Department of Clinical and Experimental Medicine, Division of Oncology, Linkoping University, Linkoping, Sweden 2 Current address: CCRI, Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung e.V., Vienna, Austria 3 Bioinformatics Infrastructure for Life Sciences (BILS) and Department of Physics, Chemistry and Biology, Linkoping University, Linkoping, Sweden 4 Department of Oncology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Correspondence to: Gizeh Perez-Tenorio, e-mail: gizeh.perez-tenorio@liu.se Keywords: EPHB2, EPH family, TaqMan array, gene expression, protein expression Received: July 03, 2015 Accepted: January 23, 2016 Published: February 8, 2016 ABSTRACT The EPH and ephrins function as both receptor and ligands and the output on their complex signaling is currently investigated in cancer. Previous work shows that some EPH family members have clinical value in breast cancer, suggesting that this family could be a source of novel clinical targets. Here we quantified the mRNA expression levels of EPH receptors and their ligands, ephrins, in 65 node positive breast cancer samples by RT-PCR with TaqMan ® Micro Fluidics Cards Microarray. Upon hierarchical clustering of the mRNA expression levels, we identified a subgroup of patients with high expression, and poor clinical outcome. EPHA2, EPHA4, EFNB1, EFNB2, EPHB2 and EPHB6 were significantly correlated with the cluster groups and particularly EPHB2 was an independent prognostic factor in multivariate analysis and in four public databases. The EPHB2 protein expression was also analyzed by immunohistochemistry in paraffin embedded material (cohort 2). EPHB2 was detected in the membrane and cytoplasmic cell compartments and there was an inverse correlation between membranous and cytoplasmic EPHB2. Membranous EPHB2 predicted longer breast cancer survival in both univariate and multivariate analysis while cytoplasmic EPHB2 indicated shorter breast cancer survival in univariate analysis. Concluding: the EPH/EFN cluster analysis revealed that high EPH/EFN mRNA expression is an independent prognostic factor for poor survival. Especially EPHB2 predicted poor breast cancer survival in several materials and EPHB2 protein expression has also prognostic value depending on cell localization.

Purpose: The phosphatidylinositol 3′-kinase/Akt pathway is frequently altered in breast cancer. PTEN, a phosphatase that opposes the effect of phosphatidylinositol 3′-kinase, can be mutated or lost, whereas the PIK3CA gene is mutated. These have been proposed as alternative mechanisms, and their clinicalpathology significance is under discussion. In this study, we aimed to explore whether PIK3CA mutations and PTEN loss are mutually exclusive mechanisms, correlate with other known clinicopathologic markers, or have clinical implication in breast cancer. Experimental Design: Exons 9 and 20 of the PIK3CA gene were analyzed in 270 breast tumors, and mutations were detected by single-stranded conformational analysis followed by sequencing. The expression of PTEN was evaluated by immunohistochemistry in 201 tumors. Results: PIK3CA mutations were found in 24% of the tumors and associated with estrogen receptor+ status, small size, negative HER2 status, high Akt1, and high cyclin D1 protein expression. PTEN was negative in 37% of the cases and PTEN loss was associated with PIK3CA mutations (P = 0.0024). Tumors presenting PTEN loss or both alterations were often estrogen receptor+, small in size, and HER2−. PIK3CA mutations predicted for longer local recurrence-free survival. Moreover, PTEN loss by itself or combined with mutated PIK3CA tended to confer radiosensitivity. In addition, the patients with high S-phase fraction had longer recurrence-free survival if they carried mutations in the PIK3CA gene and/or had lost PTEN, whereas the same alterations were associated with shorter recurrence-free survival among patients with low S-phase fraction. Conclusions: PIK3CA mutations and PTEN loss were not mutually exclusive events and associated with similar prognostic factors.

Detection of signals in the mammalian target of rapamycin (mTOR) and the estrogen receptor (ER) pathways may be a future clinical tool for the prediction of adjuvant treatment response in primary breast cancer. Using immunohistological staining, we investigated the value of the mTOR targets p70-S6 kinase (S6K) 1 and 2 as biomarkers for tamoxifen benefit in two independent clinical trials comparing adjuvant tamoxifen with no tamoxifen or 5 years versus 2 years of tamoxifen treatment. In addition, the prognostic value of the S6Ks was evaluated. We found that S6K1 correlated with proliferation, HER2 status, and cytoplasmic AKT activity, whereas high protein expression levels of S6K2 and phosphorylated (p) S6K were more common in ER-positive, and low-proliferative tumors with pAKT-s473 localized to the nucelus. Nuclear accumulation of S6K1 was indicative of a reduced tamoxifen effect (hazard ratio (HR): 1.07, 95% CI: 0.53–2.81,P=0.84), compared with a significant benefit from tamoxifen treatment in patients without tumor S6K1 nuclear accumulation (HR: 0.42, 95% CI: 0.29–0.62,PP=0.92). In addition, high protein expression of S6K1, independent of localization, predicted worse prognosis in a multivariate analysis,P=0.00041 (cytoplasm),P=0.016 (nucleus). In conclusion, the mTOR-activated kinases S6K1 and S6K2 interfere with proliferation and response to tamoxifen. Monitoring their activity and intracellular localization may provide biomarkers for breast cancer treatment, allowing the identification of a group of patients less likely to benefit from tamoxifen and thus in need of an alternative or additional targeted treatment.

Breast cancer is a heterogeneous disease and new clinical markers are needed to individualise disease management and therapy further. Alterations in the PI3K/AKT pathway, mainly PIK3CA mutations, have been shown frequently especially in the luminal breast cancer subtypes, suggesting a cross-talk between ER and PI3K/AKT. Aberrant PI3K/AKT signalling has been connected to poor response to anti-oestrogen therapies. In vitro studies have shown protein tyrosine phosphatase, non-receptor type 2 (PTPN2) as a previously unknown negative regulator of the PI3K/AKT pathway. Here, we evaluate possible genomic alterations in the PTPN2 gene and its potential as a new prognostic and treatment predictive marker for endocrine therapy benefit in breast cancer. PTPN2 gene copy number was assessed by real-time PCR in 215 tumour samples from a treatment randomised study consisting of postmenopausal patients diagnosed with stage II breast cancer 1976-1990. Corresponding mRNA expression levels of PTPN2 were evaluated in 86 available samples by the same methodology. Gene copy loss of PTPN2 was detected in 16 % (34/215) of the tumours and this was significantly correlated with lower levels of PTPN2 mRNA. PTPN2 gene loss and lower mRNA levels were associated with activation of AKT and a poor prognosis. Furthermore, PTPN2 gene loss was a significant predictive marker of poor benefit from tamoxifen treatment. In conclusion, genomic loss of PTPN2 may be a previously unknown mechanism of PI3K/AKT upregulation in breast cancer. PTPN2 status is a potential new clinical marker of endocrine treatment benefit which could guide further individualised therapies in breast cancer. Funding Agencies|Swedish Cancer Society [13 0435]; Swedish Research Council [A0346701]

Akt/PKB is a serine/threonine protein kinase that regulates cell cycle progression, apoptosis and growth factor mediated cell survival in association with tyrosine kinase receptors. The protein is a downstream effector of erbB-2 with implications in breast cancer progression and drug resistance in vitro. We aimed to examine the role of Akt-1 in breast cancer patients, by determining whether the expression (Akt-1) and/or activation (pAkt) were related to prognostic markers and survival. The expression of erbB-2, heregulin β1 and Bcl-2 was also assessed by flow cytometry or immunohistochemistry. This study comprised 93 patients, aged

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Introduction]: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. [Methods]: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. [Results]: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. [Conclusions]: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer., This work was supported by grants from the Eugenio Rodríguez Pascual Foundation (2012, to MAP), the Government of Catalonia (2009-SGR283, to AV and MAP), the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R01 DK015556, to JAK), the Red Cooperative Research Thematic Network on Cancer (RTICC) (12/0036/0002 to XRB and 12/0036/0008 to XRB and MAP) and the Spanish Ministry of Health, Fund for Health Research–Institute of Health Carlos III (11/00951 to AU and 12/01528 to MAP).

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

Introduction Akt is a signalling modulator for many cellular processes, including metabolism, cell proliferation, cell survival and cell growth. Three isoforms of Akt have been identified, but only a few studies have concerned the isoform-specific roles in the prognosis of breast cancer patients. The aim of this study was to investigate the prognostic value of v-akt murine thymoma viral oncogene homologue 1 (Akt1) and v-akt murine thymoma viral oncogene homologue 2 (Akt2) in oestrogen receptor positive (ER+) and oestrogen receptor negative (ER–) breast cancer with long-term follow-up. Material and methods The expression of Akt in tumour tissue was analysed with immunohistochemistry in a cohort of 272 postmenopausal patients with stage II breast cancer. The median follow-up time was 19 years. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using the Cox’s proportional hazards model. Results The risk of distant recurrence was reduced for patients with ER+ tumours expressing Akt2 compared to patients with no Akt2 expression (HR = 0.49, 95% CI 0.29–0.82, p = 0.007). When adjusting for important clinical tumour characteristics and treatment, Akt2 was still an independent prognostic factor (HR = 0.38, 95% CI 0.21–0.68, p = 0.001) and the association remained long-term. The prognostic value of Akt2 increased with higher oestrogen receptor levels from no effect among patients with ER– tumours to 68% risk reduction for the group with high ER-levels (P for trend = 0.042). Akt1 showed no significant prognostic information. Conclusion Our results indicate that Akt2 expression is associated with a lower distant recurrence rate for patients with ER+ tumours and that this association remains long-term. The prognostic value of Akt2 increases with higher oestrogen receptor expression, motivating further mechanistic studies on the role of Akt2 in ER+ breast cancer. Funding Agencies|Swedish Cancer Society||Swedish Research Council

The mammalian target of rapamycin (mTOR) and its substrates S6K1 and S6K2 regulate cell growth, proliferation, and metabolism through translational control. RPS6KB1 (S6K1) and RPS6KB2 (S6K2) are situated in the commonly amplified 17q21-23 and 11q13 regions. S6K1 amplification and protein overexpression have earlier been associated with a worse outcome in breast cancer, but information regarding S6K2 is scarce. The aim of this study was to evaluate the prognostic and treatment predictive relevance of S6K1/S6K2 gene amplification, as well as S6K2 protein expression in breast cancer. S6K1/S6K2 gene copy number was determined by real-time PCR in 207 stage II breast tumors and S6K2 protein expression was investigated by immunohistochemistry in 792 node-negative breast cancers. S6K1 amplification/gain was detected in 10.7%/21.4% and S6K2 amplification/gain in 4.3%/21.3% of the tumors. S6K2 protein was detected in the nucleus (38%) and cytoplasm (76%) of the tumor cells. S6K1 amplification was significantly associated with HER2 gene amplification and protein expression. S6K2 amplification correlated significantly with high S6K2 mRNA levels, ER+ status and CCND1 amplification. S6K1 and S6K2 gene amplification was associated with a worse prognosis independent of HER2 and CCND1. S6K2 gain and nuclear S6K2 expression was related to an improved benefit from tamoxifen among patients with ER+, respectively ER+/PgR+ tumors. In the ER+/PgR- subgroup, nuclear S6K2 rather indicated decreased tamoxifen responsiveness. S6K1 amplification predicted reduced benefit from radiotherapy. This is the first study showing that S6K2 amplification and overexpression, like S6K1 amplification, have prognostic and treatment predictive significance in breast cancer. The original publication is available at www.springerlink.com: Gizeh Perez-Tenorio, Elin Karlsson, Marie Ahnström, Birgit Olsson, Birgitta Holmlund, Bo Nordenskjöld, Tommy Fornander, Lambert Skoog and Olle Stål, Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer, 2011, Breast Cancer Research and Treatment, (128), 3, 713-723. http://dx.doi.org/10.1007/s10549-010-1058-x Copyright: Springer Science Business Media http://www.springerlink.com/

P21WAF1/Cip1 (p21) translocates to the cytoplasm inducing cell cycle progression and survival upon Akt/PKB activation. We studied whether heregulin beta1 (HRGbeta1), that activates the PI3K/Akt and MAPK pathways, also misallocates p21. We also explored whether HRGbeta1 interferes with the effects of tamoxifen. The clinical material studied helped us to clarify whether p21 was associated with phosphorylated Akt, recurrence-free survival and response to tamoxifen. MCF-7 cells treated with HRGbeta1 -/+ E2 were analyzed by flow cytometry to observe how the different compounds affected tamoxifen-induced cell cycle arrest and apoptosis. Total cell lysate and nuclear and cytoplasmic fractions were used to detect p21, phospho-Akt and other proteins by Western blotting. Immunofluorescence was used to visualize p21+ cells upon HRGbeta1 and E2 stimulation. The localization of p21 in breast cancer was studied by immunohistochemistry in frozen tumor sections from 280 patients. In MCF-7 we found that HRGbeta1 counteracted the inhibition of p21 expression by tamoxifen and caused p21 cytoplasmic accumulation. HRGbeta1 partially counteracted the cytostatic effect of tamoxifen but abrogated its cytotoxic effect. The clinical material revealed that nuclear p21 (P=0.022) and cytoplasmic p21 (P=0.00001) were associated with phospho-Akt. Based on p21 cell location, we identified 3 subgroups of ER+ patients: the p21N+/C- group for whom tamoxifen was needed otherwise the survival was poor (P=0.0082), the p21N+/C+ or p21N-/C- group, that responded to tamoxifen (P=0.034), and the p21C+/N- group, that might not benefit from this treatment (P=0.7). In conclusion, HRGbeta1 inhibits tamoxifen-induced apoptosis, contributes to p21 cytoplasmic expression while the cellular localization of p21 interacts with the benefit from tamoxifen treatment.

Radiotherapy is widely used in the treatment of breast cancer and reduces the risk of loco-regional recurrence. Overexpression of the erbB2 receptor occurs in 20-30% of all breast cancers, and seems to be involved in chemotherapeutic resistance of breast cancer cells and radioresistance of lung cancer cells. The hypothesis of this study was that erbB2 confers resistance to radiation-induced apoptosis in breast cancer cells through the phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway. Two human breast cancer cell lines were used, BT-474 and MCF-7. BT-474 cells overexpress erbB2 and have mutated p53, while MCF-7 have normal expression of erbB2 and functional p53. The cells were treated with the PI3-K inhibitor wortmannin or the erbB receptor ligand heregulin-beta1, which is expressed by both malignant and stromal cells in vivo. After pharmacological treatment, the cells were irradiated with 10 Gy gamma-radiation. Consistent with the p53 status in the cell lines, gamma-radiation caused G1 arrest in MCF-7 cells, but not in BT-474 cells. 10 Gy gamma-radiation increased apoptosis by on an average 76% (95% CI, 44-109%) in MCF-7. Treatment of MCF-7 with heregulin-beta1 decreased apoptosis by 66% (95% CI, 48-84%) compared to the untreated controls. In BT-474 cells, wortmannin in combination with radiation resulted in 119% (95% CI, 76-161%) more apoptosis compared to wortmannin alone, whereas radiation alone resulted in 45% (95% CI, 15-75%) increased apoptosis. This radiosensitising effect was not seen in MCF-7. Furthermore, transfection of MCF-7 cells with constitutively active Akt made the cells more resistant against apoptosis. Taken together, our results support the hypothesis that the erbB2/PI3-K/Akt signalling pathway is involved in resistance to radiation-induced apoptosis in breast cancer cells in which this signalling pathway is overstimulated.

Introduction: Akt is a signaling modulator for many cellular processes, including metabolism, cell proliferation, cell survival and cell growth. Three isoforms of Akt have been identified, but only a few studies have concerned the isoform-specific roles in the prognosis of breast cancer patients. The aim of this study was to investigate the prognostic value of Akt1 and Akt2 in estrogen receptor positive (ER+) and estrogen receptor negative (ER−) breast cancer with long-term follow-up. Material and Methods: The expression of Akt in tumor tissue was analyzed with immunohistochemistry in a cohort of 272 postmenopausal patients with stage II breast cancer. Hazard ratios and 95% confidence intervals were estimated using the Cox's proportional hazards model. Results: The risk of distant recurrence was reduced for patients with ER+ tumors expressing Akt2 compared to patients with no Akt2 expression (HR = 0.49, 95% CI 0.29–0.82, p = 0.007). When adjusting for important clinical tumor characteristics and treatment, Akt2 was still an independent prognostic factor (HR = 0.38, 95% CI 0.21–0.68, p = 0.001) and the association remained long-term. After more than five years since diagnosis the risk reduction was 57% for patients with Akt2 positive tumors. The prognostic value of Akt2 increased with higher estrogen receptor levels from no effect among patients with ER− tumors to 68% risk reduction for the group with high ER-levels (P for trend= 0.042). Akt1 showed no significant prognostic information. Conclusion: Our results indicate that Akt2 expression is associated with a lower distant recurrence rate for patients with ER+ tumors and that this association remains long-term. The prognostic value of Akt2 increases with higher estrogen receptor expression, motivating further mechanistic studies on the role of Akt2 in ER+ breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-12.

Detecting signals in the mammalian target of rapamycin (mTOR), and the estrogen receptor (ER) pathways for prediction of treatment response may be a future clinical tool in primary breast cancer. Here, we investigated the validity and value of the mTOR targets p70-S6 kinase (S6K) 1 and 2 as biomarkers for tamoxifen sensitivity in vitro and in two independent tamoxifen randomized postmenopausal breast cancer cohorts. In addition, the prognostic value of the S6Ks was evaluated. We found S6K1 to correlate with HER2 and cytoplasmic Akt activity, whereas S6K2 and phosphorylated S6K were closer connected with ER positivity, low proliferation and nucleic p-Akt. Treatment prediction and prognosis were evaluated by immunohistochemical staining. Nuclear accumulation of S6K1 was indicative of a reduced tamoxifen treatment effect, compared with a significant benefit from tamoxifen treatment in patients without tumor S6K1 nuclear accumulation. Patients with a combination of S6K1 nuclear accumulation and S6K2 cytoplasmic accumulation in the tumor cells had no tamoxifen benefit. Also, S6K1 and S6K2 activation, indicated by p-S6K-t389 expression, was associated with low benefit from tamoxifen compared with untreated patients. In addition, high protein expression of S6K1, independent of localization, predicted worse prognosis. This was not evident for variations in S6K2 or p-S6K-t389 expression. In conclusion, the mTOR targeted kinases S6K1 and S6K2 interfere with proliferation and response to tamoxifen. Monitoring their activity and intracellular localization may provide biomarkers for breast cancer treatment, allowing for identification of a group of patients less likely to benefit from tamoxifen and thus in need of an alternative or additional treatment. Citation Format: Josefine Bostner, Elin Karlsson, Cecilia Bivik, Gizeh Perez-Tenorio, Tommy Fornander, Olle Stål. S6 kinase signaling in prognosis and tamoxifen response in two randomized breast cancer cohorts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3295. doi:10.1158/1538-7445.AM2014-3295

The Eph receptors belong to a large family of receptors tyrosine kinase. The Ephs and their ligands ephrins are aberrantly expressed in cancer. However, due their complex signaling and promiscuous interactions, a wide screening of the entire family will be important to determine their clinical value. Purpose: The Eph receptor tyrosine kinase family has been implicated in breast cancer but their role in tumor progression remains elusive. Therefore we aimed to profile the Eph receptors and their ligands ephrins in breast cancer samples to define them as potential clinical markers or therapeutic targets. Experimental design: Gene expression of Eph/ephrins was quantified by RT-PCR using a TaqMan® Micro Fluidics Cards Microarray custom designed for 21 Eph/ephrin family members: EphA1-A8, EphB1-B4 and EphB6, ephrinA1-A5, ephrinB1-B3 and 2 endogenous controls: GAPDH and HPRT1 in 70 breast cancer tumors. Results: We found that high expression of EphBs, and particularly EphB2, was associated to worse outcome among postmenopausal patients with lymph node infiltration. Additionally high EphB2 expression correlated with HER2 positive status. Conclusions: Our results suggest that the EphB receptor family may have prognostic value for postmenopausal breast cancer patients with lymph nodal infiltration. We will continue this study in a larger patient material in order to better evaluate the therapeutic impact of this family. Citation Format: Gizeh Perez-Tenorio, Anna-Maria Husa, Olle Stål. Clinical potential of the Eph/ephrin profile in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2014-3819

INTRODUCTION: mTOR and its downstream effectors the 4E-binding protein 1 (4EBP1) and the p70 ribosomal S6 kinases (S6K1 and S6K2) are frequently upregulated in breast cancer, and assumed to be driving forces in tumourigenesis, in close connection with oestrogen receptor (ER) networks. Here, we investigated these factors as clinical markers in five different cohorts of breast cancer patients. METHODS: The prognostic significance of 4EBP1, S6K1 and S6K2 mRNA expression was assessed with real-time PCR in 93 tumours from the treatment randomised Stockholm trials, encompassing postmenopausal patients enrolled between 1976 and 1990. Three publicly available breast cancer cohorts were used to confirm the results. Furthermore, the predictive values of 4EBP1 and p4EBP1_S65 protein expression for both prognosis and endocrine treatment benefit were assessed by immunohistochemical analysis of 912 node-negative breast cancers from the Stockholm trials. RESULTS: S6K2 and 4EBP1 mRNA expression levels showed significant correlation and were associated with a poor outcome in all cohorts investigated. 4EBP1 protein was confirmed as an independent prognostic factor, especially in progesterone receptor (PgR)-expressing cancers. 4EBP1 protein expression was also associated with a poor response to endocrine treatment in the ER/PgR positive group. Cross-talk to genomic as well as non-genomic ER/PgR signalling may be involved and the results further support a combination of ER and mTOR signalling targeted therapies. CONCLUSION: This study suggests S6K2 and 4EBP1 as important factors for breast tumourigenesis, interplaying with hormone receptor signalling. We propose S6K2 and 4EBP1 as new potential clinical markers for prognosis and endocrine therapy response in breast cancer.

The c-Met receptor promotes cell survival and proliferation by activating signalling pathways. c-Met is abundant in several cancers and in some cases it interferes with treatment response. In vitro, c-Met has been shown to be up-regulated and activated after radiation treatment, which renders the cells resistant to apoptosis and radiation. Therefore, it was hypothesised that high c-Met expression could influence response to radiation in breast cancer. In this study, it was aimed to determine the number of MET gene copies and c-Met protein expression in breast tumours. Tumours of 172 post-menopausal patients with primary breast cancer were analysed for MET using qPCR. Increased MET copy number (three or more copies) was found in 34% of the tumours. Five or more gene copies were found in 4.7% of the patients and indicated a significantly higher risk of developing distant metastasis. For the patients that received radiotherapy as the only postoperative treatment, the number of MET copies was associated with increased risk of distant metastasis. In terms of loco-regional recurrence, it was found that increased MET copy number was associated with worse response to postoperative radiation therapy compared with chemotherapy. c-Met protein analysis by immunohistochemistry in tumours of 208 pre-menopausal patients with primary breast cancer revealed that patients with low expression of c-Met responded better to radiotherapy compared with patients with a high c-Met expression. These results suggest that radiotherapy in combination with a Met inhibitor may be an option for patients with increased c-Met expression, to overcome radio-resistance. Citation Format: Cynthia Veenstra, Gizeh Pérez-Tenorio, Tommy Fornander, Lambert Skoog, Bo Nordenskjöld, Olle Stål. c-Met reduces response to radiation in breast cancer. [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 427. doi:10.1158/1538-7445.AM2013-427

The EphB4 is a tyrosine kinase receptor with both tumor suppressor and promoting activities. The role of EphB4 in breast cancer is not fully elucidated. EphB-receptors transmit forward and reverse signals, which together are known as bidirectional signals. EphB4 reverse signaling, mediated by the ephrin-B2 ligand expressed in endothelial cells, causes some of the oncogenic properties of the receptor. In contrast, EphB4 forward signaling suppresses tumor growth. In breast cancers with high EphB4-receptor expression the forward signaling activity is low due to low expression of ephrin-B2. In fact, the EFNB2 gene, encoding ephrin-B2, is located on chromosome 13, which is lost in more than 30% of breast cancers. Our hypothesis was that ephrin-B2 re-expression in tumors could restore EphB4-forward signal inhibiting tumor growth. To examine the effects of EphB4-ephrin-B2 co-expression in vitro, we used lentiviral vectors to stably infect MCF7 cells with ephrin-B2. We found in vitro that ephrin-B2 expression caused cell detachment, inhibited cell proliferation; focus formation and cell motility, all characteristics of tumor suppression. Likewise, in patient materials we observed that high ephrin-B2 mRNA levels correlated with better distant recurrence-free survival. Interestingly, low ephrin-B2 expression identifies the group of worse prognosis among patients with high EphB4 and HER-2 expression in terms of distant recurrence and breast cancer survival. Taken together these results support our hypothesis and suggest that ephrin-B2 may be a tumor suppressor and a novel prognostic marker in breast cancer. Citation Format: Gizeh Perez-Tenorio, Olle Stål, Elena B. Pasquale. Ephrin B2 as a tumor suppressor in breast cancer. [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 1981. doi:10.1158/1538-7445.AM2013-1981

The hepatocyte growth factor receptor (HGFR), also known as the Met receptor, promotes cell survival and proliferation by activating signaling pathways such as the PI3K/Akt and RAS/MAPK pathway. HGFR, coded by MET, has been shown to be abundant in several cancers and in some cases it interferes with treatment response. In vitro, HGFR has been shown to be upregulated and activated after radiotherapy, increasing the protection from apoptosis and thus increasing radiation resistance. In this study, it was aimed to determine the number of MET gene copies in breast tumors and correlate these findings with different clinicopathological parameters that were available in this material. Tumors of 172 patients with primary breast cancer were analyzed for MET using qPCR. Increased MET copy number (three or more copies) was found in 34% of the tumors. Five or more gene copies were found in 4.7% of the patients and indicated a significantly higher risk of developing distant metastasis. For the patients that received radiotherapy as the only postoperative treatment, the number of MET copies was associated with increased risk of distant metastasis. In terms of locoregional recurrence, it was found that increased MET copy number was associated with worse response to postoperative radiation therapy compared with chemotherapy. These results suggest that radiotherapy in combination with a Met inhibitor might be an option for patients with increased MET gene copy number to overcome radiation resistance.

Background The serine/threonine kinase Akt, or protein kinase B, has recently been a focus of interest because of its activity to inhibit apoptosis. It mediates cell survival by acting as a transducer of signals from growth factor receptors that activate phosphatidylinositol 3-kinase. Methods We analysed the expression of the isoforms Akt1 and Akt2 as well as phosphorylated Akt (pAkt) by immunohistochemistry in frozen tumour samples from 280 postmenopausal patients who participated in a randomised trial comparing cyclophosphamide–methotrexate–5-fluorouracil chemotherapy and postoperative radiotherapy. The patients were simultaneously randomised to tamoxifen or to no endocrine treatment. Results Marked staining was found in 24% of the tumours for Akt1, but in only 4% for Akt2. A low frequency of Akt2-positive cells (1–10%) was observed in another 26% of the tumours. pAkt was significantly associated with both Akt1 and Akt2 expression. Overexpression of erbB2 correlated significantly with pAkt (P = 0.0028). The benefit from tamoxifen was analysed in oestrogen receptor (ER)-positive patients. Patients with a negative status of Akt (no overexpression of Akt1, Akt2 or pAkt) showed significant benefit from tamoxifen. The relative rate of distant recurrence, with versus without tamoxifen, was 0.44 (95% confidence interval [CI], 0.25–0.79) for ER+/Akt1- patients, while it was 0.72 (95% CI, 0.34–1.53) for ER+/Akt1+ patients. The difference in rate ratio did not reach statistical significance. The rate of locoregional recurrence was significantly decreased with radiotherapy versus chemotherapy for Akt-negative patients (rate ratio, 0.23; 95% CI, 0.08–0.67; P = 0.0074), while no benefit was evident for the Akt-positive subgroup (rate ratio, 0.77; 95% CI, 0.31–1.9; P = 0.58). The interaction between Akt and the efficacy of radiotherapy was significant in multivariate analysis (P = 0.042). Conclusion Activation of the Akt pathway is correlated with erbB2 overexpression in breast cancer. The results suggest that Akt may predict the local control benefit from radiotherapy.