Anna-Lotta Hallbeck

Estrogen-induced alterations of spinal cord enkephalin gene expression.

Enkephalin-synthesizing neurons in the superficial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P<0.05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P<0.05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.

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.

TGF-alpha and ErbB2 production in synovial joint tissue: increased expression in arthritic joints.

Objective: Cell types present in synovial joint tissues and during synovitis are known to produce epidermal growth factor receptor (EGFR)/ErbB‐1/HER‐1 and the potent EGFR‐ligand transforming growth factor‐alpha (TGF‐α) in vitro. Concomitant expression of TGF‐α, EGFR, and ErbB2 gives a strong proliferative drive in vitro and in vivo. However, the presence of TGF‐α and members of the EGFR/EGFR‐ligand family has not been thoroughly investigated in joint tissue in vivo. We aimed to determine whether TGF‐α, EGFR, and ErbB2 are present in human synovial joints, especially during rheumatoid arthritis (RA). Methods: TGF‐α protein was immunodetected in knee synovial fluid (SF) collected from 23 RA patients, eight patients with other arthritic conditions, two osteoarthritis (OA) patients, and six post‐traumatic patients (control). TGF‐α mRNA and TGF‐α, ErbB2, EGFR, and CD68 immunoreactivity were detected in knee synovial biopsies (6 RA/2 OA/6 control) using in situ hybridization and immunohistochemistry. TGF‐α mRNA was determined in SF cells by reverse transcription polymerase chain reaction (RT‐PCR) and/or the Northern blot technique. Results: TGF‐α protein was found in the synovial membrane (SM) and in the majority of SF samples. TGF‐α levels were significantly higher (p<0.001) in SF of RA patients than controls, TGF‐α protein and mRNA were increased and more widespread in SM of RA patients. In addition, white blood cells collected from RA SF expressed TGF‐α mRNA. Immunoreactivity for ErbB2 was found in SM and was more widespread in RA patients than in controls. Conclusion: The presence of TGF‐α in normal SF and SM may indicate a physiological maintenance function. The increased expression of TGF‐α and ErbB2 in RA SF and SM may give rise to an abnormal growth pattern, contributing to inflammatory synovial hyperplasia.

ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells : inhibition by gefitinib (ZD1839)

Members of the epidermal growth factor (EGF) family of structurally related tyrosine kinase receptors, known as the ErbB receptors (EGFR/ErbB1/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4) and their respective ligands, have been suggested to be involved in the development and progression of malignant melanoma. Here we investigate the effects of the ErbB1 tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) on human malignant melanoma cells (RaH3 and RaH5) in vitro. ZD1839 inhibited proliferation of exponentially growing RaH3 and RaH5 cells in a dose-dependent manner with a half-maximally effective dose of 3.5 and 2.0 μmol/l, respectively. Cell growth was inhibited at 0.1 μmol/l ZD1839 in both cell lines. Maximal inhibition was accomplished at 10 μmol/l ZD1839; however, the effect was not complete as both cell lines showed a continuous slow growth during the treatment period. Flow cytometry analysis of cell-cycle distribution showed that ZD1839 treatment caused accumulation of RaH3 and RaH5 cells in the G1 phase. The growth arrest induced by ZD1839 coincided with upregulation of the cyclin-dependent kinase inhibitor p27KIP1. There was no increase in apoptosis as determined by analysis of plasma phosphatidyl serine redistribution. Western blot analysis revealed that ZD1839 substantially reduced tyrosine phosphorylation of ErbB1 as well as ErbB2 and ErbB3. This was accompanied by a concomitant decrease in Akt-phosphorylation, Erk1/2-phosphorylation, and Stat3-phosphorylation. Our results show that ZD1839 interferes with the growth of human malignant melanoma cells by cytostatic effects. These findings indicate the possible use of ErbB receptor kinase inhibitors as a novel treatment strategy in malignant melanoma.

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 trials

IntroductionmTOR 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.MethodsThe 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.ResultsS6K2 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.ConclusionThis 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 pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC(50)) was approximately 0.8 μM and by 5 μM both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 μM canertinib accumulated the cells in the G(1)-phase of the cell cycle within 24h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 μM canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses ≤ 5μM canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.

The pan-ErbB receptor tyrosine kinase inhibitor canertinib induces ErbB-independent apoptosis in human leukemia (HL-60 and U-937) cells

Epidermal growth factor (EGF) receptor tyrosine kinase inhibitors have recently been shown to display anti-neoplastic effects in human malignant myeloid cells. Our study was initiated in order to determine the effect of the pan-ErbB receptor tyrosine kinase inhibitor, canertinib (CI-1033), on growth and survival of human leukemia (HL-60 and U-937) cells. We show that treatment of HL-60 and U-937 cells with canertinib significantly inhibits growth of both cell lines in a dose-dependent manner; half maximal effective dose (IC(50)) in HL-60 and U-937 cells was approximately 2.5 microM and 1.0 microM, respectively. Treatment with 2 microM canertinib promoted a G(1) cell cycle arrest, whereas doses of 5 microM or more induced apoptosis as determined by the Annexin V method and cleavage of poly-(ADP-ribose) polymerase (PARP). HL-60 and U-937 cells lacked EGF-receptor transcript but expressed ErbB2-4 mRNA as determined by RT-PCR. However, none of the corresponding ErbB-receptor proteins could be detected by Western blot analysis. We conclude that canertinib induces apoptosis in HL-60 and U-937 cells devoid of functional ErbB1-4 receptors. Our results suggest that canertinib could be of potential clinical interest in the treatment of acute myeloid leukemia.

Interleukin-6 Enhances Transforming Growth Factor-alpha mRNA Expression in Macrophage-Like Human Monocytoid (U-937-1) Cells

We have previously reported that the human monocytoid cell line U-937-1 constitutively expresses transforming growth factor-alpha (TGF-α) and that the steady-state levels of TGF-α mRNA as well as TGF-α protein release increase when U-937-1 cells are differentiated towards monocytes/macrophages. Interleukin-6 (IL-6), which has been shown to have growth-stimulatory effects on a number of cell types, has recently been shown to enhance TGF-α expression in keratinocytes. In the present study we investigated whether TGF-α expression in macrophage-like cells could be regulated by IL-6 using U-937-1 cells as a model system of monocyte/macrophage differentiation.U-937-1 cells were differentiated with retinoic acid (RA), vitamin D3 (Vit-D3) or phorbol-12-myristate-13-acetate (PMA) for 4 days and were then treated with human recombinant IL-6 (1000 IU/ml) for up to 24 hr. Northern blot analysis revealed that cells differentiated with PMA, inducing the phenotype of a secretory macrophage, markedly increased their TGF-α mRNA levels (2.7-fold) when treated with IL-6; the response was maximal at 6 hr and remained high at 12 hr. The expression of the TGF-α gene was accompanied by release of TGF-α protein into the cell culture medium, irrespective of differentiating agent, as demonstrated by enzyme-linked immunosorbent assay (ELISA), as well as by surface expression of pro-TGF-α as determined by indirect immunofluorescent cytometry. However, the superinduction of the TGF-α gene by IL-6 in cells differentiated with PMA was not accompanied by any increase in TGF-α protein release or pro-TGF-α surface expression.We conclude that since IL-6 causes increased steady-state levels of TGF-α mRNA in macrophage-like cells, it may prime these cells for production of this growth factor. Furthermore, we have shown that the IL-6 receptor complex is functional in U-937-1 cells induced to differentiate towards a secretory macrophage by treatment with PMA.

The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells.

Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 μM caused accumulation of Jurkat cells in the G(1) cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

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.