Transcriptional Profiling of Breast Cancer Metastases Identifies Liver Metastasis-Selective Genes Associated with Adverse Outcome in Luminal A Primary Breast Cancer.

PURPOSE: The complete molecular basis of the organ-specificity of metastasis is elusive. This study aimed to provide an independent characterization of the transcriptional landscape of breast cancer metastases with the specific objective to identify liver metastasis-selective genes of prognostic importance following primary tumor diagnosis. EXPERIMENTAL DESIGN: A cohort of 304 women with advanced breast cancer was studied. Associations between the site of recurrence and clinicopathologic features were investigated. Fine-needle aspirates of metastases (n = 91) were subjected to whole-genome transcriptional profiling. Liver metastasis-selective genes were identified by significance analysis of microarray (SAM) analyses and independently validated in external datasets. Finally, the prognostic relevance of the liver metastasis-selective genes in primary breast cancer was tested. RESULTS: Liver relapse was associated with estrogen receptor (ER) expression (P = 0.002), luminal B subtype (P = 0.01), and was prognostic for an inferior postrelapse survival (P = 0.01). The major variation in the transcriptional landscape of metastases was also associated with ER expression and molecular subtype. However, liver metastases displayed unique transcriptional fingerprints, characterized by downregulation of extracellular matrix (i.e., stromal) genes. Importantly, we identified a 17-gene liver metastasis-selective signature, which was significantly and independently prognostic for shorter relapse-free (P < 0.001) and overall (P = 0.001) survival in ER-positive tumors. Remarkably, this signature remained independently prognostic for shorter relapse-free survival (P = 0.001) among luminal A tumors. CONCLUSIONS: Extracellular matrix (stromal) genes can be used to partition breast cancer by site of relapse and may be used to further refine prognostication in ER positive primary breast cancer.

Impact of the first tumor response at eight weeks on overall survival in metastatic breast cancer patients treated with first-line combination chemotherapy.

The aim of this was to determine whether the change of size observed at the first response evaluation after initiation of first-line combination chemotherapy correlates with overall survival (OS) in patients with metastatic breast cancer (MBC). The change in size of tumors derived from measurements according to Response Evaluation Criteria In Solid Tumors (RECIST) at the first evaluation on computed tomography (CT) was obtained from a multicenter, randomized phase III trial ("TEX trial," n = 287) comparing treatment with a combination of epirubicin and paclitaxel alone or with capecitabine (TEX). Cox regression and Kaplan-Meier analyses were performed to evaluate the correlations between the first change in tumor size, response according to RECIST and OS. Data from CT evaluations of 233 patients were available. Appearance of new lesions or progression of non-target lesions (new/non-target) indicated short OS by univariable regression analysis (HR 3.76, 95 % CI 1.90-7.42, p < 0.001). A decrease by >30 % at this early time point was prognostic favorable (HR 0.69, 95 % CI 0.49-0.98, p = 0.04) and not significantly less than the best overall response according to RECIST. After adjustment for previous adjuvant treatment and the treatment given within the frame of the randomized trial, OS was still significantly shorter in patients with new/non-target lesions after a median 8 weeks of treatment (HR 4.41, 95 % CI 2.74-7.11, p < 0.001). Disease progression at the first evaluation correlates with OS in patients with MBC treated with first-line combination chemotherapy. The main reason for early disease progression was the appearance of new lesions or progression of non-target lesions. These patients had poor OS even though more lines of treatment were available. Thus, these factors should be focused on in the response evaluations besides tumor size changes.

Inhibitory effect of opiates on LPS mediated release of TNF and IL-8.

Most patients with advanced cancer experience severe pain and are often treated with opiates. Cancer patients are especially susceptible to opportunistic infections due to treatment with immunosuppressive and cytostatic drugs. Since opiates have been demonstrated to have immunomodulatory effects, it is of clinical importance to evaluate potential differences between commonly used opiates with regard to their effect on the immune system. The aim of this study was to evaluate the effect of morphine, tramadol, fentanyl and ketobemidone on the functioning of the immune system with special reference to TNF and IL-8 release. Method. U-937 cells were preincubated with different concentrations of opioids followed by stimulation with LPS 100 µg/ml for three hours. The effect of opioids on the levels of cytokine mRNA was studied using RT-PCR. Erk and Akt phosphorylation was also measured by Western blot. Results. All opioids with the exception of fentanyl were capable of inhibiting TNF release from U-937 cells. Morphine had no effect on IL-8 release but the effect of other opiates was almost the same as the effect on TNF. All opioids with the exception of fentanyl were capable of inhibiting production of mRNA for TNF and IL-8. The observed effects of opiates were not always reversible by naloxone, suggesting that the effects might be mediated by other receptors or through a non-receptor mediated direct effect. Although preliminary evidence suggests the involvement of Erk and Akt pathways, further studies are needed to unravel the intracellular pathways involved in mediating the effects of opiates. Our data suggests that the order of potency with regard to inhibition of cytokine release is as follows: tramadol > ketobemidone > morphine > fentanyl. Conclusion. Further studies are needed to understand the clinical implications of the observed immunosuppressive effects of tramadol and ketobemidone and to improve opioid treatment strategies in patients with 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.

Irreversible pan-ERBB inhibitor canertinib elicits anti-leukaemic effects and induces the regression of FLT3-ITD transformed cells in mice.

Recent findings have indicated that tyrosine kinase inhibitors (TKIs) targeting the ERBB receptor family display anti-leukaemic effects, despite the lack of receptor expression on human leukaemic cells. The occurrence of activating mutations in the gene encoding FMS-like tyrosine kinase 3 (FLT3) in patients with acute myeloid leukaemia (AML) has rendered inhibition of this receptor a promising therapeutic target. Due to possibility of cross-reactivity, we investigated the effect of the irreversible pan-ERBB inhibitor canertinib (CI-1033) on leukaemic cells expressing FLT3. The drug had anti-proliferative and apoptotic effects on primary AML cells and human leukaemic cell lines expressing mutated FLT3. In several AML patient samples, a blast cell population expressing FLT3-internal tandem duplication (ITD) was eradicated by canertinib. Canertinib inhibited receptor autophosphorylation and kinase activity of both mutated and FLT3 ligand stimulated wildtype FLT3, leading to inhibition of the PI3-kinase and MAP kinase pathways. Apoptotic induction was dependent on pro-apoptotic BH3-only protein BCL2L11/BIM because siRNA silencing attenuated apoptosis. Moreover, the drug induced regression of cells expressing FLT3-ITD in a murine in vivo-transplantation model at previously described tolerated doses. These results indicate that canertinib, as an irreversible TKI, could constitute a novel treatment regimen in patients with mutated or overexpressed FLT3.

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.

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.

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 micromol/l, respectively. Cell growth was inhibited at 0.1 micromol/l ZD1839 in both cell lines. Maximal inhibition was accomplished at 10 micromol/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 p27 KIP 1. 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.

Loss of Smad3 in acute T-cell lymphoblastic leukemia.

BACKGROUND: The receptors for transforming growth factor beta (TGF-beta) and their signaling intermediates make up an important tumor-suppressor pathway. The role of one of these intermediates--Smad3--in the pathogenesis of lymphoid neoplasia is unknown. METHODS: We measured Smad3 messenger RNA (mRNA) and protein in leukemia cells obtained at diagnosis from 19 children with acute leukemia, including 10 with T-cell acute lymphoblastic leukemia (ALL), 7 with pre-B-cell ALL, and 2 with acute nonlymphoblastic leukemia (ANLL). All nine exons of the SMAD3 gene (MADH3) were sequenced. Mice in which one or both alleles of Smad3 were inactivated were used to evaluate the role of Smad3 in the response of normal T cells to TGF-beta and in the susceptibility to spontaneous leukemogenesis in mice in which both alleles of the tumor suppressor p27Kip1 were deleted. RESULTS: Smad3 protein was absent in T-cell ALL but present in pre-B-cell ALL and ANLL. No mutations were found in the MADH3 gene in T-cell ALL, and Smad3 mRNA was present in T-cell ALL and normal T cells at similar levels. In mice, the loss of one allele for Smad3 impairs the inhibitory effect of TGF-beta on the proliferation of normal T cells and works in tandem with the homozygous inactivation of p27Kip1 to promote T-cell leukemogenesis. CONCLUSIONS: Loss of Smad3 protein is a specific feature of pediatric T-cell ALL. A reduction in Smad3 expression and the loss of p27Kip1 work synergistically to promote T-cell leukemogenesis in mice.

p21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-beta-mediated G1 arrest through modulation of IL-2 responsiveness.

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8(+) T cells from DKO mice, TGF-beta is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15(Ink4b) in T cells stimulated in the presence of TGF-beta is not essential, as TGF-beta also efficiently suppressed proliferation of T cells from p15(Ink4b-/-) mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-beta, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3(-/-) T cells to the induction of growth arrest by TGF-beta. In summary, the growth suppressive effects of TGF-beta in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-beta by lowering thresholds for a maximal mitogenic response.

Gene expression profiling detects gene amplification and differentiates tumor types in breast cancer.

Global gene expression analysis using microarrays has been used to characterize the molecular profile of tumors. Gene expression variability at the mRNA level can be caused by a number of different events, including novel signaling, downstream activation of transcription enhancers or silencers, somatic mutation, and genetic amplification or deletion. Genomic amplifications are commonly observed in cancer and often include known oncogenes. The tyrosine kinase-type cell surface receptor, ERBB2, is an oncogene located on chromosome 17q21.1 that is amplified in 10-40% of breast tumors. We report for the first time that phenylethanolamine N-methyltransferase (PNMT), proteasome subunit, beta type 3 (PSMB3), ribosomal protein L19 (RPL19), and nuclear receptor subfamily 1, group D, member 1 (NR1D1) are coexpressed with ERBB2 in 34 breast cancer biopsies and also mapped within the same chromosomal location as the ERBB2 gene. Consistent with previous reports, we also observed that the steroidogenic acute regulatory protein-related gene, MLN64, and growth factor receptor bound protein 7 were coexpressed with ERBB2. Coexpression and colocalization of PNMT and MLN64 with ERBB2 suggested that the amplification of ERBB2 includes the chromosomal region harboring these genes. This hypothesis was validated in a subset of 12 biopsies. Gene amplification of ERBB2, PNMT, and MLN64 significantly correlated with increased mRNA gene expression (P < 0.05). These results suggest that gene expression profiling of breast biopsies may become a valuable method for adequately characterizing and choosing treatment modality for patients with breast cancer.