Steven Elliott

Efficiency of signalling through cytokine receptors depends critically on receptor orientation

Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM (ref. 3), 10−2 of its K d value for erythropoietin–EPOR binding site 1 (Kd ≈ 1 nM), and 10−5 of the K d for erythropoietin–EPOR binding site 2 (Kd ≈ 1 μM). Overall half-maximal binding (IC50) of cell-surface receptors is produced with ∼0.18 nM erythropoietin, indicating that only ∼6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses, but much less efficiently, requiring concentrations close to their K d values (∼0.1 μM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 Å from two crystal forms, shows that erythropoietin imposes a unique 120° angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.

Cytokine Receptor CXCR4 Mediates Estrogen-Independent Tumorigenesis, Metastasis, and Resistance to Endocrine Therapy in Human Breast Cancer

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1-CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1-CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1-mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.

Targeting NFĸB mediated breast cancer chemoresistance through selective inhibition of sphingosine kinase-2.

Resistance to chemotherapy remains a significant obstacle in the treatment of hormone- independent breast cancer. Recent evidence suggests that altered sphingolipid signaling through increased sphingosine kinase activity may be an important mediator of breast cancer drug resistance. Sphingosine kinase-1 (Sphk1) is a proposed key regulator of breast cancer tumorigenesis, proliferation and resistance. There is, however, conflicting data on the role of sphingosine kinase-2 (Sphk2) in cancer biology and resistance, with some suggesting that Sphk2 has an opposing role to that of Sphk1. Here, we studied the effects of the novel selective Sphk2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide), on human breast cancer. ABC294640 blocked both viability and survival at low micromolar IC50 concentrations in the endocrine therapy-resistant MDA-MB-231 and chemoresistant MCF-7TN-R cell systems. Treatment with the inhibitor significantly reduced proliferation, as seen in immunofluorescence staining of Ki-67 in vitro. Interestingly, pharmacological inhibition of Sphk2 induced apoptosis through the intrinsic programmed cell death pathway. Furthermore, ABC294640 also diminished NF-ĸB survival signaling, through decreased activation of the Ser536 phosphorylation site on the p65 subunit. Xenografts of MCF-7TN-R cells growing in immunocompromised mice were utilized to validate the therapeutic efficacy of the sphingosine kinase-2 inhibitor. Treatment with 50 mg of ABC294640/kg completely blocked tumor volume in this model. These results indicate that pharmacological inhibition of Sphk2 with the orally bioavailable selective inhibitor, ABC294640, has therapeutic potential in the treatment of chemo- and endocrine therapy- resistant breast cancer.

Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis

Purpose: We have identified the phytoalexin compounds glyceollins I, II, and III, which exhibit marked antiestrogenic effects on estrogen receptor function and estrogen-dependent tumor growth in vivo. The purpose of this study was to investigate the interactions among the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in ovariectomized athymic mice. Experimental Design: Four treatment groups for each cell line were used: vehicle control, 20 mg/kg/mouse/d glyceollin mixture injection, 0.72 mg estradiol (E2) implant, and E2 implant + 20 mg/kg/mouse/d glyceollin injection. Results: Treatment with glyceollin suppressed E2-stimulated tumor growth of MCF-7 cells (−53.4%) and BG-1 cells (−73.1%) in ovariectomized athymic mice. These tumor-inhibiting effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumors. In contrast to tamoxifen, the glyceollins had no estrogen-agonist effects on uterine morphology and partially antagonized the uterotropic effects of estrogen. Conclusions: These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma.

Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence

Adult human mesenchymal stem cells (hMSCs) have been shown to home to sites of breast cancer and integrate into the tumor stroma. We demonstrate here the effect of hMSCs on primary breast tumor growth and the progression of these tumors to hormone independence. Co-injection of bone marrow-derived hMSCs enhances primary tumor growth of the estrogen receptor-positive, hormone-dependent breast carcinoma cell line MCF-7 in the presence or absence of estrogen in SCID/beige mice. We also show hormone-independent growth of MCF-7 cells when co-injected with hMSCs. These effects were found in conjunction with increased immunohistochemical staining of the progesterone receptor in the MCF-7/hMSC tumors as compared to MCF-7 control tumors. This increase in PgR expression indicates a link between MCF-7 cells and MSCs through ER-mediated signaling. Taken together, our data reveal the relationship between tumor microenvironment and tumor growth and the progression to hormone independence. This tumor stroma-cell interaction may provide a novel target for the treatment of estrogen receptor-positive, hormone-independent, and endocrine-resistant breast carcinoma.

Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype

IntroductionDespite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.MethodsTo compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.ResultsProteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.ConclusionsThis study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

Antiestrogenic Effects of the Novel Sphingosine Kinase-2 Inhibitor ABC294640

Alterations in sphingolipid metabolism have been shown to contribute to the development of endocrine resistance and breast cancer tumor survival. Sphingosine kinase (SK), in particular, is overexpressed in breast cancer and is a promising target for breast cancer drug development. In this study, we used the novel SK inhibitor ABC294640 as a tool to explore the relationship between SK and estrogen (E2) receptor (ER) signaling in breast cancer cells. Treatment with ABC294640 decreased E2-stimulated ERE-luciferase activity in both MCF-7 and ER-transfected HEK293 cells. Furthermore, the inhibitor reduced E2-mediated transcription of the ER-regulated genes progesterone receptor and SDF-1. Competitive receptor-binding assays revealed that ABC294640 binds in the antagonist ligand-binding domain of the ER, acting as a partial antagonist similar to tamoxifen. Finally, treatment with ABC294640 inhibited ER-positive breast cancer tumor formation in vivo. After 15 d of treatment with ABC294640, tumor volume was reduced by 68.4% (P < 0.05; n = 5) compared with control tumors, with no marked weight loss or illness. Taken together, these results provide strong evidence that this novel SK inhibitor, which had not previously been known to interact with E2 signaling pathways, has therapeutic potential in treating ER-positive breast cancer via inhibition of both SK and ER signaling.

Effects of human mesenchymal stem cells on ER-positive human breast carcinoma cells mediated through ER-SDF-1/CXCR4 crosstalk.

BackgroundAdult human mesenchymal stem cells (hMSC) have been shown to home to sites of carcinoma and affect biological processes, including tumour growth and metastasis. Previous findings have been conflicting and a clear understanding of the effects of hMSCs on cancer remains to be established. Therefore, we set out to investigate the impact of hMSCs on the oestrogen receptor positive, hormone-dependent breast carcinoma cell line MCF-7.ResultsIn this study, we show the effects of hMSCs on cancer cells are mediated through a secreted factor(s) which are enhanced by cancer cell-hMSC contact/communication. In addition to enhanced proliferation when in co-culture with hMSCs, MCF-7 cells were found to have increased migration potential in vitro. Inhibition of ER signalling by the pure anti-oestrogen ICI 182,780 decreased the effect of hMSCs on MCF-7 cell proliferation and migration supporting a role for ER signalling in the hMSC/MCF-7 cell interaction. Additionally, hMSCs have been shown to secrete a wide variety of growth factors and chemokines including stromal cell-derived factor-1 (SDF-1). This coupled with the knowledge that SDF-1 is an ER-mediated gene linked with hormone-independence and metastasis led to the investigation of the SDF-1/CXCR4 signalling axis in hMSC-MCF-7 cell interaction. Experiments revealed an increase in SDF-1 gene expression both in vivo and in vitro when MCF-7 cells were cultured with hMSCs. SDF-1 treatment of MCF-7 cells alone increased proliferation to just below that seen with hMSC co-culture. Additionally, blocking SDF-1 signalling using a CXCR4-specific inhibitor decreased hMSC induced proliferation and migration of MCF-7. However, the combined treatment of ICI and AMD3100 reduced MCF-7 cell proliferation and migration below control levels, indicating targeting both the ER and CXCR4 pathways is effective in decreasing the hMSCs induction of MCF-7 cell proliferation and migration.ConclusionsThe sum of these data reveals the relationship between tumour microenvironment and tumour growth and progression. Better understanding of the mechanisms involved in this tumour stroma cell interaction may provide novel targets for the development of treatment strategies for oestrogen receptor positive, hormone-independent, and endocrine-resistant breast carcinoma.

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

IntroductionAcquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam).MethodsWe cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions.ResultsQuantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility.ConclusionsOur data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.

Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. We previously showed the pan-deacetylase inhibitor LBH589 induces CDH1 expression in TNBC cells, suggesting regulation of EMT. The purpose of this study was to examine the effects of LBH589 on the metastatic qualities of TNBC cells and the role of EMT in this process. A panel of breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549), drugged with LBH589, was examined for changes in cell morphology, migration, and invasion in vitro. The effect on in vivo metastasis was examined using immunofluorescent staining of lung sections. EMT gene expression profiling was used to determine LBH589-induced changes in TNBC cells. ZEB overexpression studies were conducted to validate requirement of ZEB in LBH589-mediated proliferation and tumorigenesis. Our results indicate a reversal of EMT by LBH589 as demonstrated by altered morphology and altered gene expression in TNBC. LBH589 was shown to be a more potent inhibitor of EMT than other HDAC inhibitors, SAHA and TMP269. Additionally, we found that LBH589 inhibits metastasis of MDA-MB-231 cells in vivo. These effects of LBH589 were mediated in part by inhibition of ZEB, as overexpression of ZEB1 or ZEB2 mitigated the effects of LBH589 on MDA-MB-231 EMT-associated gene expression, migration, invasion, CDH1 expression, and tumorigenesis. These data indicate therapeutic potential of LBH589 in targeting EMT and metastasis of TNBC.