James W. Antoon

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.

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.

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.

Pharmacological inhibition of sphingosine kinase isoforms alters estrogen receptor signaling in human breast cancer

Recently, crosstalk between sphingolipid signaling pathways and steroid hormones has been illuminated as a possible therapeutic target. Sphingosine kinase (SK), the key enzyme metabolizing pro-apoptotic ceramide to pro-survival sphingosine-1-phosphate (S1P), is a promising therapeutic target for solid tumor cancers. In this study, we examined the ability of pharmacological inhibition of S1P formation to block estrogen signaling as a targeted breast cancer therapy. We found that the Sphk1/2 selective inhibitor (SK inhibitor (SKI))-II, blocked breast cancer viability, clonogenic survival and proliferation. Furthermore, SKI-II dose-dependently decreased estrogen-stimulated estrogen response element transcriptional activity and diminished mRNA levels of the estrogen receptor (ER)-regulated genes progesterone receptor and steroid derived factor-1. This inhibitor binds the ER directly in the antagonist ligand-binding domain. Taken together, our results suggest that SKIs have the ability to act as novel ER signaling inhibitors in breast carcinoma.

Preferential star strand biogenesis of pre‐miR‐24‐2 targets PKC‐alpha and suppresses cell survival in MCF‐7 breast cancer cells

microRNAs (miRNA) are regulators of cellular pathways and alterations of normal miRNA expression levels have been shown to increase tumorigenesis. miR‐24 has been demonstrated as having both tumor suppressive and oncogenic properties depending on cell context. Here, we demonstrate a possible role for pre‐miR‐24‐2 as a tumor suppressor in the MCF‐7 breast cancer cell line through the preferential processing of mature miR‐24‐2* over miR‐24. Specifically, we show that the ectopic expression of miR‐24‐2* in MCF‐7 breast cancer cells results in a suppression of cellular survival both in vivo and in vitro. Notably, the overexpression of miR‐24‐2* results in a dampening of cell survival through the targeted suppression of PKCα. In addition, a similar biological change is observed in vivo where MCF‐7 cells overexpressing pre‐miR‐24‐2 have decreased tumorigenicity and tumor incidence. Taken together our data demonstrate that when overexpressed biogenesis of the pre‐miR‐24‐2 favors miR‐24‐2* in the MCF‐7 breast cancer cell line and suggests a tumor suppressive role for miR‐24‐2* observed through the inhibition of PKCα‐mediated cellular survival.

Design, Synthesis, and Biological Activity of a Family of Novel Ceramide Analogues in Chemoresistant Breast Cancer Cells

Resistance to chemotherapy and endocrine therapy is a major cause of breast cancer treatment failure. We have synthesized six novel analogues using C8-ceramide as the lead analogue and studied their effect on hormone therapy resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells. Pharmacologic intervention using these ceramide analogues inhibited clonogenic survival and induced apoptosis, with one analogue being more effective than C8-ceramide. Our results show ceramide-based therapy has therapeutic potential in treating drug resistant breast cancer.

Neurocognitive Effects of Chemotherapy and Endocrine Therapies in the Treatment of Breast Cancer: Recent Perspectives

With an estimated 207,090 patients diagnosed with breast cancer in 2010, the role of chemotherapy-induced cognitive impairment is of growing importance. Studies to determine the impact of chemotherapy-induced cognitive impairment have been hindered by difficulties in study-design, in particular, study methodology. Here, we present a review of existing studies and discuss several mechanisms for chemotherapy-induced neurocognitive impairment in breast cancer patients, such as direct neurotoxic injury, telomere shortening, oxidative stress, cytokine dysregulation, estrogen-mediated effects, and the role of certain genetic polymorphisms. Decreased estrogen levels may serve as a link between multiple mechanisms potentiating the effects of the chemotherapy-induced cognitive impairment.

Novel D: -erythro N-octanoyl sphingosine analogs as chemo- and endocrine-resistant breast cancer therapeutics.

PurposeResistance to endocrine and chemotherapies remains the primary cause of breast cancer treatment failure. We have synthesized four novel d-erythro N-octanoyl sphingosine analogs and catalogued their activity in drug-sensitive (MCF-7), endocrine-resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells.Methods3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability; colony assay was performed to determine effects on clonogenic survival and 1H NMR, 13C NMR, HPLC spectra and elemental analytical data analyses were used to determine analog identity and purity.ResultsAll four analogs inhibited both viability and clonogenic survival, with analog C exhibiting a log-fold improvement in anti-survival activity compared to the parent compound.ConclusionWith resistance to current breast cancer chemotherapies on the rise, the development of novel therapeutic targets is of growing importance. Our results show that lipid analogs have therapeutic potential in treating chemo- and endocrine-resistant breast cancer.

Altered Death Receptor Signaling Promotes Epithelial-to-Mesenchymal Transition and Acquired Chemoresistance

Altered death receptor signaling and resistance to subsequent apoptosis is an important clinical resistance mechanism. Here, we investigated the role of death receptor resistance in breast cancer progression. Resistance of the estrogen receptor alpha (ER)-positive, chemosensitive MCF7 breast cancer cell line to tumor necrosis factor (TNF) was associated with loss of ER expression and a multi-drug resistant phenotype. Changes in three major pathways were involved in this transition to a multidrug resistance phenotype: ER, Death Receptor and epithelial to mesenchymal transition (EMT). Resistant cells exhibited altered ER signaling, resulting in decreased ER target gene expression. The death receptor pathway was significantly altered, blocking extrinsic apoptosis and increasing NF-kappaB survival signaling. TNF resistance promoted EMT changes, resulting in a more aggressive phenotype. This first report identifying specific mechanisms underlying acquired resistance to TNF could lead to a better understanding of the progression of breast cancer in response to chemotherapy treatment.