Leyre Brizuela

Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug

Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.[Mol Cancer Ther 2009;8(4):809–20]

Sphingosine Kinase 1: A New Modulator of Hypoxia Inducible Factor 1α during Hypoxia in Human Cancer Cells

Here, we provide the first evidence that sphingosine kinase 1 (SphK1), an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids, modulates the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1-dependent stabilization of HIF-1alpha levels is mediated by the Akt/glycogen synthase kinase-3beta signaling pathway that prevents its von Hippel-Lindau protein-mediated degradation by the proteasome. The pharmacologic and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1alpha and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung), suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia, giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences.

FTY720 (Fingolimod) Sensitizes Prostate Cancer Cells to Radiotherapy by Inhibition of Sphingosine Kinase-1

Radiotherapy is widely used as a radical treatment for prostate cancer, but curative treatments are elusive for poorly differentiated tumors where survival is just 15% at 15 years. Dose escalation improves local response rates but is limited by tolerance in normal tissues. A sphingosine analogue, FTY720 (fingolimod), a drug currently in phase III studies for treatment of multiple sclerosis, has been found to be a potent apoptosis inducer in prostate cancer cells. Using in vitro and in vivo approaches, we analyzed the impact of FTY720 on sphingolipid metabolism in hormone-refractory metastatic prostate cancer cells and evaluated its potential as a radiosensitizer on cell lines and prostate tumor xenografts. In prostate cancer cell lines, FTY720 acted as a sphingosine kinase 1 (SphK1) inhibitor that induced prostate cancer cell apoptosis in a manner independent of sphingosine-1-phosphate receptors. In contrast, γ irradiation did not affect SphK1 activity in prostate cancer cells yet synergized with FTY720 to inhibit SphK1. In mice bearing orthotopic or s.c. prostate cancer tumors, we show that FTY720 dramatically increased radiotherapeutic sensitivity, reducing tumor growth and metastasis without toxic side effects. Our findings suggest that low, well-tolerated doses of FTY720 could offer significant improvement to the clinical treatment of prostate cancer.

Activation of Sphingosine Kinase-1 in Cancer: Implications for Therapeutic Targeting

Sphingolipid metabolites are critical to the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation, cell survival and angiogenesis. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because its produces the pro-survival and pro-angiogenic sphingosine 1-phosphate and decreases the amount of both ceramide and sphingosine, the pro-apoptotic sphingolipids. Moreover, its gene is oncogenic, its mRNA is overproduced in several solid tumors, its overexpression protects cells from apoptosis, and its activity is down-regulated by anti-cancer treatments. Therefore, the sphingosine kinase-1/sphingosine 1-phosphate signaling pathway appears to be a target of interest for therapeutic manipulation.

Critical Role for Sphingosine Kinase-1 in Regulating Survival of Neuroblastoma Cells Exposed to Amyloid-β Peptide

We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid β (Aβ) peptide (25-35). Upon incubation with Aβ, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Aβ-induced cell death. SphK1 overexpression impaired the cytotoxicity of Aβ, whereas SphK1 silencing by RNA interference mimicked Aβ-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against Aβ toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-β1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of Aβ-treated cells.

Sphingosine Kinase-1 Is Central to Androgen-Regulated Prostate Cancer Growth and Survival

Background Sphingosine kinase-1 (SphK1) is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival. Methodology/Principal Findings Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT) to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway—by negatively impacting SphK1 activity—could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity. Conclusions/Significance We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer.

Hypoxia, therapeutic resistance, and sphingosine 1-phosphate.

Hypoxia, defined as a poor oxygenation, has been long recognized as a hallmark of solid tumors and a negative prognostic factor for response to therapeutics and survival of patients. Cancer cells have evolved biochemical mechanisms that allow them to react and adapt to hypoxia. At the cellular level, this adaptation is under the control of two related transcription factors, HIF-1 and HIF-2 (hypoxia-inducible factor), that respond rapidly to decreased oxygen levels to activate the expression of a broad range of genes promoting neoangiogenesis, glycolysis, metastasis, increased tumor growth, and resistance to treatments. Recent studies have identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway-which elicits various cellular processes including cell proliferation, cell survival, or angiogenesis-as a new regulator of HIF-1 or HIF-2 activity. In this review, we will focus on how the inhibition/neutralization of the SphK1/S1P signaling could be exploited for cancer therapy.

Osteoblast-derived sphingosine 1-phosphate to induce proliferation and confer resistance to therapeutics to bone metastasis-derived prostate cancer cells

Sphingosine 1‐phosphate (S1P) plays important roles in cell proliferation, differentiation or survival mainly through its surface G‐protein‐coupled receptors S1P1−5. Bone represents the major site of metastasis for prostate cancer (CaP) cells, which rely on bone‐derived factors to support their proliferation and resistance to therapeutics.

Biochemical Methods for Quantifying Sphingolipids: Ceramide, Sphingosine, Sphingosine Kinase-1 Activity, and Sphingosine-1-Phosphate

Sphingolipids (ceramide, sphingosine, and sphingosine-1-phosphate) are bioactive lipids with important biological functions in proliferation, apoptosis, angiogenesis, and inflammation. Herein, we describe easy and rapid biochemical methods with the use of radiolabeled molecules ((3)H, (32)P) for their mass determination. Quantitation of sphingosine kinase-1 activity, the most studied isoform, is also included.

Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1

The bioactive metabolite sphingosine-1-phosphate (S1P), a product of sphingosine kinases (SphKs), mediates diverse biological processes such as cell differentiation, proliferation, survival and angiogenesis. A fluorinated analogue of S1P receptor agonist has been synthesized by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion as the key reaction. In vitro activity of this S1P analogue is also reported.