S1P in chemo- and radio-resistance

The sphingolipid metabolites, ceramide, sphingosine and sphingosine 1-phosphate (S1P) play crucial roles as signaling molecules in physio(patho)logy. Two of these sphingolipids, ceramide and S1P, have received particularly significant attention since the mid-1990s as critical mediators of cell death or survival. Ceramide is the central molecule in sphingolipid metabolism and, produced through de novo synthesis and/or the hydrolysis of sphingomyelin, has been shown to mediate apoptotic cell death in response to a wide array of anticancer treatments (click here for review). Chemotherapy and radiotherapy elicit an increase in endogenous ceramide levels, occurring prior to the first biochemical signs of apoptosis, namely mitochondrial release of apoptogenic proteins such as cytochrome c and activation of effector caspases. Moreover, addition of exogenous short-chain ceramides causes apoptosis in numerous cancer cell lines. Sphingosine, the catabolite of ceramide, has also been shown to be generated in cells undergoing apoptosis, and addition of sphingosine recapitulates the biological effects of ceramide in cancer cells (click here for review). Notwithstanding, ceramide and sphingosine are interconvertible metabolites and it is extremely difficult to decipher their individual roles in mediating apoptosis. In contrast to ceramide (and sphingosine), sphingosine 1-phosphate (S1P) promotes cell survival in response to apoptotic stresses that induce ceramide generation in vitro, ex vivo and in vivo. The opposing directions of ceramide- and S1P-mediated signaling gave birth to the concept of a ceramide/S1P biostat, and the postulate that the ratio between these two lipids could determine cell fate ( Cuvillier et al., Nature, 1996).
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A master regulator of this ceramide/S1P balance is
sphingosine kinase-1 (SphK1), the main enzyme that phosphorylates sphingosine to form S1P. SphK1 is a key enzyme in the sphingolipid metabolism because it serves the dual function of modulating ceramide and S1P levels: it produces the pro-growth, antiapoptotic messenger S1P but also decreases intracellular levels of the pro-apoptotic ceramide, counterbalancing its effect.

In agreement with the general idea that ceramide is a mediator of apoptosis, lack of generation of ceramide has been linked to tumor-cell resistance Radiation-resistant and chemo-resistant cell lines do not generate ceramide after irradiation or chemotherapy, a defect that could be bypassed by addition of cell-permeable ceramides.

Interestingly, a correlation was originally established between SphK1 activity and resistance to irradiation in prostate cancer cells. It has been demonstrated that SphK1 activity was not affected by ionizing radiations in radio-resistant LNCaP cells, whereas SphK1 was markedly inhibited in radio-sensitive TSU cells (Nava, Cuvillier et al.,
Cancer Res, 2000).

Based on this seminal work, our Lab has been studying the contribution of the ceramide/S1P balance controlled by SphK1 in various cancer cell models including leukemia and prostate cancer cell and animal models.

SphK1 activity is a sensor of ‘sensitivity/resistance’ in leukemia

The multidrug-resistant (MDR) phenotype, commonly observed in malignant acute myeloid leukemia (AML) cells exposed to anthracyclines or plant- based alkaloids, remains a major obstacle to successful chemotherapy. The role of SphK1 signaling in susceptibility to antineoplastic agents of either sensitive or MDR acute myeloid leukemia (AML) cells was examined. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60/VP16 cells overexpressing MRP1 and MDR1, respectively. Chemosensitive HL-60 cells displayed SphK1 inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP16 had sustained SphK1 activity and did not produce ceramide during treatment. Enforced expression of SphK1 in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a SphK1 inhibition and apoptosis both prevented by SphK1 overexpression. Furthermore, the sphingosine kinase inhibitor F-12509a led to ceramide accumulation, decrease in S1P content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that could be prevented by adding S1P or overexpressing SphK1. F-12509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.
We also examined the involvement of SphK1 in susceptibility to imatinib of either sensitive or resistant chronic myeloid leukemia (CML) cells. Imatinib-sensitive LAMA84-s displayed marked SphK1 inhibition coupled with increased content of ceramide and decreased S1P. Conversely, no changes in the sphingolipid metabolism were observed in LAMA84-r treated with imatinib. Overcoming imatinib resistance in LAMA84-r with farnesyl- transferase or MEK/ERK inhibitors as well as with cytosine arabinoside led to SphK1 inhibition. Overexpression of SphK1 in LAMA84-s cells impaired apoptosis and inhibited the effects of imatinib on caspase-3 activation, cytochrome c and Smac release from mitochondria through modulation of Bim, Bcl-xL and Mcl-1 expression. Pharmacological inhibition of SphK1 with F-12509a or its silencing by siRNA induced apoptosis of both imatinib-sensitive and -resistant cells, suggesting that SphK1 inhibition was critical for apoptosis signaling. We also show that imatinib-sensitive and -resistant primary cells from chronic myeloid leukemia patients can be successfully killed in vitro by the F-12509a inhibitor. These results uncover the involvement of SphK1 in regulating imatinib-induced apoptosis and establish that SphK1 is a downstream effector of the Bcr-Abl/Ras/ERK pathway inhibited by imatinib but upstream regulator of Bcl-2 family members.

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Bonhoure et al, Leukemia, 2006

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Bonhoure et al, Leukemia, 2008

SphK1 activity is a chemotherapy sensor in prostate cancer cells and animal models

The role of SphK1 as an indicator of tumor cell sensitivity or resistance was also examined in vitro and in vivo in prostate cancer. The differential effects of the topoisomerase inhibitor camptothecin and the anti-microtubule agent docetaxel were assessed on human PC-3 and LNCaP prostate cancer cells. A strong inhibition of SphK1 and elevation of the ceramide/S1P ratio was seen only in cell lines sensitive to these drugs. This difference in effect was confirmed in an orthotopic PC-3/GFP model established in nude mice. Docetaxel induced stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin; moreover, this was accompanied by smaller tumor volumes and a reduced frequency and number of metastases. These results were the first in vivo demonstration of SphK1 as a sensor during chemotherapy. Note that the limited efficacy of camptothecin in PC-3 and do- cetaxel in LNCaP cells was associated with a transient paradoxical increase in SphK1 activity, suggesting that SphK1 stimulation compromises chemotherapy success. As anticipated, the pharmacological inhibition of SphK1 strongly sensitized prostate cancer cells to the effects of camptothecin and docetaxel, both in cell culture and in vivo, suggesting that the SphK1/S1P pathway could be used as a potential target for developing novel chemosensitizers.

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Orthotopic implantation of prostate cancer cells.

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Visualization of prostate of metastases (eg. lung metastases).

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In green, visualization of prostate cancer and periaortic lymph nodes 5 weeks after implantation (GFP-tagged PC-3 cells).
In yellow, the bladder.

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Pchejetski et al, Cancer Res, 2005

FTY720 (Fingolimod) sensitizes to irradiation by decreasing S1P levels

Sphingolipids have been associated as important components of irradiation-induced apoptosis of human prostate cancer cells. Abundant evidence suggests that ceramide and sphingosine are critical mediators of ionizing radiation-induced apoptosis (Nava et al., 2000, Cancer Res.) whereas S1P can protect from the deleterious effects of irradiation including in animal models (Morita et al., 2000, Nature Med.).
We have shown that a sphingosine analogue FTY720 (Fingolimod), a prominent drug now used for treatment of multiple sclerosis, is 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. FTY720 acted as a SphK1 inhibitor that induced apoptosis in a manner independent of S1P receptors. In contrast, irradiation did not affect SphK1 activity in prostate cancer cells yet synergized with FTY720 to inhibit SphK1. In mice bearing orthotopic or subcutaneous prostate cancer tumors, we showed 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 might offer significant improvement to clinical treatment of prostate cancer.

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Pchejetski et al, Cancer Res, 2010

The manipulation of S1P lyase (SPL) activity modulates the response to chemo- and radiotherapy

Our recent studies demonstrate and uncover the mechanisms by which the knockdown by siRNA strategies of S1P-generating enzyme SphK1 or the up-regulation of S1P lyase (SPL) - the enzyme that degrades S1P - strongly sensitizes to irradiation, whereas knockdown of SPL by maintaining a hight content of intracellular S1P induces radioresistance pointing out the essential role of S1P to protect against ionizing radiations.
In multiple prostate cancer cell models, silencing SPL increased S1P content and enhanced survival after irradiation by decreasing expression of γ-H2AX, DNA-PKcs-P and ATM-P, proteins involved in the sensing and reparation of DNA double-strand breaks. On the contrary, enforced expression of SPL sensitized cancer cells to radiation by tilting the ceramide/S1P balance towards cell death.

We also report that cancer cells overexpressing SPL struggle to survive to the SPL-mediated degradation of pro-survival S1P and generation of pro-apoptotic ceramide by enhancing S1P production through increased SphK1. Accordingly, combined S1P lowering strategy based on SphK1 silencing and SPL overexpression further amplified radiosensitivity.
The sensitization induced by SPL overexpression is likely to be attributed to its effect on the ceramide/S1P balance (increase in ceramide and decrease in S1P) rather than increased formation of reaction products of SPL as hexadecenal and palmitic acid failed to sensitize to radiation.

Similarly overexpression of SPL was able to sensitize prostate cancer cells to chemotherapy.

Overall, our clinical observation showing that SPL is down-regulated in prostate cancer while SphK1 is up-regulated suggests that a therapeutic approach combining the inhibition of S1P production associated with a stimulation of its degradation should have a favorable therapeutic index, notably in combination with radio- or chemotherapy.

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Brizuela et al,
Mol Cancer Ther, 2012