The previous studies using leukemia-derived transformed cell lines revealed many of the basic effectors of ceramide and S1P signaling in leukemia cells and established their generally opposing actions in the context of leukemia. More recent studies have examined the effects of sphingolipid signaling in human primary leukemia cells and animal models of the disease. Findings in these systems have provided more substantial evidence strengthening the physiological relevance of sphingolipid signaling in hematopoietic malignancies.
For example, microarray analysis demonstrated that an S1P5
receptor is upregulated in human T-cell LGL leukemia 76
. LGL leukemia cells appear to derive from a clonal expansion of terminally differentiated, antigen-primed, competent cytotoxic T lymphocytes. As such, patients with LGL leukemia have autoimmune conditions such as rheumatoid arthritis 77
. Recent studies have implicated PDGF and IL-15 as key signaling factors responsible for the pathogenesis and activation of LGL 78
. Interestingly, multiple genes involved in the sphingolipid metabolic pathway were found to be differentially expressed in these tumors 79
. Among the genes identified in this study were SphK1, acid ceramidase, and neutral sphingomyelinases. Inhibition of acid ceramidase or SK, or treatment with FTY720, a sphingosine analog that functions as an immunomodulatory drug by binding to and promoting internalization of S1P1
, induced apoptosis or sensitized the cells to Fas-induced apoptosis. Thus, dysregulation of the S1P signaling may contribute to the effects of upstream growth factor activation in LGL, such as by facilitating the escape of activated T cells from Fas-mediated cell death.
A central role for SphK1 in the development of erythroleukemia was revealed when microarray analysis was used to compare gene expression in nontumorigenic and tumorigenic proerythroblasts from a transgenic mouse model of erythroleukemia 80
. Using this approach, SphK1 upregulation was found to be a recurrent oncogenic event underlying the tumorigenic phenotype in proerythroblasts. When erythroleukemic cells were forced to overexpress SphK1, they exhibited a proliferative advantage, increased clonogenicity, tumorigenicity when implanted in nude mice, and resistance to apoptosis in response to serum-deprivation. This protection from stress was mediated through an ERK- and PI3K-dependent pathway. In vitro
suppression of SK activity either by expressing a dominant negative SphK1 or through pharmacological inhibition blocked the proliferation and stress resistance of erythroleukemia cells.
In chronic myelogenous leukemia (CML), the tyrosine kinase ABL is in a constitutively active state, resulting in unrelenting growth signaling. The drug imatinib, which inhibits ABL tyrosine kinase activity, is the first example of a new class of chemotherapy agents that target a mutant enzyme that is characteristic of a particular cancer cell. This distinguishes imatinib from the many nonspecific agents that target DNA replication and cell cycle progression and thereby affect all or most rapidly dividing cell populations. The development of imatinib mesylate for CML has revolutionized CML treatment. However, resistance has been a problem in achieving complete killing of tumor cells 81
. A recent study examined the mechanisms for resistance to imatinib-induced apoptosis using a pair of isogenic human K562 CML cell lines, one of which is resistant to imatinib and the other sensitive. In these cells, ceramide and S1P were found to have opposing effects on imatinib-induced apoptosis 82
. Imatinib treatment induced the generation of endogenous C18 ceramide, resulting in enhanced apoptosis in the sensitive cell line, whereas decreased apoptosis was observed in imatinib-resistant cells that failed to generate C18 ceramide, exhibited elevated SphK1 expression and had an increased ratio of S1P/C18 ceramide. Importantly, partial knockdown of SphK1 in the resistant cells increased their sensitivity to imatinib. In a separate study, cell lines derived from CML patients in blast crisis were used to investigate the role of SphK1 in imatinib resistance 83
. Overexpression of SphK1 resulted in resistance to imatanib-induced cell death, concomitant with decreased levels of cytochrome c and Smac/DIABLO expression. Further, SphK1 activity was significantly decreased with imatanib treatment. Recently, continuous exposure of K562 cells to gradually increasing concentrations of imatinib was used to select an imatinib-resistant cell line. This line exhibited upregulation of both BCR-ABL and SphK1. SphK1 was found to be upregulated in an AKT2-dependent manner that promoted imatinib resistance. Both BCR-ABL upregulation and imatinib resistance could be reversed by SphK1 knockdown and by pharmacological inhibition of SK activity using the sphingosine analog N,N-dimethylsphingosine (DMS) 84
. These studies suggest that modulating the sphingolipid rheostat could be exploited to address the problem of drug resistance in long-term treatment of CML.
In an effort to investigate mechanisms of chemotherapy resistance in acute myeloid leukemia (AML) cells, the role of SphK1 was explored in AML cells demonstrating resistant to the chemotherapeutic agents doxorubicin or etoposide 83
. As in CML, elevated SphK1 expression levels correlated with survival and inhibition of apoptosis in AML, and these findings were associated with a block in mitochondrial cytochrome c translocation to the cytoplasm (see ).
The predominance of published studies analyzing S1P and ceramide signaling in cancer have focused on the intracellular events controlling their biosynthesis and degradation. However, recent reports have begun to establish a role for S1P receptors in hematopoietic malignancy as well. For example, the S1P1
receptor has recently been implicated in myeloid tumor progression via a mechanism involving signal transducer and activator of transcription 3 (STAT3) signaling 85
. The STAT3 protein is a transcription factor that is activated by many cytokines and plays a central role in inflammation, cell growth and apoptosis 86
. The binding of IL-6 cytokine family members to the gp130 receptor results in STAT3 phosphorylation by Janus kinase 2 (JAK2), and constitutive activation of this signaling pathway has been implicated as a causative factor in cancer. STAT3 signaling has been shown to be important in determining sensitivity of leukemia cells to apoptosis 87
. Recently, STAT3 was shown to be involved in a feedback loop with S1P1 85
was found to be a transcriptional target of STAT3, and STAT3 positive tumor cells exhibited elevated S1P1
expression. In addition, S1P1
signaling led to activation of STAT3 and increased IL-6 via upregulation of JAK2 activity, thereby resulting in an auto-stimulatory loop. Unlike transient activation of STAT3 by IL-6, S1P1
signaling was shown to persistently activate this pathway through its influence on tumor cells and the tumor microenvironment (see ). Importantly, the effects of this auto-activating signaling loop promoted tumor growth and metastatic spread, whereas inhibition of S1P1
signaling prevented these effects. These results provide new insight into how S1P signaling may contribute fundamentally to leukemogenesis. They simultaneously raise the possibility that S1P signaling may be an Achilles heel for some leukemias in which the pathway is dysregulated.