Lnc-HZ14 promotes LLPS-mediated SPHK1 aggrephagy degradation to suppress trophoblast cell proliferation in unexplained recurrent miscarriage

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

<div>Abstract<p><b>Purpose:</b> The present study was to investigate the clinical significance of sphingosine kinase 1 (SPHK1), an oncoenzyme, in the development and progression of gastric cancer.</p><p><b>Experimental Design:</b> mRNA and protein levels of SPHK1 expression in normal gastric epithelial cells, gastric cancer cell lines, and paired gastric cancer lesions and the adjacent noncancerous tissues were examined using reverse transcription-PCR and Western blotting. Immunohistochemistry was employed to analyze SPHK1 expression in 175 clinicopathologically characterized gastric cancer cases. Statistical analyses were applied to derive prognostic and diagnostic associations.</p><p><b>Results:</b> Levels of SPHK1 mRNA and protein were higher in gastric cancer cell lines than in normal gastric epithelial cells. SPHK1 protein level was up-regulated in gastric cancer lesions compared with that in the paired adjacent noncancerous tissues. Gastric cancer tissues from 115 of 175 (65.7%) patients revealed high level of SPHK1 protein expression in contrast to the undetectable or marginally detectable expression of SPHK1 in the adjacent noncancerous gastric tissues. Significantly different expression levels of SPHK1 were found in patients at different clinical stages (<i>P</i> = 0.003), T classification (<i>P</i> = 0.035), and M classification (<i>P</i> = 0.020). Patients with higher SPHK1 expression had shorter overall survival time, whereas those with lower SPHK1 expression survived longer. Further multivariate analysis suggested that SPHK1 up-regulation was an independent prognostic indicator for the disease.</p><p><b>Conclusions:</b> SPHK1 protein could be a useful marker for the prognosis of gastric cancer. Further study on the potential use of SPHK1 as a therapeutic target is also warranted.</p></div>

The present study was to investigate the clinical significance of sphingosine kinase 1 (SPHK1), an oncoenzyme, in the development and progression of gastric cancer. mRNA and protein levels of SPHK1 expression in normal gastric epithelial cells, gastric cancer cell lines, and paired gastric cancer lesions and the adjacent noncancerous tissues were examined using reverse transcription-PCR and Western blotting. Immunohistochemistry was employed to analyze SPHK1 expression in 175 clinicopathologically characterized gastric cancer cases. Statistical analyses were applied to derive prognostic and diagnostic associations. Levels of SPHK1 mRNA and protein were higher in gastric cancer cell lines than in normal gastric epithelial cells. SPHK1 protein level was up-regulated in gastric cancer lesions compared with that in the paired adjacent noncancerous tissues. Gastric cancer tissues from 115 of 175 (65.7%) patients revealed high level of SPHK1 protein expression in contrast to the undetectable or marginally detectable expression of SPHK1 in the adjacent noncancerous gastric tissues. Significantly different expression levels of SPHK1 were found in patients at different clinical stages (P=0.003), T classification (P=0.035), and M classification (P=0.020). Patients with higher SPHK1 expression had shorter overall survival time, whereas those with lower SPHK1 expression survived longer. Further multivariate analysis suggested that SPHK1 up-regulation was an independent prognostic indicator for the disease. SPHK1 protein could be a useful marker for the prognosis of gastric cancer. Further study on the potential use of SPHK1 as a therapeutic target is also warranted.

<div>Abstract<p><b>Purpose:</b> The present study was to investigate the clinical significance of sphingosine kinase 1 (SPHK1), an oncoenzyme, in the development and progression of gastric cancer.</p><p><b>Experimental Design:</b> mRNA and protein levels of SPHK1 expression in normal gastric epithelial cells, gastric cancer cell lines, and paired gastric cancer lesions and the adjacent noncancerous tissues were examined using reverse transcription-PCR and Western blotting. Immunohistochemistry was employed to analyze SPHK1 expression in 175 clinicopathologically characterized gastric cancer cases. Statistical analyses were applied to derive prognostic and diagnostic associations.</p><p><b>Results:</b> Levels of SPHK1 mRNA and protein were higher in gastric cancer cell lines than in normal gastric epithelial cells. SPHK1 protein level was up-regulated in gastric cancer lesions compared with that in the paired adjacent noncancerous tissues. Gastric cancer tissues from 115 of 175 (65.7%) patients revealed high level of SPHK1 protein expression in contrast to the undetectable or marginally detectable expression of SPHK1 in the adjacent noncancerous gastric tissues. Significantly different expression levels of SPHK1 were found in patients at different clinical stages (<i>P</i> = 0.003), T classification (<i>P</i> = 0.035), and M classification (<i>P</i> = 0.020). Patients with higher SPHK1 expression had shorter overall survival time, whereas those with lower SPHK1 expression survived longer. Further multivariate analysis suggested that SPHK1 up-regulation was an independent prognostic indicator for the disease.</p><p><b>Conclusions:</b> SPHK1 protein could be a useful marker for the prognosis of gastric cancer. Further study on the potential use of SPHK1 as a therapeutic target is also warranted.</p></div>

A comparison of endometrial and subendometrial vascularity assessed by three-dimensional ultrasonography and power Doppler angiography between healthy fertile women and women with unexplained primary recurrent miscarriage

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.

Background: Sphingosine kinase 1 (SPHK1) is over-expressed in multiple cancers including breast and colon cancer. SPHK1 catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). S1P promotes tumorigenesis by inhibiting apoptosis and increasing cell proliferation and angiogenesis. Thus SPHK1 has been evaluated as a therapeutic target in cancer. The antidiabetic drug, metformin, has been reported to have protective effects in several cancers, including ovarian cancer. However, molecular mechanisms mediating the anti-tumor effects of metformin are not fully understood. In this study, we demonstrate that SPHK1 is a novel target of metformin and may predict metformin response in ovarian cancer. Methods: The S1P signaling cascade was profiled in several ovarian cancer cell lines. Four different ovarian cancer cell lines were treated with 1mM and 5mM metformin and the mRNA and protein levels of SPHK1 were measured by qRT-PCR and western blotting respectively. Next, the Tyk-nu, HeyA8, SNU119, Kuramochi cell lines were stably transfected to overexpression SPHK1 and metformin sensitivity was measured using MTT proliferation assays. Metabolomic analysis was performed on serum from ovarian cancer patients using metformin for diabetes and compared controls (IRB 13248A). Serum samples were analyzed using a Q-Exactive orbitrap mass spectrometer coupled to a Dionex ultimate UHPLC. Results: In all cell lines tested, metformin suppresses SPHK1 mRNA and protein levels. Analysis of the S1P signaling pathway showed that metformin sensitive cell lines (Tyknu and HeyA8) have high SPHK1 and low S1P lyase (SGPL1) expression, the opposite is true in metformin resistant cell lines (Kuramochi and SNU119). At the same time, S1P receptor 1 (S1PR1) is the main S1P receptor among five S1P receptors (S1PR1-S1PR5) in metformin sensitive cancer cells, however S1PR1 is not dominant in metformin resistant cancer cells. Overexpression of SPHK1 upregulates S1PR1 and increases metformin sensitivity in both metformin sensitive and resistant cell lines. The in vitro findings are augmented by preliminary metabolomic analysis of patient samples that revealed that ovarian cancer patients using metformin for diabetes have lower serum levels of S1P compared to controls. Conclusions: The findings of this study indicate that SPHK1 is a novel metformin target in ovarian cancer. Specifically, high SPHK1 expression sensitizes cells to metformin and S1P signaling profiles predicts metformin response. Based on these findings we propose that sphingolipid signaling be evaluated as a metformin-response signature in ongoing clinical trials of the drug as adjuvant treatment for ovarian cancer (NCT02122185). Citation Format: Tatsuyuki Chiyoda, Xiaojing Liu, Ernst Lengyel, Jason Locasale, Iris Romero. Sphingosine kinase 1 as a mediator and predictor of metformin's protective effect in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A68.

Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and gamma-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1)

To characterize the expression of sphingosine kinase-1 (SPHK1) in human astrocytomas and to investigate the association between SPHK1 expression and progression of astrocytomas. The expression of SPHK1 in normal human astrocytes, astrocytoma cell lines, and four pairs of matched astrocytoma tissues and their adjacent normal brain tissues were detected by quantitative reverse transcription-PCR and Western blot. In addition, SPHK1 protein expression was examined in 243 cases of histologically characterized astrocytomas by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations. SPHK1 in astrocytoma cell lines was elevated at both mRNA and protein levels, and the SPHK1 mRNA and protein were significantly up-regulated by up to 6.8- and 40-fold, respectively, in primary astrocytomas compared with those in the adjacent noncancerous brain tissues. Immunohistochemical analysis showed that 100 of 243 (41.2%) paraffin-embedded archival astrocytoma biopsies exhibited high expression of SPHK1. Statistical analysis suggested that the up-regulation of SPHK1 was significantly correlated with the histologic grade of astrocytoma (P=0.000) and that patients with high SPHK1 level exhibited shorter survival time (P<0.001). Multivariate analysis revealed that SPHK1 up-regulation might be an independent prognostic indicator for the survival of patients with astrocytoma. SPHK1 might represent a novel and useful prognostic marker for astrocytoma and play a role during the development and progression of the disease.

Numerous long noncoding RNAs (lncRNAs) have been discovered, however only a small number of lncRNAs have been explored with respect to their function and little is known how they operate at chromatin. In this study, the function of an E2F1-regulated lncRNA, termed KHPS1, has been investigated. KHPS1 is transcribed in antisense orientation from the SPHK1 (Sphingosine kinase 1) promoter which in sense orientation directs transcription of SPHK1 mRNA. The results demonstrate that KHPS1 activates SPHK1 transcription by recruiting histone acetyltransferase p300 and the transcription factor E2F1 to the SPHK1 enhancer. Binding of p300 and E2F1 is required for transcription of an enhancer-derived RNA, eRNASphk1. Transcription of eRNA-Sphk1 evicts CTCF, a factor that insulates the SPHK1 enhancer from the SPHK1 promoter and facilitates SPHK1 expression. Importantly, the direct association of KHPS1 with a homopurine stretch upstream at the SPHK1 enhancer is essential for SPHK1 expression. Binding of KHPS1 to the SPHK1 enhancer is mediated via Hoogsteen base pairing, forming RNA-DNA-DNA triplex structure. Tethering KHPS1 and associated p300 and E2F1 to the SPHK1 enhancer is a prerequisite for activation of eRNA-Sphk1 transcription and expression of SPHK1. The functional relevance of triplex formation was further studied using reporter plasmids which mimic KHPS1-dependent transcription activation of SPHK1. Deletion or mutation of the triplex forming region (TFR) attenuated recruitment of p300 and E2F1 and compromised transcription of eRNA-Sphk1. Replacement of the TFR by foreign triplex-forming motifs from lncRNAs MEG3 or Fendrr functionally replaced the TFR of SPHK1, i.e. activated eRNA-Sphk1 transcription. Ectopic KHPS1 with the TFR of MEG3 targeted E2F1 and p300 to the MEG3 target gene TGFBR1, underscoring the functional relevance of triplex-forming sequences. Genomic deletion of the TFR or intervention of KHPS1 binding to DNA by ectopic TFR-containing RNA decreased SPHK1 expression and impaired cell viability. Collectively, the results unravel a triplexdependent regulatory feed-forward mechanism, involving lncRNA-mediated activation of eRNA which enhances expression of its target gene. The results underscore the pivotal role of triplex formation in transcription control, supporting a model whereby lncRNAs tethered to specific loci serve as sequence-specific molecular anchors.

Acetylation of sphingosine kinase 1 regulates cell growth and cell-cycle progression

Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.