• Open Access

Diacylglycerol kinases are essential for hepatocyte growth factor-dependent proliferation and motility of Kaposi’s sarcoma cells

Authors


To whom correspondence should be addressed. E-mail: mprat@med.unipmn.it

Abstract

Hepatocyte growth factor (HGF) is involved in the pathogenesis of Kaposi’s sarcoma (KS), the most frequent neoplasia in patients with AIDS, characterized by proliferating spindle cells, infiltrating inflammatory cells, angiogenesis, edema, and invasiveness. In vitro, this factor sustains the biological behavior of KS derived cells, after activation of its receptor and the downstream MAPK and AKT signals. In other cell types, namely endothelial and epithelial cells, movement, proliferation, and survival stimulated by HGF and other growth factors and cytokines depend on diacylglycerol kinases (DGK). In an effort to identify new intracellular transducers operative in KS cells, which could represent therapeutic targets, we investigated the role of DGK in KS cell movement and proliferation by treating cells with the DGK pharmacological inhibitor R59949. We report that R59949 strongly inhibits HGF-induced KS motility, proliferation, and anchorage-independent growth with only a partial effect on cell adhesion and spreading. R59949 does not affect cell survival, HGF receptor activation, or the classical MAPK and AKT signalling pathways. Furthermore, we carried out an siRNA screen to characterize the DGK isoforms involved in KS motility and anchorage independent growth. Our data indicate a strong involvement of DGK-δ in KS motility and of DGK-ι in anchorage-independent growth. These results indicate that DGK inhibition is sufficient to impair in vitro KS cell proliferation and movement and suggest that selected DGK represent new pharmacological targets to interfere with the malignant properties of KS, independently from the well-known RAS/MAPK and PI3K/AKT pathways. (Cancer Sci 2011; 102: 1329–1336)

Kaposi’s sarcoma is the most frequently observed neoplasm arising in patients with AIDS, but it is also reported in a number of other clinical conditions, including renal transplantation, chronic use of glucocorticoids, autoimmune diseases, and lymphoproliferative disorders, and is endemic in Central Africa.(1) The same biological characteristics are present in the different KS types, namely, spindle cell proliferation, inflammatory cell infiltration, angiogenesis, edema, and invasiveness (see reviews(2,3)).

Multiple concomitant pathogenetic factors are involved in this complex disease. The human herpes virus type 8 has been identified as the necessary, although not sufficient, causative agent and indeed other cofactors, such as HIV, host-derived inflammatory cytokines (IL-6, IL-10), and growth factors (VEGF, basic fibroblast growth factor, HGF), are also required for the development of the disease.(4–7)

A pathogenetic role for HGF and its receptor Met is suggested by the following findings: (i) KS cells express Met and often also HGF;(8–10) (ii) an autocrine HGF loop sustains the proliferation of human KS cells(11) and of spindle-shaped cells derived from KS-like lesions developed in BKV/tat transgenic mice;(9) (iii) the main biological features of this neoplasm (of endothelial origin, highly vascularized and invasive) are compatible with the biological properties of HGF;(12,13) and (iv) in vitro HGF stimulation of a KS cell line activates the ERK-1/2 MAPK and PI3K pathways(14) and elicits a series of biological effects, including cell migration, proliferation, and invasiveness.(10) Moreover, by means of Met receptor agonist mAbs, proliferation and invasiveness were shown to require a sustained and long-lasting activation of the Met receptor and MAPK.(14)

Despite numerous studies, KS continues to be an incurable disease, but the targeting of signaling pathways has emerged as a promising approach(15,16) In the effort to identify new possible targets for KS control we have investigated the involvement of DGK in KS cell transformation. Diacylglycerol kinase plays a central role in lipid signaling by phosphorylating DG to generate PA, thus downregulating DG signal transduction and promoting PA signaling. Diacylglycerol kinase regulates a wide variety of cellular functions by binding effectors containing the cysteine-rich C1 domain, including many PKC isoforms, PKD, Unc-13, chimaerin, the Rap exchange factors CalDAG-GEF, and also proteins lacking the C1 domain, such as the Ca2+-permeable transient receptor potential channel.(17) The product of DGK, PA, regulates a number of signaling proteins such as phosphatidylinositol-4-phosphate 5-kinase, Raf-1 kinase, atypical PKC, p47phox and sphingosine kinase. It is noteworthy that chimaerins are activated by both PA and DG and that the translocation of PKCε to the plasma membrane requires the simultaneous production of PA and DG.(18) The importance of these second messengers implies a tight control of their interconversion by DGK, thus justifying the functional and structural diversity of this family, comprising 10 distinct enzymes grouped into five classes, each featuring distinct regulatory domains and a highly conserved catalytic domain preceded by two C1 domains.(19) An increasing body of evidence indicates that DGK activity is regulated by extracellular factors: DGK-α is activated by HGF and VEGF in endothelial and epithelial cells;(20,21) DGK-ζ is activated by gonadotropin-releasing hormone;(22) and DGK-θ is activated by nerve growth factor (NGF) in PC12 cells.(23) Furthermore, all DGK are recruited by β-arrestin to the activated muscarinic receptor.(24) Some DGK isoforms could be involved in cell transformation: DGK-α is activated by v-Src and NPM-ALK oncogenes and is required for matrix invasion, proliferation, and survival of transformed cells,(21,25,26) whereas DGK-ι is required for v-Ha-Ras induced tumor formation in vivo.(27)

We have investigated the role of DGK in the different biological responses elicited by HGF in the SLK KS cell line using R59949, a pharmacological inhibitor rather specific for class I DGK comprising DGK-α, -β, and -γ.(28) We also used an siRNA panel to identify the DGK isoforms involved in KS cell migration and anchorage-independent growth.

Materials and Methods

Reagents, antibodies and cell culture.  Recombinant HGF was from Preprotech (Rocky Hill, NJ, USA). R59949 was from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO, which was added in the same amounts in the controls and never exceeded 0.1%. DO-24 and DL-21 anti-Met mAbs were purified from ascitic fluid by ammonium sulfate precipitation and affinity chromatography on protein A–Sepharose 4B.(29,30) Antibodies specific for ERK, P-ERK, AKT, and P-AKT were from Cell Signaling Technologies (Danvers, MA, USA). Cell culture medium and reagents were from Invitrogen, (Carlsbad, CA, USA). SLK cells were originally isolated from a KS biopsy from a patient who underwent immunosuppressive therapy after kidney transplantation.(31) KS-IMM cells were already described.(10) 293T was from Invitrogen. Cells were maintained in DMEM, 10% FCS, penicillin (100 U/mL), streptomycin (100 μg/mL), and fungizone (0.25 μg/mL) in a humidified atmosphere of 5% CO2 air.

Cell migration assays.  For wound healing assay, cells (2.5 × 105/well) were grown in 24-well plates, allowed to reach confluence, then further incubated in DMEM 0.2% FCS for 18 h. The monolayers were then wounded with a plastic pipette, washed with PBS and incubated for 24 h in the same spent medium with or without HGF and R59949. Cells were fixed with 11% glutaraldehyde and stained with 0.1% crystal violet in 20% methanol, as described.(10) Images were taken by a digital camera.

For migration on gelatin, cells (2 × 105/50 μL) were seeded in the upper chamber of a modified Boyden chamber (Neuroprobe, Gaitherburg, MD, USA). The under surface of a PVDF filter (8 μm pores; Nucleopore, Pleasanton, CA, USA) was coated with 0.1% gelatin. The lower chamber was filled with serum-free medium with or without HGF and incubated for 18 h. Where indicated, 10 μM R59949 was added to both the upper and the lower chambers. Filters were then removed, stained with Diff-Quik (Baxter Diagnostic, McGaw Park, IL, USA) and migrated cells were counted using an inverted microscope with a high-power oil immersion objective (Zeiss, Milan, Italy).

Matrigel morphogenetic assay.  Cells (2 × 104/well) were plated on Matrigel (Becton Dickinson, Milan, Italy) (growth factor-free, 0.2 mL/well) precoated 48-well plates. After 48 h incubation with or without the indicated treatments, the 3D organization of the cells was examined under an inverted phase contrast photomicroscope (Zeiss) and photographed.

Adhesion assay.  Cells (105/well), detached with 10 mM EDTA in PBS, washed and resuspended in serum-free DMEM, with or without R59949, were plated on 96-well plates precoated with 10 μg/mL fibronectin (Chemicon, Prodotti Gianni Spa, Milano, Italy), 100 μg/mL collagen I (Sigma-Aldrich), 10 μg/mL BSA or 100 μg/mL polylysine and let adhere for 45 min. Non-adhered cells were washed away with PBS, adhered cells were fixed in 4% formaldehyde, stained with crystal violet, and solubilized in DMSO. Absorbance was read at 570 nm.

Spreading assay.  Cells were detached, washed, and resuspended in serum-free DMEM, with or without R59949, and were plated (3 × 104/well) on 24-well plates precoated with 10 μg/mL fibronectin or 100 μg/mL collagen I for experimentally defined times (40 and 60 min, depending on the coating protein). Non-adhered cells were washed away by PBS. Adhered cells were fixed in 4% formaldehyde, observed by a 20× objective in phase-contrast microscopy and five pictures were taken for each well (Zeiss). In each well more than 150 cells were scored and the ratio of spreaded versus total number was used to calculate the percentage of spreading.

Cell proliferation assay.  Cells (2.5 × 103/microtiter well) were plated in 10% FCS for 18 h, starved in serum-free medium for 18 h, then treated with HGF (100 ng/mL) or FCS (10%), with or without R59949, for 72 h. The BrdU incorporation was measured by ELISA as suggested by the manufacturer (GE Healthcare, Milan, Italy).

Anchorage-independent growth.  Cells (5 × 103/well) were seeded in 12-well plates in semisolid medium (0.3% Seaplaque agar in DMEM 1% or 2% FCS), with or without R59949. Fresh medium with or without R59949 was added weekly; after 2–3 weeks colonies were stained with MTT, photographed with a VersaDoc imager and counted with Quantity One colony counting software (Bio-Rad Laboratories, Milan, Italy).

Caspase-3 activity assay.  Cells (2 × 105/well) were seeded in 12-well plates in 10% FCS for 18 h, then incubated in serum-free medium with or without R59949 (1 μM or 10 μM ) or staurosporin (1 μM) for 30 h. Specific caspase-3 activity was measured with a fluorimetric assay (Caspase 3 Assay kit; Sigma-Aldrich).

Phosphoprotein quantification.  Quiescent confluent cells in 12-well plates were pretreated or not with R59949 (10 μM, 30–180 min) and stimulated with the HGF or the Met receptor agonist mAb DO-24 (10 nM, 30 min), rinsed with cold PBS, lysed in DIM buffer (50 mM PIPES [pH 7.4], 300 mM saccharose, 100 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 100 μM ZnCl2), containing 1% Triton X-100, 1 mM ortho-vanadate and a cocktail of protease inhibitors (Sigma-Aldrich) for 10 min, then extracts were clarified. They were separated in SDS-PAGE, transferred to PVDF filters (GE Healthcare) and blotted with different antibodies. Similarly, stimulated cells were lysed with the Bio-Plex cell lysis kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. Protein concentration was determined with BCA (Pierce Thermo Fisher Scientific, Rockford, IL, USA), samples were then diluted to 0.5 mg/mL and frozen. P-ERK1/2, P-Jnk, P-p38, and P-AKT levels were quantified by Bio-Rad Laboratories at the Molecular Biology Center (Torino, Italy) using the Bio-Plex phosphoprotein assay. Positive controls were run in parallel for each phosphoprotein. For each condition three samples were quantified in duplicate.

Determination of human DGK isoforms using RT-PCR.  RNA was extracted from 2 × 106 growing SLK, 293FT, and HL60 cells using the ChargeSwitch Total RNA Cell kit (Invitrogen). RNA aliquots (200 ng) were retrotranscribed using the RT-kit (Applied Biosystems, Austin, TX, USA). One-tenth of the cDNA obtained was amplified using Go-Taq Master Mix (Promega, Milan, Italy) and the appropriate primers (Table S1). The PCR conditions were: 95°C for 2 min, 30 cycles at 95°C for 45 s, 60°C for 45 s, 72°C for 45 s, and at the end 72°C for 5 min. The amplified products were resolved by 2% agarose gel electrophoresis, stained with ethidium bromide and documented with the GelDoc system (Bio-Rad Laboratories). Amplified products match the expected molecular weight.

For mRNA relative quantification, total RNA from cultured cells was extracted by TRIzol reagent (Applied Biosystems) and retrotranscribed with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time PCR was carried out with the ABI7200 Sequence Detection System (Applied Biosystems) using the following assays: Hs00176278_m1 (DGK-α); Hs01114141_m1 (DGK-δ); Hs00177537_m1 (DGK-ε); Hs01632414_s1 (DGK-ζ); Hs00410739_m1 (DGK-η); Hs01092337_m1 (DGK-θ); Hs00177993_m1 (DGK-ι); and Hs00939627_m1 (β-glucuronidase) as endogenous control.

RNA interference.  SLK cells were transfected with Oligofectamine with Plus additive (Invitrogen). The siRNA was chemically synthesized as double-stranded RNA (Applied Biosystems). The sequence was designed using a Cenix BioScience (http://www.cenix-bioscience.com/) algorithm, considering Tm, nucleotide content of the 3′ overhangs, nucleotide distribution over the length of the siRNA, and presence and location of mismatches to off-target genes. A key step in the algorithm is a stringent analysis of each siRNA sequence to maximize target specificity. Preliminary experiments with fluorescently labeled oligonucleotides indicated a transfection efficiency of at least 60% and a permanence in SLK cells for at least 3 days (data not shown).

For the wound-healing experiments, cells (105/well) were seeded in 24-well plates for 24 h and the monolayers were transfected in serum-free media. At 24 h, monolayers were wounded, washed with PBS, and incubated for an additional 24 h in the same medium with or without HGF (100 ng/mL). In this condition, wounding is not complete and was quantified counting cells that had migrated along 2 mm of the wound. For the soft agar assay, cells were transfected and after 18 h subjected to the soft agar assay for 1 week in 10% serum or 3 weeks in 2% serum.

Statistical analysis.  Data are shown as the mean ± SEM. For statistical analysis, Student’s t-test was used. Experiments shown are representative of three or more carried out, except for the Bio-Plex phosphoprotein analysis and siRNA screens, which were repeated twice.

Results

Diacylglycerol kinase inhibitor impairs HGF-induced motility of KS cells.  The DGK inhibitor R59949 was shown to impair the HGF-induced endothelial cells migration and in vitro angiogenesis, mainly by inhibiting DGK-α.(20,21) We investigated whether this inhibitor also impairs the migration and 3D organization of KS cells, which were shown to migrate in an HGF-dependent manner.(10)

In a wound healing assay, carried out with quiescent confluent cells incubated with low serum, R59949 at a concentration of 10 μM significantly impaired wound healing induced by 50 nM HGF (Fig. 1a). Moreover, in a Boyden chamber assay in serum-free medium used to better quantify cell migration, HGF (100 ng/mL) induced a relevant SLK cell migration, which was completely abrogated by 10 μM R59949 (Fig. 1b). Interestingly, 10 μM R59949 did not affect the very strong migration induced by 10% FCS, possibly due to sequestration of the inhibitor by serum proteins.(32)

Figure 1.

 R59949 inhibits hepatocyte growth factor (HGF)-induced motility of Kaposi’s sarcoma cells. (a) Monolayers of confluent quiescent SLK cells in 0.2% FCS were wounded and incubated with (+) or without (−) HGF (50 ng/mL), ±R59949 (1 μM) for 24 h. Cells were then fixed and stained. (b) SLK cell migration in Boyden chambers, with chemoattractants (50 ng/mL HGF or 10% FCS) in the lower compartments, ±10 μM R59949 in both compartments. Values are the means of six replicates ± standard error. *< 0.05, Student’s t-test. (c) Cells were plated on growth factor-deprived Matrigel and stimulated with HGF (100 ng/mL) ± 10 μM R59949 for 48 h. Cells were photographed with contrast phase microscopy. (d) Migration of Kaposi-derived KS-IMM cells in Boyden chambers, with chemoattractants (100 ng/mL HGF) in the lower compartments, ±10 μM R59949 in both compartments. Values are the means of four replicates ± standard error. *< 0.05, Student’s t-test. Representative of three independent experiments.

We also tested the effect of R59949 on the ability of SLK cells to form tubular-like structures on Matrigel, an assay used to test in vitro pro- or anti-angiogenic properties of different molecules.(33) When plated on Matrigel and stimulated with HGF (100 ng/mL), SLK cells connected and organized in tubular-like structures, and this activity was severely impaired by 10 μM R59949 (Fig. 1c). We also assayed the effect of R59949 on the motility of an independently generated Kaposi-derived cell line, KS-IMM.(10) In these cells, 10 μM R59949 significantly reduced HGF-induced cell migration both in wound healing (data not shown) and Boyden chamber assays (Fig. 1d).

Taken together, these data indicate that R59949-sensitive DGK activity is required for HGF-induced KS cell migration and morphogenesis on Matrigel.

Adhesion properties of SLK cells depend on DGK activity.  In the tumor progression toward a metastatic phenotype, cell adhesion to the extracellular matrix plays a pivotal role, as cells must loose their normal adhesion properties and contextually acquire new ones to home and give origin to secondary tumors. Two aspects of SLK cell adhesion were analyzed: the ability to interact with different ECM molecules and the ability to spread on them. The former depends on the pattern of the cell surface integrins and their activation state. The latter recapitulates the molecular events required to primary tumor cells to home to different body sites, when metastasis occurs.

Preliminary time-course experiments, where cells detached by EDTA were let adhere in serum-free medium to fibronectin and collagen (not shown), indicated that maximal difference between control and R59949 treated cells was observed at 45 min. Indeed, adhesion of SLK cells onto both fibronectin and collagen I was significantly reduced by 10 μM R59949 (Fig. 2a). In the controls, adhesion to polylysine, mediated by its positive charge attracting negatively charged cell surface phospholipids and glycosaminoglycans, was not affected, and cell adhesion to BSA was negligible.

Figure 2.

 R59949 partially inhibits adhesion and spreading of SLK cells on fibronectin and collagen I. (a) Cells were seeded on the different matrices with (+) or without (−) R59949 (1 or 10 μM). After 45 min, non-adhered cells were washed away and adhered cells were fixed, stained with crystal violet, and solubilized with DMSO. Absorbance was read at 570 nm in a microplate reader. The values on the Y-axis represent the mean of absorbance units (AU) of quadruplicates, ± standard error. (b) Cells were allowed to adhere on fibronectin for 40 min or on collagen I for 60 min ± 10 μM R59949, then non-adherent cells were washed away. The values on the Y-axis represent the percentage of the spreaded cells relative to total cells in five microscopic fields. Values are the means of three replicates ± standard error. *< 0.05, Student’s t-test. Representative of three independent experiments.

Spreading occurs concomitantly with adhesion with slightly different kinetics, depending on the coating matrix. Plated cells were periodically controlled and fixed when approximately 50% of the cells in the untreated controls were spread, namely 40 min in the case of fibronectin and 60 min in the case of collagen I. Spreading was significantly affected by 10 μM R59949 treatment: it was inhibited by 40% on fibronectin and by 20% on collagen I (Fig. 2b). As in the chemotaxis assay, 1 μM R59949 was ineffective (not shown).

Altogether, these data demonstrate that DGK activity, sensitive to 10 μM R59949 treatment, is required for SLK cells to achieve adhesion and spreading onto common ECM molecules.

Diacylglycerol kinase activity required for proliferation and anchorage-independent growth of KS cells.  Like most neoplastic cells, SLK cells show a decreased requirement for growth factors,(31,34) reflected in a consistent basal DNA synthesis, which increases in the presence of HGF (100 ng/mL) or 10% serum. R59949 significantly impaired HGF-induced DNA synthesis in a dose-dependent manner (Fig. 3a,b). Interestingly, R59949 at any concentration did not induce cell detachment or relevant caspase-3 activation, a hallmark of apoptosis which, as expected, was induced by staurosporine, used as control (Fig. 3c).(21)

Figure 3.

 R59949 inhibits hepatocyte growth factor (HGF)-induced anchorage-dependent and anchorage-independent proliferation of Kaposi’s sarcoma cells without promoting apoptosis. (a,b) SLK cells (2.5 × 103) were plated in the presence of FCS for 18 h, then incubated for 30 h in serum-free medium with (+) or without (−) HGF (100 ng/mL), ±R59949 (1 μM in (a), 10 μM in (b)) or 10% FCS. Incorporation of BrdU was quantified in the last 6 h. Values are the means of four replicates ± standard error. *< 0.05, Student’s t-test. (c) SLK cells (2 × 105) were plated in the presence of FCS for 18 h, then incubated in serum-free medium ± R59949 (1 and 10 μM) or staurosporin (1 μM). After 30 h, caspase-3 activity was assayed with a fluorigenic substrate. Values are the means of three replicates ± standard error. AU, absorbance units. *< 0.05, Student’s t-test. (d) SLK cells were seeded in 0.3% SeaPlaque agar with 2% FCS ± 10 μM R59949 and replenished once a week with DMEM 2% FCS. Cells were photographed and counted after 3 weeks. Values are the means of the triplicates ± standard error. *< 0.05, Student’s t-test. (e) Kaposi-derived KS-IMM cells were seeded in 0.3% SeaPlaque agar with 1% FCS ± R59949 and replenished once a week with DMEM 1% FCS. After 2 weeks colonies were stained and counted. Values are the mean of the triplicates ± standard error. *< 0.05, Student’s t-test. Scale bar = 5 mm.

Kaposi’s sarcoma cells are tumorigenic in mice.(35) The assay best featuring this property in vitro is the anchorage-independent proliferation. Indeed SLK cells are very resistant to anoikis, or detachment-induced cell death (not shown), and when plated in soft agar, they gave rise to colonies clearly visible after 21 days. The colonies were inhibited in a dose-dependent manner by treatment with R59949, already significantly effective at 1 μM and giving maximal inhibition at 5–10 μM (Fig. 3d). The same kind of experiment was carried out on KS-IMM cells, which proliferated rapidly even in the absence of growth factors (data not shown) and formed detectable colonies in soft agar after only 14 days. With these cells, R59949 inhibited colony formation in a dose-dependent manner and almost completely at 5–10 μM (Fig. 3e).

These data show that at the low doses inhibiting class I DGK, R59949 impairs the growth of KS cells both in adhesion and in the absence of adhesion without promoting cell death.

Effects of DGKα inhibition on HGF receptor signal transduction.  From the above data it is clear that pharmacological inhibition of DGK severely affects the transduction of HGF chemotactic and proliferative signals in SLK cells. We thus investigated whether R59949 treatment affected the HGF receptor signaling pathways activated in KS cells and mediating proliferation and movement.(14,36) Preliminary Western blot analysis on total cell extracts indicated that maximal phosphorylation of ERK-1/2 and AKT was observed at 15–30 min of stimulation with HGF and that 15 min pretreatment with 10 μM R59949 did not significantly affect the activation of these signalling pathways (Fig. 4a). We also verified the effect of prolonged treatment with 10 μM R59949 on the levels and phosphorylation of HGF receptor and transducers. Pretreatment with R59949 had no significant effect, except for a marginal inhibition of AKT activation (Fig. 4b). As Western blot analyses are rather qualitative, we evaluated HGF-induced signal transduction using the BioPlex phosphoprotein assay, a system that allows the quantitative and simultaneous evaluation of the phosphorylation status of multiple transducers. In these experiments, cells were stimulated with the mAb DO-24 (10 nM), already shown to be a full agonist of the HGF receptor in different cell systems.(14,30,37) Both ERK-1/2 and AKT were significantly phosphorylated after 30 min of stimulation, whereas p38 and Jnk showed only a small increase in their phosphorylation (Fig. 4c–f). Also using this quantitative system, ERK-1/2 and AKT phosphorylation was not significantly affected by 30 min pretreatment with 10 μM R59949, although a slight decrease was detected (Fig. 4a,b).

Figure 4.

 Effects of R59949 on hepatocyte growth factor (HGF) receptor signal transduction. (a) SLK cells were treated with (+) or without (−) 10 μM R59949 for 30 min before being stimulated or not with HGF (100 ng/mL) for the indicated time. Cells were then lysed and analyzed using Western blotting for phosphorylated and total Erk-1/2 and Akt levels. (b) SLK cells were treated with or without 10 μM R59949 for 180 min before being stimulated or not with HGF (100 ng/mL) for 15 min. Cells were then lysed and analyzed using Western blotting for HGF receptor levels and for phosphorylated and total Erk-1/2 and Akt levels. (c–f) SLK cells were treated with or without 10 μM R59949 for 30 min before being stimulated or not with the HGF receptor agonist mAb DO-24 (10 nM) for 30 min. Cells were then lysed and, after adjusting protein concentration to 0.5 μg/mL, phosphorylation of Erk-1/2 (a), Akt (b), p-38 (c), and Jnk (d) was measured using the BioPlex method. Data are the means of triplicates ± standard error. *< 0.05, Student’s t-test.

These data demonstrate that in SLK cells the DGK inhibitor only marginally affect the ras-ERK-1/2 MAPK and PI3K-AKT pathways, suggesting that DGK are involved in alternative pathways mediating HGF-induced motility and proliferation.

SLK cells express multiple DGK, contributing to malignant phenotype.  Reverse transcription-PCR experiments were then carried out on mRNA prepared from SLK cells with specific primers to investigate which DGKs are expressed on SLK cells. It was found that among class I DGK, the class for which R59949 displays more specific inhibitory activity at low concentration, only DGK-α was expressed, although at low levels, when compared to DGK isoforms of other classes. Moreover, SLK cells expressed all other tested classes of DGK, except the more recently identified κ isoform (Fig. 5a).

Figure 5.

 Diacylglycerol kinase (DGK) isoforms expressed in SLK cells and their involvement in wound healing. (a) RT-PCR was carried out with primers for different DGK isoforms on the SLK cell line. 293FT and HL-60 were used as positive controls. rRNA18S was used as loading control. (b) SLK confluent monolayers were transfected with the indicated siRNA and after 24 h in 0% serum were wounded and incubated without (−) or with hepatocyte growth factor (HGF; 100 ng/mL) for 24 h. After fixing and staining, cells migrated along 2 mm of the wound were counted. Data are the means ± standard error of four replicates from two independent experiments. *< 0.05, Student’s t-test.

Experiments with DGK-specific siRNA were then carried out to confirm the role of these enzymes in the malignant phenotype of KS cells and to try to identify which isoform/s is/are involved. We first designed a panel of isoform-specific siRNA, comprising three specific siRNA for each DGK isoform, to grant that at least one sequence would interfere with the target, and a random sequence not matching any human RNA as control. Each siRNA was validated for its ability to specifically downregulate the target mRNA measured by quantitative RT-PCR. After 24 h of transfection, 18 siRNA (90%) out of the 20 tested decreased their target mRNA by at least one-third, with only one not specifically interfering with an off-target isoform (Table S2). Interference was transient and decreased at 48 h, probably because of the high proliferation rate of transformed cells. We also noticed an increase in DGK-ι expression, possibly representing a compensatory response (Table S2).

The role of specific DGK isoforms on cell migration was investigated in a wound healing assay. An SLK cell monolayer was transfected with isoform-specific siRNA before wounding and allowed 24 h of healing in the presence or absence of HGF (100 ng/mL). In this experimental setup wound healing is not complete and was quantified by counting cell migration along 2 mm of the wound. All three siRNA targeting DGK-δ severely impaired HGF-induced migration, strongly indicating that this isoform plays an essential role in SLK migration. Moreover, single siRNAs targeting DGK-ζ, η, and θ isoforms impaired cell migration, suggesting they may play a role in HGF-promoted migration.

In order to address the role of DGK isoforms on anchorage-independent growth, cells were transfected with isoform-specific siRNAs and their growth rate was assayed in a short protocol (10% serum, 1 week) or the previously used protocol (2% serum, 3 weeks). Two out of three siRNA against DGK-ι significantly reduced soft agar growth (Table 1) in both experimental setups, strongly indicating that DGK-ι is required for anchorage-independent growth. Single effective siRNA targeting the DGK-α, ζ, η, and θ isoforms were also observed, suggesting that they may contribute to anchorage-independent proliferation.

Table 1.   Diacylglycerol kinase (DGK)-specific siRNA inhibit anchorage-independent proliferation of Kaposi’s sarcoma cells
TargetsiRNA ID1 week in 10% FCS
(colonies, % of control siRNA)
3 weeks in 2% FCS
(colonies, % of control siRNA)
  1. Data are the mean ± SEM of triplicate experiments. *< 0.05 versus control siRNA, Student’s t-test. n.d., not done.

DGK-αDGK-α1118 ± 285 ± 18
DGK-αDGK-α289 ± 4*10 ± 7*
DGK-δ39723131 ± 339 ± 9*
DGK-δ139966104 ± 10n.d.
DGK-δ13996792 ± 7n.d.
DGK-ε43699 ± 571 ± 26
DGK-ε435108 ± 14n.d.
DGK-ε43498 ± 12n.d.
DGK-ζ432119 ± 773 ± 23
DGK-ζ13999690 ± 4*n.d.
DGK-ζ139997105 ± 5n.d.
DGK-η129948103 ± 474 ± 13*
DGK-η43673106 ± 12n.d.
DGK-η129949113 ± 13n.d.
DGK-θ145795125 ± 2584 ± 18
DGK-θ12469 ± 4*n.d.
DGK-θ160990 ± 11n.d.
DGK-ι57285 ± 5*64 ± 8*
DGK-ι571107 ± 8n.d.
DGK-ι13762572 ± 1*n.d.

Discussion

Kaposi’s sarcoma is a multifactorial neoplasia, in which growth factors, inflammatory cytokines, and proteins encoded by human herpes virus type 8 and HIV-1, namely G protein coupled receptor and Tat, respectively, together contribute to the onset, progression, and maintenance of the disease and to its characteristic vascular phenotype.(6) In particular, HGF and its Met receptor are involved in the pathogenesis of KS,(8–11) whose biological features (high vascularization and invasiveness) are compatible with the biological properties of HGF, which also induces or upregulates the expression of VEGF, the most important endothelial growth factor.(38) The different biological aspects of the disease are sustained by the multiplicity of signal pathways, which are activated by HGF and other cytokines/growth factors, namely the ras-ERK-1/2 and the AKT-PI3 kinase pathways.(14)

Recent studies have revealed that DGK isozymes play pivotal roles in transducing signals involved in development, neural and immune responses, cytoskeleton reorganization, and carcinogenesis.(18) Here we have shown that DGK-mediated conversion of DG to PA is required for major biological features of the transformed phenotype of KS cells, namely migration, proliferation, and anchorage-independent growth, as all of these activities are impaired by the pharmacological inhibitor R59949. Diacylglycerol kinase enzymes play an essential role in KS cell proliferation. R59949 strongly reduces their high spontaneous proliferation observed in routine cultures and completely abolishes their HGF-induced proliferation without affecting cell viability, suggesting that DGK activity is required for cell cycle progression (Fig. 3). Furthermore, R59949 strongly reduces anchorage-independent growth, the in vitro hallmark of cell transformation. The indication that a DGK sensitive to R59949 is essential for KS growth, even in the absence of adhesion, suggests that these enzymes are promising targets for pharmacological intervention on KS cells.

SLK cells express several DGK isoforms, namely α, δ, ε, ζ, η, θ, and ι. DGKα is strongly inhibited by R59949,(20) whereas DGK-δ, ε, and θ are poorly inhibited by R59949(39–41) and DGK-ζ is resistant to R59949.(42) No information is available on the R59949 sensitivity of DGK-η and ι, but their homology to DGK-δ and ζ suggests they may be resistant.

We observed that multiple siRNAs against DGK-ι reduced significantly, but not completely, KS anchorage-independent growth, a key feature of tumorigenic cells. Consistently, in vivo lack of DGK-ι strongly impairs PMA driven and H-Ras driven skin tumorigenesis.(27) We observed similar effects with single siRNA targeting DGK-α, ζ, η, and θ, however, those isolated hits may represent either false positives in the screen, or the involvement of these isoforms in anchorage-independent growth (Table 1). The involvement of multiple DGK, some of them poorly inhibited by R59949, might explain the observation that low R59949 concentrations (1 μM), which effectively inhibit only class I DGK, partially reduced proliferation and anchorage-independent growth, whereas higher concentrations (10 μM) have much more dramatic effects (Fig. 3).

Conversely, higher concentrations (10 μM) of R59949 are required to inhibit HGF-induced cell migration and morphogenesis on Matrigel. In addition, at the same concentrations, R59949 decreases cell adhesion and spreading onto fibronectin and collagen suggesting that an impairment in integrin mediated adhesion and signaling could underlie the defects in cell movement. A similar R59949-induced defect in cell migration and matrix invasion has been reported in endothelial cells(20,21) and in transformed epithelial cells.(25,43) The higher R59949 concentration required to impair cell movement suggests the involvement not only of DGK-α but also of other DGK isoforms less sensitive to R59949.(28) We observed that three independent siRNA targeting DGK-δ abolished HGF-induced migration of SLK cells, clearly indicating that DGK-δ plays a role in SLK cell migration (Fig. 5). Also in this case, the presence of single effective siRNA against DGK-ε, η, and θ suggests the involvement of these multiple isoforms in cell migration. Indeed both DGK-α and DGK-ζ contribute to the regulation of Rac1 activity, establishing a link between PA formation and actin cytoskeleton dynamics in growth factor stimulated cells.(25,44)

By consuming DG and producing PA, DGK affect membrane recruitment and modulate the activity of several signaling proteins, such as classical and atypical PKC, Ras GDP release factor, Rac1, phosphatidylinositol-4-phosphate 5-kinase, Raf-1 kinase, mTOR, and p47phox.(17,18) Thus, acting both at the plasma membrane and in the nucleus, DGK are involved in multiple cell responses such as the control of cell proliferation, survival, and motility.(18,19,45) For example, by positively regulating PKC-ζ and inducing nuclear factor-κB nuclear translocation, DGKα was reported to counteract apoptosis induced by tumour necrosis factor-α.(46) In contrast, other DGK isoforms reside or translocate to the nucleus where they contribute to the nuclear phosphatidylinositol cycle.(45) Interestingly, nuclear DGK-θ produces the PA necessary for the transcriptional activity of steroidogenic factor 1,(47) suggesting that DGK might also directly control cell transcriptional activity. At the cell periphery, DGK-δ has been previously linked to the control of tyrosine kinase signaling, as knock-out studies have shown that the absence of DGK-δ activity promotes PKC-mediated EGF receptor downregulation.(48) What is less clear is whether other DGK isoforms(21,26,49) are regulated by extracellular signals, and their specific role in tyrosine kinase signaling.

In this context we found that the activation of AKT and ERK-1/2, two signal transducers essential for KS cell response to growth factors in general, and to HGF in particular,(14) are only minimally perturbed by the R59949 inhibitor. Furthermore, prolonged R59949 treatment did not affect HGF receptor expression or signaling capability. This observation suggests that DGK are downstream effectors of HGF signaling and do not cross-talk with the ras-ERK-1/2, PI3K-AKT, or p38 pathways. This opens the possibility that DGK could be an alternative and additional therapeutic target, which is extremely important in view of the fact that drug resistance is a common finding in oncology.

Acknowledgments

This work was supported by grants from: (i) ISS, Programma Nazionale sull_AIDS, Progetto “Patologia, clinica e terapia dell’AIDS” (Rome, Italy) to MP; (ii) MIUR, Progetto “Aspetti clinici e patogenetici del sarcoma di Kaposi.” to MP and PRIN 2008 to AG; (iii) AIRC project 5392 to AG; (iv) CARIPLO 2010-0737 to AG; (v) Regione Piemonte (Ricerca Finalizzata) to MP and AG; (vi) Regione Piemonte through L.R. 4/2006 for brain drain containment, and D.R. 392-2007 to FC. We thank S. Cutrupi, S. Nocera, and E. Ruffo for their involvement in aspects of this study.

Disclosure Statement

The authors have no conflicts of interest to declare.

Abbreviations
DG

diacylglycerol

DGK

diacylglycerol kinase

HGF

hepatocyte growth factor

IL

interleukin

KS

Kaposi’s sarcoma

PA

phosphatidic acid

PKC

protein kinase C

PI3-K

phosphatidylinositol 3-kinase

VEGF

vascular endothelial growth factor

Ancillary