Abstract
UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a key role in biosynthesis of vitamin K2 and coenzyme Q10 using geranylgeranyl diphosphate (GGPP). However, the mechanism by which UBIAD1 participates in tumorigenesis remains unknown. This study show that UBIAD1 interacts with H-Ras, retains H-Ras in the Golgi apparatus, prevents H-Ras trafficking from the Golgi apparatus to the plasma membrane, blocks the aberrant activation of Ras/MAPK signaling, and inhibits the proliferation of bladder cancer cells. In addition, GGPP was required to maintain the function of UBIAD1 in regulating the Ras/ERK signaling pathway. A Drosophila model was employed to confirm the function of UBIAD1/HEIX in vivo. The activation of Ras/ERK signaling at the plasma membrane induced melanotic masses in Drosophila larvae. Our study suggests that UBIAD1 serves as a tumor suppressor in cancer and tentatively reveals the underlying mechanism of melanotic mass formation in Drosophila.
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Introduction
Ras proteins are essential components of the intracellular signaling cascades that regulate various fundamental cellular activities such as proliferation, apoptosis, differentiation, and senescence1,2. One of the central paradoxes involving Ras signal transduction is how Ras, which acts as a simple binary switch, can generate many different biological outputs3,4,5. The intracellular trafficking cycle of Ras partially accounts for this phenomenon6. Ras interacts dynamically with specific microdomains of the plasma membrane and other internal cell membranes. These different membrane microenvironments modulate Ras signal output, highlighting the complex interplay between Ras location and function7. For example, Ras signaling in the plasma membrane is rapid and transient, whereas that in the Golgi apparatus is delayed and sustained8. Ras activates Raf-MEK-ERK in the plasma membrane and leads to cellular growth6. Cellular differentiation is induced when Ras activates Raf or PI3K in the Golgi apparatus9,10.
H-Ras, which accounts for all Ras mutations in bladder carcinoma6, is also common in thyroid cancer, salivary carcinoma11, epithelial myoepithelial cancer and kidney cancer12. After being synthesized in cytosolic ribosomes, H-Ras undergoes farnesylation at the cysteine of the C-terminal CVLS motif in the cytosol, followed by proteolysis of the VLS sequence in the endoplasmic reticulum (ER). However, as farnesylation of Ras provides only a weak signal for membrane interaction, another motif in the HVR (hypervariable region) of Ras is required for membrane association. Two palmitoylation groups at the cysteine, adjacent to the farnesylated cysteine of H-Ras, are the second membrane-anchoring signal. Palmitoylation primarily occurs at the Golgi surface, and palmitoylated H-Ras then aggregates to the plasma membrane via a secretory pathway for eventual insertion into the plasma membrane13. In addition, palmitoylated H-Ras undergoes dynamic de/re-acylation with depalmitoylation leading to detachment from the plasma membrane to facilitate transfer to the Golgi apparatus14,15. Bladder carcinoma with H-Ras mutation16 ranks ninth in cancer incidence worldwide17. Considering that H-Ras induces melanoma development in mice18 and melanotic spots appear in heix/ubiad1 mutant Drosophila19, we focused on establishing the relationship between UBIAD1 and H-Ras.
UBIAD1 (also known as TERE1), a cancer suppressor gene in bladder carcinoma20,21, encodes a class of UbiA prenyltransferases22 and participates in the regulation of cholesterol23,24,25,26. UBIAD1 is widely expressed in various human tissues20 and organelles, including the endothelial reticulum19,22, Golgi apparatus27,28, and mitochondria29. Interestingly, UBIAD1, with both side-chain cleavage and its prenylation activity, is the first enzyme responsible for human vitamin K biosynthesis22,30. Moreover, UBIAD1 is a nonmitochondrial CoQ10-forming enzyme with a specific cardiovascular antioxidant function via regulation of eNOS activity in zebrafish27. Given that UBIAD1 participates in various biological processes, its downregulation or mutation can induce the development of diseases such as cancer20,31, Schnyder crystalline corneal dystrophy (SCCD)32, Parkinson’s disease29, and cardiovascular disease27.
Recent research on this tumor suppressor show that UBIAD1 protects the cholesterol biosynthetic enzyme HMG-CoA reductase from degradation33,34,35 and plays an important role in vascular cell calcification36. DNA methylation of ubiad1 has significant negative associations with EGFR/KRAS mutations in lung adenocarcinoma37. Furthermore, considering that UBIAD1 is downregulated in bladder and prostate carcinomas, and its overexpression inhibits tumor cell proliferation21,38. We previously reported that UBIAD1 knockdown activates the Ras/MAPK signaling pathway39.
Here, we report that UBIAD1 interacts with H-Ras, increases the retention of H-Ras in the Golgi apparatus, inhibits the aberrant activation of Ras/ERK signaling at the plasma membrane and consequently suppresses the proliferation of bladder cancer cells.
Results
UBIAD1 inhibited the activation of the Ras/MAPK signaling pathway
In previous studies, UBIAD1 downregulation has been shown to induce the activation of the Ras/MAPK signaling pathway39, and UBIAD1 has inhibited the growth of bladder (Fig. 1a-c)20 and prostate cancers21. However, the underlying mechanism and relationship between UBIAD1 and Ras/MAPK signaling have not been clearly elucidated. Thus, we examined ERK signaling, following the graded overexpression of UBIAD1 and found dose-dependent inhibition of ERK phosphorylation (p-ERK) in T24 cells (Fig. 1d and Supplementary Fig. S1a, b). To further explore the functional role of UBIAD1 in Ras/ERK signaling, we employed shRNA to knock down endogenous UBIAD1. Phosphorylation of ERK, MEK and c-Raf significantly increased when UBIAD1 was knocked down (Fig. 1e and Supplementary Fig. S1c, d). A rescue assay was performed to confirm the specificity of the silencing effect of UBIAD1-shRNA. Activation of Ras/MAPK signaling by knocking down UBIAD1 was abrogated by UBIAD1 (Supplementary Fig. S1e). In addition, an increase in p-ERK was prevented by the green fluorescence protein-Ras-binding domain (GFP-RBD), which efficiently bound to Ras in the GTP-bound state to competitively inhibit Ras activity (Fig. 1f and Supplementary Fig. S1f). These results indicate that UBIAD1 suppresses Ras activation. UBIAD1 is not expressed in bladder tumors20, and H-Ras mutations, which affect MAPK pathways, are associated with bladder carcinoma40. Therefore, UBIAD1 function might be related to H-Ras. To verify this hypothesis, HEK293T cells were cotransfected with H-Ras (or H-RasG12V) and UBIAD1. UBIAD1 inhibited both H-Ras-induced and H-RasG12V-induced p-ERK (Fig. 1g), which indicates that UBIAD1 is a negative regulator of H-Ras.
We used Drosophila as an animal model to further study and confirm the function of UBIAD1 in vivo. P-ERK levels were increased in heix (the homologous gene of ubiad1) Drosophila mutants (one P-element allele: heixk11403 and one ethylmethansulfonate allele: heix1). Neither heixk11403 nor heix1 express the HEIX protein29. These findings are consistent with a previous study reporting that heix regulates expression of yan, which is mediated by Ras19. Overexpression of the heix gene in heix mutants decreased phosphorylated ERK levels and led to the subsequent disappearance of melanotic masses (Fig. 1h, i, and Supplementary Fig. S1g). Moreover, melanotic masses in heix mutants vanished after U0126 treatment (MEK inhibitor), suggesting that melanotic mass results from abnormal activation of Ras/ERK signaling (Fig. 1j).
UBIAD1 inhibited H-Ras trafficking from the Golgi apparatus to the plasma membrane
Considering that UBIAD1 is a Golgi-localized protein (Supplementary Fig. S2a)28 that acts on H-Ras, we investigated whether UBIAD1 could alter the localization of H-Ras in the Golgi apparatus. When H-Ras (or H-RasG12V) was overexpressed in HEK293T cells, H-Ras was widely localized in the plasma membrane with little traces in the Golgi apparatus, which is consistent with previous reports41,42. However, when coexpressed with UBIAD1-EGFP in HEK293T and T24 cells, the localization of H-Ras in the Golgi apparatus significantly increased (Fig. 2a and Supplementary Fig. S2b, c). We determined whether overexpression of the protein is responsible for the accumulation of H-Ras in the Golgi apparatus. The results showed that UBIAD1 increased the retention of H-Ras in the Golgi apparatus after treatment with cycloheximide, which blocks protein synthesis (Fig. 2a). UBIAD1 increased endogenous pan-Ras retention in the Golgi apparatus of T24 cells (Fig. 2b). UBIAD1 also promoted the localization of endogenous H-RasG12V marked with GFP-RBD in the Golgi apparatus of T24 cells (Supplementary Fig. S2d). Alternatively, when UBIAD1 was deficient in HEK293T cells, high amounts of Ras were transported to the plasma membrane (Fig. 2c and Supplementary Fig. S2e). These data indicate that UBIAD1 regulates the localization of H-Ras (or H-RasG12V) in the Golgi apparatus.
An EGF (Epidermal Growth Factor) assay was performed to analyze the possible connection between the retention of H-Ras in the Golgi apparatus and the decrease in p-ERK. Under unstimulated conditions, GFP-RBD was not associated with any intracellular structures, including the plasma membrane9. By contrast, when cells were treated with EGF, GFP-RBD first aggregated in the plasma membrane (Ras activation in the plasma membrane followed by Ras/ERK signaling) and then in the Golgi apparatus43. However, when GFP-RBD and H-Ras were coexpressed with UBIAD1, UBIAD1 prevented the transport of H-Ras to the plasma membrane under short-term treatment with EGF (Fig. 3a). UBIAD1 decreased p-ERK levels only under short-term treatment with EGF, indicating that ERK phosphorylation was inhibited by UBIAD1 in the plasma membrane (Fig. 3b). Interestingly, when UBIAD1 was deficient, large amounts of Ras were transported to the plasma membrane, which was abrogated by 2BP (2-bromopalmitate, an inhibitor of palmitoyltransferase, which is necessary for Ras transport to the plasma membrane44) (Fig. 3c). Ras inhibitors (FTI-277, Salirasib) and palmitoyltransferase inhibitors (2BP, tunicamycin) prevented ERK phosphorylation in the absence of UBIAD1 (Fig. 3d). This result indicates that the ERK signaling induced by knockdown of UBIAD1 initiated in the plasma membrane.
To confirm that the melanotic mass formation in heix mutants was related to Ras signaling, an inhibition assay was performed, fewer traces of melanotic masses were detected in the heix mutants when treated with FTI-277, Salirasib, 2BP, tunicamycin (Fig. 3e). These data, combined with cellular experimental results, suggest that large quantities of Ras could be transported to the plasma membrane to activate Ras/ERK signaling. Consequently, the activate Ras signaling induced melanotic masses in Drosophila, which is consistent with previous reports19,45.
UBIAD1 interacted with H-Ras in the Golgi apparatus
Considering that UBIAD1 directly acts on Ras signaling and trafficking, we investigated the possibility that UBIAD1 interacts with H-Ras in the Golgi apparatus. When UBIAD1 and H-Ras were overexpressed in HEK293T cells, both molecules colocalized in the Golgi apparatus (Fig. 4a). Consistently, a small portion of the staining pattern of endogenous UBIAD1 overlapped with endogenous Ras in HEK293T cells (Supplementary Fig. S3a).
To provide evidence that UBIAD1 forms a complex with H-Ras, a fluorescence resonance energy transfer (FRET) assay was employed to detect this molecular interaction. Significant FRET signals were detected with 405-515 microscope channels when HEK293T cells were cotransfected with UBIAD1 and H-Ras (Fig. 4b). To determine the possibility of false positives induced by high expression of the protein, we measured FRET signals of UBIAD1/apoE and UBIAD1/Golgi-marker as controls (Supplementary Fig. S3b). FRET between UBIAD1 and apoE, which have confirmed interactions38, served as the positive control, and FRET between UBIAD1-GFP and Golgi-BFP was the negative control. To further confirm interaction between UBIAD1 and H-Ras, bimolecular fluorescence complementation (BIFC) and coimmunoprecipitation (co-IP) assays were performed. BIFC signals were detected only in HEK293T cells cotransfected with UBIAD1 and H-Ras (Supplementary Fig. S3c). In addition, immunoprecipitation of UBIAD1 could pull-down H-Ras (Fig. 4c), and vice versa (Supplementary Fig. S3d, e). Ectopically expressed UBIAD1 interacted with Ras but not with Rab11B (a small GTP-binding protein in the Golgi apparatus) in T24 cells (Supplementary Fig. S4a). UBIAD1 strongly interacted with H-RasG12V compared to its interaction with H-Ras (Supplementary Fig. S4b, c). This finding can explain the more significant effects of UBIAD1 on H-RasG12V than on H-Ras in inhibiting of the Ras/ERK signaling (Fig. 1g). Furthermore, endogenous Ras and UBIAD1 interacted with each other in HEK293T cells (Fig. 4d). A small portion of HEIX was located in the Golgi apparatus of the blood vasculature (Supplementary Fig. S5), and endogenous HEIX interacted with Ras in wild-type Drosophila larvae (Fig. 4e). These data show that UBIAD1 interacts with H-Ras.
UBIAD1 interacted with the C-terminus of H-Ras
In normal cells, H-Ras is farnesylated by FTase in the cytoplasm. Then, the VLS of H-Ras is removed by Ras-converting enzyme (Rce1), and the terminal prenylated cysteine residue is methylated by isoprenylcysteine methyltransferase (Icmt) in the ER. Finally, the complex is palmitoylated by palmitoyltransferase in the Golgi apparatus and transported to the plasma membrane46. To assess the effect of H-Ras trafficking on the interaction between UBIAD1 and H-Ras, we performed knockdown and inhibition assays. We found that interaction between UBIAD1 and Ras decreased following knock down of FTNA (α-domain of FTase and GGTase) (Fig. 5a) and Icmt (Fig. 5b), respectively, but increased with 2-BP (the inhibitor of palmitoyltransferase) (Fig. 5c), indicating that UBIAD1 interacts with H-Ras in the Golgi apparatus.
To determine the binding region of H-Ras for UBIAD1, a truncated H-Ras protein was fused with GFP. As shown in Fig. 5d and Supplementary Fig. S4d, the C-terminus of H-Ras interacted with UBIAD1. To confirm the contribution of the UBIAD1/H-Ras complex to Ras signaling, the C-terminus of H-Ras (GFP-HRas-CT) was used as a competitive inhibitor to block interaction between UBIAD1 and H-Ras. GFP-HRas-CT decreased interaction between UBIAD1 and H-Ras, and increased the level of p-ERK (Fig. 5e). In addition, UBIAD1 could not decrease the H-Ras-induced or H-RasG12V-induced p-ERK in cells overexpressing H-Ras-CT (Fig. 5f). GFP-H-Ras-CT blocked the inhibition of UBIAD1 in T24 cell viability (Fig. 5g) and proliferation (Fig. 5h, i). Hence, UBIAD1/HEIX physically interacts with Ras, and this interaction inhibits Ras signaling and T24 cell proliferation.
Geranylgeranyl pyrophosphate (GGPP) was required for interaction between UBIAD1 and H-Ras in the Golgi apparatus
UBIAD1 utilizes GGPP as a source of geranylgeranyl side-chains during biosynthesis of MK-422 and CoQ1027. Statins (simvastatin) can inhibit the mevalonate pathway via 3-hydroxy-3-methylglutaryl-coenzyme A reductase, leading to a simultaneous decrease in the production of FPP (farnesyl pyrophosphate) and GGPP27,33,47,48. In our study, we found that statins abrogated the influence of UBIAD1 on the Ras/ERK signaling pathway (Supplementary Fig. S6a, b). However, statins were also able to disrupt the function of Ras because FPP is necessary for the post-translational modification of Ras (Supplementary Fig. S6c). Therefore, statins can block the function of both UBIAD1 and Ras, consistent with those of previous studies40,47. This event is prevented by knocking down geranylgeranyl diphosphate synthase (GGPPS), which synthesizes GGPP from FPP49. Thus, knockdown of GGPPS decreased interaction between UBIAD1 and H-Ras, which was abrogated by supplementary GGPP (Fig. 6a). Moreover, a lack of GGPPS abolished UBIAD1-induced H-Ras retention in the Golgi apparatus (Fig. 6b and Supplementary Fig. S7). To further confirm whether GGPP was required for the proper function of UBIAD1 on Ras/ERK signaling and cell proliferation, p-ERK, cell viability and cell proliferation were evaluated under a lack of GGPPS. As shown in Fig. 6c–f, GGPPS knockdown attenuated the decrease in p-ERK level and abrogated the inhibition of cell viability and cell proliferation after UBIAD1 transfection for 24 h. This result suggests that GGPPS knockdown interfered with the ability of UBIAD1 to regulate Ras/ERK signaling. Moreover, supplementary GGPP rescued the effect of GGPPS knockdown (Fig. 6a–f and Supplementary Fig. S7), indicating that GGPP contributes to the function of UBIAD1 in the Ras/ERK signaling pathway.
Mutations of UBIAD1 induced a loss of UBIAD1 function in Ras/MAPK signaling in the Golgi apparatus
Our study showed that UBIAD1 induced H-Ras retention in the Golgi apparatus via GGPP. UBIAD1 mutants were then used to confirm this observation. UBIAD1N102S is not able to bind to GGPP and be transported to the Golgi apparatus47,50, and UBIAD1RPWS does not localize in the Golgi apparatus28. Figure 7a shows that UBIAD1 mutants did not localize in the Golgi apparatus, which is consistent with previous studies28,33. Interestingly, H-Ras was located in the Golgi apparatus in the presence of UBIAD1WT, while it was located in the plasma membrane in the presence of UBIAD1N102S and UBIAD1RPWS. UBIAD1 mutation did not affect ERK phosphorylation (Fig. 7b) and inhibit T24 cell viability (Fig. 7c). In addition, mutations of UBIAD1 did not influence T24 cell proliferation (Fig. 7d, e), indicating that UBIAD1 mutation trigger its loss of function in regulating the Ras/ERK signaling pathway. These findings demonstrate that UBIAD1 inhibits H-Ras in the Golgi apparatus via GGPP.
Discussion
In this study, it is reported that UBIAD1 interacts with the C-terminus of H-Ras, regulates the plasma membrane-Golgi cycle of H-Ras, and limits activation of H-Ras signaling in the plasma membrane which consequently inhibits cell growth.
Ras mutations are found in approximately 25% of human tumors51, and Ras oncogenic activity is dependent on the protein’s association with the inner face of the plasma membrane. Therefore, preventing Ras from trafficking to the plasma membrane is an important direction for anti-cancer and anti-Ras drug discovery52.
Farnesyl pyrophosphate is added to the CAAX cysteine of Ras by farnesyltransferase, and the AAX amino acids of the CAAX motif are substrates for an endoprotease designated Rce1. Following Rce1-mediated proteolysis, the C-terminal prenylcysteine of Ras is methylesterified by Icmt53. Due to the different HVR domains, H-Ras, N-Ras and K-Ras4A are transported to the Golgi apparatus, whereas K-Ras4B is transported to the plasma membrane after being phosphorylated by PKC54. H-Ras, N-Ras and K-Ras4A are palmitoylated by palmitoyl acyltransferases on the surface of the Golgi apparatus and then are transported to the plasma membrane55. The G-P peptidyl-prolyl bond of H-Ras at position 178–179 undergoes cis-trans isomerization catalyzed by FKBP12 and then HRas is transported back to the Golgi apparatus15, representing plasma membrane-Golgi cycling of H-Ras.
FTIs have been developed based on the essential role of the farnesyl lipid modification for all subsequent post-translational modifications and for RAS oncogenic activity53. Although FTIs inhibit H-Ras-driven growth of cancer cells, they have no effect on cancer driven by N-Ras or K-Ras because NRas and KRas are modified with the related geranylgeranyl isoprenoid in the presence of FTIs56. Inhibition of palmitoylation or increasing the activation of FKBP12 can limit activation of Ras in the plasma membrane, leading to the suppression of cell growth52.
One identified target for anti-Ras drugs is the prenyl-binding protein phosphodiesterase δ (PDEδ), which binds to the C-terminus of Ras and facilitates the transit of Ras proteins to either the Golgi apparatus or the recycling endosomes57. PDEδ inhibitor treatment shifts Ras from the plasma membrane to the endomembranes, inhibits Ras activation in the plasma membrane and suppresses the growth of cancer cells58. In principle, targeting PDEδ would overcome the concerns encountered with FTIs.
Here, we found that UBIAD1 interacted with the C-terminus of H-Ras, which is consistent with the binding data for FKBP12 and PDEδ15,58. UBIAD1 inhibited H-Ras trafficking from the Golgi apparatus to the plasma membrane and inhibited the proliferation of bladder cancer cells. Ras translocates to the plasma membrane in the absence of UBIAD1, leading to the formation of cancer. Therefore, UBIAD1 has a similar function as PDEδ inhibitors or FKBP12 in regulating Ras trafficking between the plasma membrane and the Golgi apparatus (or ER). UBIAD1 may be a potential target for regulating Ras trafficking.
Ras is an important protein implicated in cell proliferation circuits, and can influence other cell circuits, such as motility, cytostasis, differentiation, and viability circuits4,5. Ras sends different signal outputs in various regions such as the plasma membrane, Golgi apparatus, ER and mitochondria59. These events may occur through several factors, such as the different MAPK scaffolds employed by the plasma membrane and the Golgi apparatus3. Ras/ERK signaling activated by Ras in the plasma membrane induces cell growth, and Ras in the Golgi can regulate the Raf/ERK, PI3K/AKT/mTOR or PLC-γ-Ca2+ signaling pathway, leading to cell differentiation59,60. PAQR9/10 bound to H-Ras in the Golgi retains H-Ras in the Golgi and induces cell differentiation9. ENOS activates N-Ras in the Golgi and induces T-cell dependent apoptosis61. Additionally, FKBP12 promotes H-Ras trafficking from the plasma membrane to the Golgi, activates H-Ras in the Golgi, and induces cell differentiation under EGF treatment15. Our findings show that UBIAD1 prevents H-Ras trafficking from the Golgi to the plasma membrane. UBIAD1 is essential in embryonic mouse development30, and heix is specifically expressed in a small cluster of hemocytes within the Drosophila embryonic head mesoderm, a developmental zone of hemocytes19. These findings suggest a new function of the UBIAD1 protein in cell differentiation during biological development. UBIAD1 may regulate the balance between cell proliferation and differentiation by mediating plasma membrane- Golgi cycling of H-Ras.
UBIAD1 suppresses tumor growth in bladder cancer20, prostate cancer21, and renal carcinoma31. UBIAD1 can inhibit the cell cycle and cell proliferation by regulating cholesterol and SXR target genes in the ER or mitochondria26,62. Here, the results demonstrate that UBIAD1 inhibits cell growth by regulating Ras/ERK signaling in the Golgi apparatus, suggesting that UBIAD1 is involved in the inhibition of cell proliferation. UBIAD1 is a membrane-associated protein with different functions in the ER and Golgi apparatus27,33,47, and GGPP is necessary during the transport of this protein from the ER to the Golgi apparatus63. When GGPP is insufficient, the transport of UBIAD1 to the ER under treatment with statins or GGPPS knockdown induces HMG-CoA reductase synthesis of GGPP and cholesterol, causing cell proliferation33. By contrast, transport of UBIAD1 to the Golgi apparatus in the presence of GGPP causes the protein to bind to H-Ras, which consequently prevents the trafficking of H-Ras from the Golgi apparatus to the plasma membrane and inhibits cell growth. UBIAD1 mutants lack the ability to use GGPP and are therefore located in the ER instead of the Golgi apparatus, inducing cholesterol generation and SCCD disease33. These data show that UBIAD1 mutation or wild-type UBIAD1 without GGPP are localized in the ER and both types lose their functionality, consistent with previous studies. Therefore, UBIAD1 is in a dynamic balance between the ER and the Golgi apparatus to regulate cell growth.
The establishment of carcinoma is a complex process. Ectopic expression of H-Ras can induce melanoma in mice18, and a lack of heix can also cause melanotic mass formation in Drosophila larvae19. Therefore, H-Ras and UBIAD1 may be associated with melanoma formation. Our data show that large amounts of H-Ras aggregate in the plasma membrane and activate Ras/ERK signaling, causing melanotic mass formation under heix-deficient conditions. The mechanism responsible for melanotic mass formation in Drosophila may be similar to that in mice. Melanotic masses, the most obvious feature of heix mutation in Drosophila19, can be subdivided into melanotic nodules engaging the hemocyte-mediated encapsulation and melanizations that are not encapsulated by hemocytes64. Loss of function of heix or gain of function of Ras leads to an increased proliferation of hemocytes19,45. Hemocyte proliferation is closely correlated with the formation of melanotic mass, which can be attributed to an increase in the number of crystal cells associated with melanization65,66. Our study show that heix mutant leads to activation of Ras signaling on the plasma membrane. Therefore, the formation of melanotic mass in a heix mutant may result from Ras-induced proliferation of hemocytes. Activated Ras reportedly increases macropinocytosis and autophagy and directs glucose metabolism into hexosamine biosynthetic pathways by upregulating many key enzymes involved in glycolysis51. Therefore, UBIAD1/HEIX knockdown induces Ras/MAPK signaling and might reprogram metabolism, leading to tumorigenesis. In addition, it is reported that the melanotic mass formation in Drosophila is linked to a hemocyte-mediated immune response64. Previous studies report that loss of heix function not only activates the Ras signaling pathway but also leads to the activation of immune-related pathways (Toll, JAK/STAT, IMD pathways)19,66. Therefore, besides the Ras pathway, the formation of melanotic mass in heix mutants may be influenced by immune-related pathways.
In summary, UBIAD1 bind to H-Ras, regulates the H-Ras intracellular trafficking cycle, inhibits Ras/MAPK signaling, suppreses bladder cancer cell proliferation and plays important roles in melanotic mass formation in Drosophila. Our findings confirm the tumor suppressor function of UBIAD1 in cellular circuits in melanotic mass formation in Drosophila.
Methods
Cell culture and cell transfection
Human bladder cancer cells T24 and human embryonic kidney cells HEK293T were obtained from American Type Culture Collection. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (FBS).T24 cells were cultured in minimum essential medium (MEM, Hyclone) supplemented with 10% FBS. All cultures were maintained at 37 °C in a humidified incubator with 5% CO2. Transient transfection of HEK293T and T24 cells was performed with LipofectamineTM 200028.
Real-time quantitative PCR analysis
Total RNA was extracted from HEK293T cells with TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using the first-strand cDNA synthesis kit (Eastep RT Master Mix kit Promega, USA). Real-time quantitative PCR was performed using the double-stranded DNA dye SYBR Green (Roche, Switzerland) to quantify the amount of gene expression. Gene expression assays were performed using procedures recommended by ABI Biosciences, and data were analyzed using ΔΔCt values. Primer pairs for UBIAD1 were used according to a previous study67
(UBIAD1-F:5'-GTGTGCCTCCTACGTGTTGGCC-3'; UBIAD1-R:5'-AAATTACCGGCCCCGTGCACAG-3').
The mRNAs levels were normalized to ACTIN.
The primer pairs of ACTIN:
(ACTIN-F:5'-CGCGAGAAGATGACCCAGAT-3'
ACTIN-R:5'-GTACGGCCAGAGGCGTACAG-3').
Plasmid constructs
The plasmid pOTB-UBIAD1 was purchased from Open Biosystems Inc. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described28. For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector. BFP-H-Ras was constructed by fusing H-Ras at the N-terminus of BFP into the pcDNA3.1 vector. GFP-H-Ras and its mutation were cloned into pEGFP-C1. Flag-H-Ras and its mutation were constructed by fusing the Flag tag to the N-terminus of H-Ras and inserting into pcDNA3.1. The plasmid 3Flag-UBIAD1 was constructed by fusing the 3Flag tag to the N-terminus of UBIAD1 and inserting into pcDNA3.1. pGFP-RBD was kindly provided by Dr. Mark R. Philips. Full-length human UBIAD1 and Apo E cDNA fragment were separately cloned into the pEGFP-N1 vector to create UBIAD1-EGFP and Apo E-EGFP. UBIAD1 was cloned into pcDNA3.1 to generate pcDNA3.1-UBIAD1. UBIAD1-BFP, Golgi-BFP and Apo E-BFP were constructed by fusing BFP with the C-terminus of UBIAD1 and Apo E and inserting into pcDNA3.1. All vectors for the BIFC assay were constructed using pcDNA3.1.
Immunofluorescence (IF) and confocal microscopy
HEK293T cells were mounted onto the polylysine-slides and were 4% formaldehyde fixed (10 min) to permeabilize cells. Cells were incubated in 1% BSA (Bovine Serum Albumin)/10% normal goat serum in 0.1% PBS (Phosphate Buffer Saline)-Tween for 2 h to block non-specific protein-protein interactions. Cells were then incubated with primary antibodies overnight at 4 °C. After several washes with cold PBS, the slides were incubated with secondary antibodies for 2 h at room temperature. Fluorescence was observed by confocal microscopy (FV1000, Olympus) using 488 argon ion laser for GFP/488, a 543 He-Ne laser for RAF/549, and a 405 diode laser for BFP/DAPI. FLUOVIEW (Olympus) was used as the image acquisition software. Images were acquired, processed and analyzed with Image J (National Institutes of Health).
EGF treatment and inhibitor assay
Cells were starved for 6 h, cultured with DMEM and stimulated with 100 ng/ml EGF for different periods of time. The inhibitors U0126 (Medchem Express, USA, #HY12031), FTI-277 (Sigma, USA, #F9803), Salirasib (Medchem Express, USA, #HY14754), 2BP (Sigma, USA, #238422), tunicamycin (Medchem Express, USA, #HY13585), and simvastatin (Cayman, USA, #10010344) were dissolved in DMSO (Dimethyl sulfoxide). The working concentrations for cells and Drosophila were as follows: U0126 (10 μM), FTI-277 (10 μM), Salirasib (35 μM), 2BP (150 μM), tunicamycin (10 μM), and simvastatin (10 μM). FPP (#63250) and GGPP (#63330) were purchased from Cayman.
Antibodies
The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA). Antibodies used for experiments with Drosophila were as follows: p-ERK (sc-7976) and ERK2 (sc-153) from Santa Cruz (US), pan-Ras (#3339) from Cell Signaling Technology (USA). HEIX antibodies were synthesized by GL Biochem (Shanghai, China), as previously described19.
Immunoprecipitation and immunoblotting
An immunoprecipitation assay was performed 48 h after cells were transfected with plasmids. Cells were lysed in lysis buffer (50 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 0.5 mM EDTA and 0.1% NP-40, supplemented with protease inhibitor cocktail (CST #5871S) and phosphorylated protease inhibitor cocktail (BOSTER #AR119)), frozen and thawed once. Cell lysates and specific antibodies were incubated at 4 °C with continuous mixing overnight. Fifty microliters protein G-coupled beads (Millipore, USA, LSKMAGG02) were prepared (according to the supplier’s instructions), and the mixture containing the pre-formed antibody-antigen complex was added to the beads. After 3 h of incubation at 4 °C, the supernatant was removed, and the beads were washed five times with cold PBS containing 0.1% Tween 20. Following the final wash, 60 μl sample buffer was added and the reaction was heated at 90 °C. The sample was detected by western blotting as previously described by Xia and collaborators39. In short, proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12% gel) and transferred to PVDF (Polyvinylidene fluoride) membrane. The membranes were subsequently blocked with 5% fat-free milk dissolved in TBS (Triethanolamine Buffer Saline) containing 0.1% Tween for 3 h at room temperature and then incubated overnight with primary antibodies at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibody, proteins were detected using enhanced chemiluminescence (ECL). Band intensity was quantified with Quantity One software.
RNA interference
shRNA vector-pGPU6/GFP/Neo was constructed by GenePharma (Suzhou, China). sh-UBIAD1:26 5'-CACCGTAAGTGTTGACAATTACCGGTTCAAGAGACCGGTAATTGTCAACACTTACTTTTTTG-3'.
sh-NC: 5'-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTAACGTGACACGTTCGGAGAATTTTTTG-3'.
sh-FNTA:68 The shRNA vector into PGH1/RFP/Neo were constructed by GenePharma. 5'-CACCGATCCGGTGCCGCAGAATGATTCAAGAGATCATTCTGCGGCACCGGATTTTTTTG-3’. sh-NC: 5'-CACCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTTG-3'.
si-RNA oligonucleotides were constructed by GenePharma (Suzhou, China).
si-UBIAD1:39 5'-GUAAUUUGGUCAACACUUATT-3'
si-GGPPS:49 5'-GUCCCACUGAAGAAGAAUA-3'.
si-Icmt:69 5'-CCAUAGCUUAUAUUCUCAAdTdT-3'.
si-NC: 5'-UUCUCCGAACGUGUCACGUTT-3'.
Cell viability assay and BrdU cell proliferation ELISA assay
T24 cells were plated in 96-well plates at a density of approximately 105 cells per well; after 24 h, the cells were transfected with plasmids. Cell viability was evaluated using the MTT (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, #G3580, Promega, US) assay, according to the manufacturer’s protocol.
T24 cells were plated in 96-well plates at a density of approximately 103 cells per well; after 24 h, the cells were transfected with plasmids. Cell proliferation was evaluated using the BrdU cell proliferation ELISA kit (colorimetric) (ab126556, Abcam, US), according to the manufacturer’s protocol.
Drosophila stocks and medicine treatment
The stocks used in this study were previously described19. heixk11403/Df and heixk11403/heix1 are mutations of heix. For the P-element allele heixk11403, the insertion site is between bases 2L: 16299582 and 2L: 16299583 (transcription initiation area) and is expected to induce a non-expression (Bloomington stock 11031). For the ethylmethansulfonate (EMS) allele heix1, an AR144 mutation resulting from a G to A base change in the last residue of the only intron and is expected to result in an aberrant splicing event (Bloomington stock 3600). The deficiency allele Df(2 L)RA5/CyO (Bloomington stock 6915). The control is wild-type (w1118), and heixk11403/Df; tub P > heix is the rescue type.
Food for Drosophila was prepared in a tube on the first day, and treatments were added to the food surface on the second day. Virgin flies were placed in the tube on the third day, and crossing was performed on the fourth day. The flies were removed from the tube 1 week later.
Statistical analysis
Results are presented as the mean ± standard deviation (SD). Statistical data comparisons among groups were performed using a non-parametric, Student’s t-test, p < 0.05 was considered statistically significant. Each experiment was performed at least in triplicate.
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Acknowledgements
We are thankful to Dr. Mark Phillips for kindly providing us the RBD-GFP construct. We particularly thank Drs. Samira Zohra Midoun, Zhengxing Wu, Xin Liu, and Mingkun Yang for their technical assistance. We thank Stacy Hsu and American Journal Experts for English editing. This project was supported in part by the Grants from NSFC (P. R. China, No. 30971608, 81272210, 81773202), 863 Grant of Ministry of Science and Technology (P. R. China, No. 2007AA09Z449), NSF of the Hubei Province (P. R. China, 2009CDB074), Research Grant for Wuhan Science and Technology Bureau (P.R.China) (Grant No. 2017060201010145) and Research Grant for Chinese Universities from the Central Chinese Government (P.R.China) (Grant No. 2015YGYL007).
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L.H. and Z.X. planned the project. Z.X. performed the experiments and analyzed the data. Z.X. wrote the manuscript. F.D. and Z.X performed the T24 cells experiments. Maytham Abdulkadhim Dragh and Z.X performed the Drosophila experiments. H.Lu, Y.X., and H. Liang provided the material. All authors reviewed the manuscript.
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Xu, Z., Duan, F., Lu, H. et al. UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras. Cell Death Dis 9, 1170 (2018). https://doi.org/10.1038/s41419-018-1215-4
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DOI: https://doi.org/10.1038/s41419-018-1215-4
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