Site-specific glycoproteomic analysis revealing increased core-fucosylation on FOLR1 enhances folate uptake capacity of HCC cells to promote EMT.

Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous.
Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches.
Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells. Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells. © The author(s).

Introduction

Hepatocellular carcinoma (HCC), which ranks sixth among the most common malignancies and fourth in mortality among all kinds of cancers in the world, causes about 841,000 new cases and 782,000 deaths annually 1. Although improved early diagnosis and surgical treatment in recent years have led to elevated survival of patients with HCC, the 5-year survival rate is still less than 20% 2. The main cause of such poor prognosis and high mortality is the recurrence and metastasis of HCC 3, 4.

Epithelial-mesenchymal transition (EMT), which is mainly involved in the transient de-differentiation of epithelial cells into mesenchymal phenotypes, has been recognized as a major trigger for cancer metastasis and invasion in recent years 5-8. According to previous studies, EMT is involved in the metastasis of HCC 9, melanoma 10, breast cancer 11, and esophageal cancer 12. With the help from genomics and proteomics tools, many EMT-related transcription factors, genes, miRNAs and proteins have been reported 6, 13-15, which provide valuable data for us to truly understand the intrinsic changes with EMT. In spite of that, the detailed molecular mechanism for EMT is still ambiguous.

Glycosylation is one of the most extensive and important protein post-translational modifications and participates in many essential biological processes including the cell growth, molecular recognition, signal transduction, immune defense, and other biological processes in normal tissue. The alteration of glycosylation has been proved to be associated with the progression and metastasis of many cancers, such as bladder 16 and breast cancers 17. Especially, the abnormality of N-glycan on the cell surface has been proved to be associated with the metastasis and EMT of HCC 18, 19. One example of EMT-related glycan changes is that the increased core-fucosylation on N-glycans catalyzed by the glycosyltransferase FUT8 20. These studies, however, mainly focus on the analysis of released glycans by using glycomics or lectin microarray approaches 16-19, which are unable to link glycans with their related glycoproteins 19. Therefore, the related functional glycoproteins and detailed mechanism are still largely unknown. In recent years, site-specific glycosylation analysis has been achieved by directly analyzing intact glycopeptides using mass spectrometry 21-25, by which both glycan alterations and their attached glycoprotein information can be clearly profiled. We reason that this glycoproteomic technology has great chance to recover the possible connections between altered glycans and glycosylated proteins associated with EMT of cancer cells, and even identify some new site-specific glycan changes related to EMT to facilitate a better understanding of the EMT mechanism.

In this study, site-specific glycosylation alterations in the EMT process of HCC were systematically investigated using glycoproteomic methods (Figure ), and the possible roles of EMT-related glycoproteins and site-specific glycans were further investigated by molecular biological approaches. Using these approaches, we revealed that the cellular uptake capacity of folates can be enhanced by increased core-fucosylation on FOLR1 especially at the glycosite Asn-201 to promote the EMT of HCC, which demonstrated a specific role of protein glycosylation in the EMT progress of HCC.

Results

HGF triggers EMT-like changes in two hepatocellular carcinoma cell lines

In order to systematically investigate the alterations of site-specific glycosylation in the EMT process of HCC, two widely used HCC cell lines SMMC-7721 and HepG2 were treated by HGF to induce their EMT 26-28, and their whole cellular proteins were harvested at six time points after HGF-treatment (0, 6, 12, 24, 48, and 72 h). Then, the trypsin digested peptides from whole cellular proteins of different time points were labeled by TMT reagents and pooled into one sample for global proteomic analysis with LC-MS/MS. The intact glycopeptides were enriched from pooled samples and analyzed by LC-MS/MS for intact glycopeptide identification and quantification (Figure ).

The HGF-induced EMT was first evaluated by expression levels of the epithelial marker E‑cadherin and mesenchymal marker N‑cadherin 29. With 10 ng/mL HGF treatment, the mRNA level of E-cadherin decreased to less than 25% within 6 h, and to < 1% after 24 h in both cell lines. The mRNA level of N-cadherin significantly increased within 12 h, and up to 9-fold and 25-fold after 72 h treatment in SMMC-7721 and HepG2 cells, respectively (Figure ). Based on the expression of EMT markers, the whole EMT process could be divided into three major stages, including epithelial (E: 0 h), intermediate (I: 6 h and 12 h), and mesenchymal stages (M: 24 h, 48 h, and 72 h) (Figure ). These results demonstrated that HGF treatment caused up-regulated expression of N-cadherin and down-regulated expression of E‑cadherin in a time-dependent manner in both cell lines, indicating the successful induction of EMT by HGF on both cell lines within three days.

The HGF-induced EMT was also confirmed by the relatively high cell growth rates of both HGF-treated cell lines in FBS-free medium, a condition that the untreated cells could not survive. The number of both treated cells increased from 30% to approximately 90% within three days (, while the untreated cells decreased from 30% to almost 0% during the same time period (data not shown).

Site-specific glycosylation profiling in two HCC cell lines

From SMMC-7721 and HepG2 cell lines, we were able to identify 1474 and 1682 unique N-linked intact glycopeptides (still with N-glycan attached) within 1% false discover rate (FDR), respectively (Figure and. In total, 2306 unique intact glycopeptides, including 848 in common (36.7%), were identified in these two cell lines within 1% FDR (Figure ). These intact glycopeptides represented 891 glycosites from 657 glycoproteins, modified with 283 glycan structures (159 compositions) (. Up to 80 glycans were identified at the glycosite Asn-69 (NACCST69N#TSQEAHK) of folate receptor alpha (FOLR1). The 283 N-glycans contained 16 high mannose, 121 hybrid, 138 complex, and eight paucimannose subtypes of glycans (Figure ). Three pairs of representative MS/MS spectra, each containing a spectrum from 20% HCD fragmentation and a spectrum from 37% HCD fragmentation, were provided to demonstrate the principle of glycan type determination in the intact glycopeptide analysis (-S5).

Quantitative intact glycopeptide analysis revealing increased core-fucosylation with EMT

We first performed quantitative proteomic analysis during the EMT process of both cell lines. The relative abundance of each protein was calculated as the ratio of protein expression at the I or M stages versus that of E stage. Among all protein groups identified from SMMC-7721 (2,403 proteins) and HepG2 (1,964 proteins) cell lines, over 99.7% of them were changed within two folds in the I and M stages ( and). Even though a 1.5-fold change was used as the cutoff, only six increased proteins were identified in the I or M stage compared with the initial E stage in both cell lines (). Although more in-depth proteomic analysis may lead to larger numbers of changed proteins identified, the present results clearly indicated that the majority of proteins remained unchanged during the EMT process of both HCC cell lines. Gene ontology (GO) analysis indicated all six proteins were involved in lipids and cholesterol biosynthetic process, in which five of these proteins could interact with each other (-D). These results suggested that increased synthesis of lipids and cholesterol might be involved in the EMT process induced by HGF, which is consistent with the simulative role of lipid and cholesterol to EMT in prostate cancer 30, breast cancer 31 and ovarian cancer 32. This also agrees with a previous study showing that lipids and cholesterol were harmful metabolites to the liver and their up-regulation could promote the development and growth of HCC 33.

We then analyzed the quantitative glycoproteomic data to investigate whether glycosylation was changed at the intact glycopeptide level. The distribution of log2(I/E) and log2(M/E) ratios revealed significant alterations of glycopeptides with approximately 14% and 9% glycopeptides changed more than two folds in the I and M stages of SMMC-7721 cell line, respectively (). Compared with SMMC-7721, fewer glycopeptides were altered (3% and 2% for I and M stages, respectively) in HepG2 cell line (Figure ). Overall, these results demonstrated a more significant change at glycosylation level than that of protein level during EMT.

In order to quantify the alterations of intact glycopeptides, we selected 607 unique glycopeptides detected in both cell lines with PSMs ≥ 5. Among them, glycopeptides modified by high mannose glycans accounted for the largest proportion, followed by complex and hybrid glycans (Figure ). We further analyzed structures of complex glycans using our in-house StrucGP software and found that core-fucosylated glycans accounted for the largest proportion of N-glycans, followed by glycans with terminal sialic acids and di-antennary glycans (Figure ). Quantification results showed that 20 intact glycopeptides from 9 proteins had more than two-fold increases at I or M stage in both cell lines (Figure ). Among them, four intact glycopeptides were modified with core-fucosylation, including HLA-C modified by N4H5F1S1 at the glycosite Asn-110, CD63 modified by N4H5F1 at the glycosite Asn-130, and FOLR1 modified by N4H5F1S1 and N4H5F3 at the glycosites Asn-69 and Asn-201, and all of them were elevated more than 2-fold at the I and/or M stages in both cell lines (Figure and. Notably, two of them were identified from FOLR1. These results implied that the site-specific core-fucosylation on FOLR1 might play an important role in HGF-stimulated EMT process in HCC cell lines. In addition, these four altered glycopeptides were manually inspected to ensure the correctness of their glycan structure identifications (Figure ).

Core-fucosylation of FOLR1 is upregulated in HCC cells during EMT process

To examine the relationship between the site-specific glycosylation on FOLR1 and HGF-stimulated EMT process, we mapped N-linked glycans on each glycosite of FOLR1 based on our glycoproteomics data. A total of 51 unique intact glycopeptides were identified from FOLR1, which were comprised of three glycosites (69N#TS, 161N#WT, and 201N#YS) and 31 glycans (). Among these glycopeptides, one was modified with paucimannose glycan, 13 were modified with high-mannose glycans, and the other 37 were modified with 23 hybrid or complex glycans (Figure ). We interestingly found that core-fucosylation was accounted for 62.7% of all glycans on identified intact glycopeptides, including 7 glycans with both core- and branch-fucosylation. The majority of core-fucosylated intact glycopeptides were up-regulated during EMT in SMMC-7721 cell line, and many were also increased in HepG2 cell line (Figure and). In addition, many core-fucosylated intact glycopeptides of FOLR1 were also up-regulated in HGF-stimulated Hep3B cells (Figure and).

We further measured whether core-fucosylation on FOLR1 was also up-regulated in TGF-β1-treated HCC cells. Three cell lines were also treated with 10 ng/mL TGF-β1, and their whole cellular proteins were harvested at three time points (0, 24 and 72 h). Western blot analysis showed that the E-cadherin protein level was decreased, while the N-cadherin protein level was significantly increased in all three TGF-β1-treated cell lines, indicating the successfully induction of EMT by TGF-β1 (). After LC-MS/MS analysis, we found that most of the core-fucosylation of FOLR1 were also up-regulated in TGF-β1-treated SMMC-7721 and Hep3B cells, especially the glycosylation of glycosite Asn-201 (Figure and), which was consistent with the results of HGF treatment. In fact, we also harvested and analyzed TGF-β1-treated HepG2 cells by LC-MS/MS. Unfortunately, the intact glycopeptides from FOLR1 were not identified, which most likely was due to the low expression level of FOLR1 in HepG2 cells ().

A representative pair of spectra from an up-regulated glycopeptide modified by the glycan HexNAc4Hex5Fuc3 is shown in Figure -D. In the spectra, the peptide sequence VS201N#YSR was identified based on the b/y ions of the intact glycopeptide fragmented by the high HCD energy (Figure ), while the glycan structure was determined based on the B/Y ions of the intact glycopeptide fragmented by the low HCD energy (Figure ). These results indicated that FOLR1 is a highly core-fucosylated glycoprotein and many core-fucosylated glycans were increased with the EMT in both HCC cell lines, further proved that core-fucosylation of FOLR1 might be involved in the EMT progress of HCC cells.

Core-fucosylation is mainly regulated by FUT8 during EMT

It is well-known that FUT8 is the sole enzyme to generate a-1, 6-fucosylated structures (core-fucosylation) on N-glycans 34. To investigate the role of core-fucosylation in the process of HGF or TGF-β1-induced EMT, we first detected the expression of FUT8 at both mRNA and protein expression levels in HGF-treated cells using qRT-PCR and western blot, respectively. The results indicated that FUT8 was significantly increased at both mRNA and protein levels within 24 h of HGF treatment. Especially, the mRNA levels of FUT8 were increased up to 8-fold after 72 h treatment in both SMMC-7721 and HepG2 cells (Figure ). Similarly, the expression levels of FUT8 protein were significantly up-regulated in three TGF-β1-treated cell lines (). These results indicated that FUT8 was positively correlated with the increase level of core-fucosylated glycans with EMT.

To demonstrate the relationship between FUT8 and EMT, we further over-expressed FUT8 in three cell lines in the absence of HGF and TGF-β1 treatment (Figure ). As shown in Figure , overexpression of FUT8 significantly increased the protein and mRNA expression level of N-cadherin, which is a key marker of EMT, while significantly decreased the protein and mRNA expression of E-cadherin, indicating that up-regulation of FUT8 could trigger the EMT of HCC cell lines, a similar effect as the HGF or TGF-β1 treatment. We next investigated whether aberrant FUT8 expression promoted the migration and invasion of three cell lines using trans-well invasion assay and crystal violet stain. As expected, overexpression of FUT8 consistently increased the invasive ability of three cell lines (Figure ).

To further consolidate the above observations, we silenced endogenous FUT8 in three cell lines by short hairpin RNAs targeting FUT8 (shFUT8) (Figure ). Western blot and qRT-PCR results showed that knockdown of FUT8 could cause a significant increase of E-cadherin and a significant decrease of the N-cadherin in stable cell lines in the absence or presence of HGF treatment (Figure ), revealing that the EMT of three cell lines induced by HGF could be relieved by knockdown of FUT8. To further assess the role of core-fucosylation in migration and invasion of HCC cells, we established stable cell lines of FUT8 knockdown in three cell lines. Then, the effect of FUT8 knockdown on migration and invasion of HCC cells were determined in three cell lines through matrigel trans-well invasion assay. Consistent with our observations, the invasive ability of the stable cell line of FUT8 knockdown was significantly reduced (Figure ). In addition, the mass spectrometry data showed that overall core-fucosylation were significantly increased in FUT8 overexpressed SMMC-7721 and HepG2 cells (Figure ), and decreased in FUT8 knockdown cell lines (Figure ). And the core-fucosylation rate of FOLR1 were also significantly increased with FUT8 over-expression, and decreased with FUT8 knockdown in SMMC-7721 cells (Figure ). These results provided strongly evidence that increased core-fucosylation is mainly regulated by FUT8 during EMT of HCC cell lines.

Core-fucosylation enhances the folate uptake capacity of FOLR1

To further examine the effects of FUT8 overexpression and knockdown on FOLR1 expression and core-fucosylation of the attached N-glycans, we measured the mRNA and protein expressions of FOLR1 in HGF-treated SMMC-7721 and HepG2 cells by using qRT-PCR and quantitative proteomic approaches, respectively. The results showed that FOLR1 had no significant alteration at mRNA and protein levels in both HCC cell lines treated by HGF (Figure ). In addition, both overexpression (Figure ) and knockdown (Figure ) of FUT8 didn't affect the expression of FOLR1 at mRNA and protein levels, but positively affected the overall core-fucosylation of proteins (Figure -G) as well as core-fucosylation on FOLR1 (Figure ).

Previous studies have shown that out of three functional folate receptors in human (FOLR1, FOLR2, and FOLR3), only FOLR1 exists on the apical surface of polarized epithelial cells, mediating mainly cellular uptake of folate by binding and releasing folate from outside to inside of cells 35. When incubating FUT8 over-expressed cell lines with 300 μM FITC-labeled folates (FITC-FA), we found that the amount of green fluorescence of folate was increased compared with that of control (Figure ). Conversely, the amount of green fluorescence of folate was significantly reduced by Cy3-labeled siFUT8 compared with that of control cells (Figure ). These results indicated that FUT8 can positively regulate uptake capacity of folate in HCC cells, possibly via the increased core-fucosylation on FOLR1.

Furthermore, we measured the effect of different concentrations of folate on the status of HCC cell lines. Different concentrations (100~1200 μM) of folate did not affect the mRNA (-B) and protein (Figure ) expression of FUT8 in SMMC-7721 and HepG2 cell lines. Folate increased the expression of N-cadherin and decreased the expression of E-cadherin both in a dose-dependent manner (Figure ), indicating that “super-physiological” dose of folate solely can directly promote the EMT of HCC cells.

Glycosylation on Asn-201 of FOLR1 is most essential for uptake capacity of folate

To further determine which glycosite of FOLR1 is critical for cellular uptake ability of folate, we mutated asparagine residues to alanine one by one at three N-glycosites on FOLR1, and constructed five stable cell lines of RFP, FOLR1 WT, FOLR1 N69A, N161A and N201A, respectively. The qPCR and western blot results showed that the mRNA (Figure ) and protein levels (Figure ) of FOLR1 were significantly increased in four stable cell lines including FOLR1 WT, N69A, N161A and N201A compared with the RFP, respectively. After treated with HGF for 24 h, the EMT progressions were partially blocked in all three glycosite-mutated cell lines in SMMC-7721 and HepG2 cells, according to the mRNA (-D) and protein (Figure ) alterations of E-cadherin and N-cadherin. Among three glycosites, the blocking effect of the mutation at the glycosite Asn-201 was the most significant.

To further evaluate effects of site-specific glycosite mutations of FOLR1 on uptake capability of folate, four stable cell lines were incubated with 300 μM FITC-FA. Compared with FOLR1 WT cells, the amount of green fluorescence from folate was significantly decreased in FOLR1 N69A, N161A and N201A cells (Figure -G). These results suggested that the folate uptake capacity of FOLR1 was maximally affected by site-specific glycosylation of FOLR1, in particular the glycosylation of glycosite Asn-201. In addition, these results further confirmed that the folate uptake capacity of HCC cells could be enhanced by the core-fucosylation of FOLR1.

A proposed novel pathway for HGF-induced EMT of HCC cells

Based on above results, we concluded that HGF treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins, especially FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity of HCC cells to promote the progression of EMT (Figure ). Within this pathway, our glycoproteomic data provided substantial evidence to reveal that the folate uptake capacity of FOLR1 can be positively regulated by site-specific core-fucosylation attached at FOLR1, which established a direct connection between the overexpression of FUT8 (and increased core-fucosylation) induced by HGF or TGF-β1 and the EMT of cancer cells stimulated by “super-physiological” dose of folate, which were only reported disjointly in previous studies 36-38.

Discussion

EMT has been recognized as an important step toward the invasion and metastasis enhancement of HCC for decades. In previous studies, many alterations including the up-regulation of FUT8 39, increased core-fucosylation 40, excessive demands of folate, and the abnormal abundance of FOLR1 36-38 have been associated with tumor occurrence and development. It is also known that core-fucosylation is normally regulated by the expression level of FUT8, and folates were transported into the cells mainly by the cell surface folate receptor FOLR1. However, it seems that these two are independent stimulation methods for promoting the EMT of cancers. In this study, by systematically screening the dynamic changes of site-specific N-glycans in three HGF or TGF-β1 treated HCC cell lines, complemented by molecular biology approaches, we provided solid evidences to show that the folate uptake capacity of FOLR1 is enhanced by its increased site-specific core-fucosylation, and therefore links these two stimulation factors into one complete pathway 20.

Core-fucosylation, which was synthesized by the glycosyltransferase FUT8, has long been recognized as an important modification on N-linked glycans. It plays essential roles in modulating the affinity activity of many cell surface receptors with their ligands 41-46. The abnormity of core-fucosylation has been associated with many diseases, including congenital disorder of glycosylation 47, melanomas 39, and breast cancer 48. In recent studies, it has been reported that core-fucosylation is also elevated in liver tumors compared to normal liver tissues 20, and the up-regulation of FUT8 can promote HCC metastasis 49. The detailed functions and underlying mechanisms of core-fucosylation in the progression of HCC and HGF/TGF-β1-stimulated EMT, however, remain unknown. In this study, we also observed the increase of core-fucosylated glycans (and FUT8) in HGF/TGF-β1-treated HCC cells by using our site-specific glycoproteomics method. In addition, direct analysis of intact glycopeptides allowed us to further identify FOLR1 as an important target of elevated FUT8 for core-fucosylation during EMT, which is the key to link up-regulation of FUT8 with increased folate uptake for EMT promotion.

Among three known functional folate receptors, FOLR1 and FOLR2 are located at the plasma membrane of the human cells, while FOLR3 is a secreted protein 35, 50-54. FOLR1 is mainly displayed on the cell surface of epithelial cells, while FOLR2 is usually expressed in the latter stages of normal myelopoiesis and in the placenta, spleen, and thymus 55, 56. As an important cell-surface glycoprotein on epithelial cells, FOLR1 can bind folate with high affinity to mediate its cellular uptake for cell metabolism as well as DNA synthesis and repair 56. It has relatively high affinity for folate and folate analogs at neutral pH, and a lower affinity at slightly acidic pH after endocytosis to facilitate the release of folates. FOLR1 has restricted expression in normal tissues but is highly expressed in specific malignant tumor mainly to meet the folate demand of rapidly dividing cells 35-38, 55. It therefore has been used as an important target for cancer therapy 35, 50. In this study, we further confirmed that high concentration of folate can promote the EMT of HCC cells. In addition to providing excessive amounts of folate in the cell culture medium, we showed that the elevated folate uptake of the HCC cell could also be achieved by up-regulating the core-fucosylation at all glycosites of FOLR1, preferably at the glycosite Asn-201 (but not increasing the FOLR1 protein expression). In a previous study, Chen et al 55 reported that the de-glycosylated form of FOLR1 (only a single HexNAc moiety attached at each glycosylation site) had a similar folate-binding affinity with the fully glycosylated protein. This implies that the enhancement of folate uptake capacity by increased site-specific core-fucosylation on FOLR1 may mainly occur at the folate release and/or FOLR1 transportation steps.

Conclusion

In conclusion, with site-specific glycoproteomics and molecular biology approaches, we demonstrated that HGF/TGF-β1 significantly up-regulated the expression of FUT8, which led to the increasing of core-fucosylation on FOLR1. This further increased the cellular uptake of folate and promoted the EMT of HCC cells. With glycosite mutations, we further identified Asn-201 as the most critical core-fucosylated glycosite for folate uptake and EMT progression. Based on these results, the core-fucosylation of FOLR1 especially at the glycosite Asn-201 may represent another promising marker and therapeutic target for HCC monitoring and treatment.