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Pak2-mediated phosphorylation promotes RORγt ubiquitination and inhibits colonic inflammation

SUMMARY Dysregulated interleukin-17 (IL-17) expression and its downstream signaling is strongly linked to inflammatory bowel diseases (IBDs). However, the molecular mechanisms by which the function of RORγt, the transcription factor of IL-17, is regulated remains elusive. By a mass spectrometry-based approach, we identify that Pak2, a serine (S)/threonine (T) kinase, directly associates with RORγt. Pak2 recognizes a conserved KRLS motif within RORγt and phosphorylates the S-316 within this motif. Genetic deletion of Pak2 in Th17 cells reduces RORγt phosphorylation, increases IL-17 expression, and induces severe colitis upon adoptive transfer to Rag1−/− mice. Similarly, reconstitution of RORγt-S316A mutant in Rorc−/− Th17 cells enhances IL-17 expression and colitis severity. Mechanistically, we demonstrate that Pak2-mediated phosphorylation causes a conformational change resulting in exposure of the ubiquitin ligase Itch interacting PPLY motif and degradation of RORγt. Thus, we have uncovered a mechanism by which the activity of RORγt is regulated that can be exploited therapeutically.

INTRODUCTION
Interleukin-17 (IL-17) is a proinflammatory cytokine that is produced by a variety of immune cells, including the Th17 subset of helper CD4 + T cells, δ T cells, and innate lymphoid cells (Honda and Littman, 2016;Kumar et al., 2021b;Zhou and Sonnenberg, 2020). IL-17 signals through the IL-17 receptor, composed of IL-17RA and IL-17RC receptor subunits (Gaffen, 2009). The binding of IL-17 to its receptor activates the target cells, such as epithelial cells, endothelial cells, and fibroblasts, and induces the expression of CXCL1, CXCL2, and CXCL8, which attract myeloid cells such as neutrophils and promote inflammation (Gaffen, 2009). IL-17 is critically essential for host defense against fungal and bacterial infections. However, dysregulated IL-17 expression and downstream signaling are involved in several human diseases, including inflammatory bowel disease (IBD) (McGeachy et al., 2019;Patel and Kuchroo, 2015).
The orphan nuclear receptor RORγt is a transcription factor crucial for IL-17 expression (Ivanov et al., 2006;Zhang et al., 2008). RORγt consists of a ligand-independent activation function 1 helix, a DNA-binding domain, a flexible hinge domain, and a C-terminal ligandbinding domain (Kumar et al., 2021b). The zinc finger motifs within the DNA-binding domain recognize the ROR response elements within the IL-17 promoter to induce IL-17 expression (Kumar et al., 2021b). Given the critical role of IL-17 in inflammatory diseases, RORγt has emerged as an attractive target for pharmacological interventions (Beringer et al., 2016). However, a clear understanding of the regulation of RORγt is currently lacking, which is absolutely necessary to target RORγt effectively.
Crosstalk between different posttranslational modifications plays a critical role in regulating the activity of transcription factors (Hunter, 2007). We have demonstrated earlier that the E3 ligase Itch targets RORγt for ubiquitination, and Itch deficiency results in spontaneous rectal prolapse as a result of severe spontaneous colitis (Kathania et al., 2016). Here, we show how Pak2-mediated phosphorylation facilitates RORγt ubiquitination to prevent excessive Th17 response. A clear understanding of this regulatory pathway could lead to the development of safe and highly selective modulators of RORγt as a targeting strategy for inflammatory diseases driven by IL-17.

Pak2 interacts and phosphorylates RORγt
To gain molecular insights into the regulation of RORγt, we immunoprecipitated RORγt from the cell lysate of in vitro-generated Th17 cells and subjected it to mass spectrometry (MS) analysis. Through this approach, we identified Pak2 as a RORγt-binding protein (Figures 1A and S1A). We also precipitated RORγt from the cell lysate of CD4 + T cells sorted from colonic lamina propria lymphocytes (cLPLs) of mice treated with DSS and subjected it to MS analysis and found Pak2 as a RORγt-binding protein ( Figure S1B). To validate the MS results, we transiently transfected 293T cells with plasmids encoding Myc-tagged Pak2 and FLAG-tagged RORγt. Cell lysates were immunoprecipitated with either anti-FLAG or anti-Myc antibodies. Samples immunoprecipitated with the anti-Myc antibody contained FLAG-RORγt, and vice versa ( Figure S1C), suggesting that Pak2 and RORγt physically interact with each other. To determine whether this interaction occurred between endogenous proteins in primary cells, we performed immunoprecipitation assays with anti-Pak2 and anti-RORγt antibodies using the lysate of in vitro-generated Th17 cells and found an interaction between RORγt and Pak2 ( Figure 1B). Similar results were obtained in CD4 + T cells sorted from cLPLs of mice treated with DSS ( Figure S1D). We also performed glutathione S-transferase (GST) pull-down assays using recombinant Pak2 protein. GST-RORγt, but not GST alone, precipitated recombinant Pak2 ( Figure 1C), suggesting a direct interaction.
conserved KRLS motif in RORγt, which could be potentially phosphorylated ( Figure  1D). To test if Pak2 phosphorylates RORγt, we knocked down Pak2 in in vitro-generated Th17 cells using short hairpin RNA (shRNA) lentiviral particles and found that inhibiting Pak2 reduced the phosphorylation of RORγt. As seen in Figure 1E, knocking down Pak2 reduced RORγt phosphorylation. Further, we confirmed that Pak2 phosphorylates RORγt by performing an in vitro kinase assay. As shown in Figure 1F, incubation of RORγt with wild-type Pak2, but not a Pak2 kinase-dead mutant (Pak2-ΔK278R), resulted in RORγt phosphorylation. Similarly, incubation of the RORγt-ΔS316A mutant with wild-type Pak2 exhibited a defect in phosphorylation. These data suggest that Pak2 phosphorylates RORγt at Serine 316 (S316). To further confirm these results, we reconstituted CD4 + T cells isolated from Pak2 f/f CD4 Cre mice with either wild-type Pak2 or Pak2-ΔK278R by lentivirus transduction. We found RORγt phosphorylation in Pak2 −/− cells reconstituted with wildtype Pak2 but not the Pak2-ΔK278R mutant ( Figure 1G). Similarly, reconstitution of Rorc −/− CD4 + T cells with wild type, but not the RORγt-ΔS316A mutant, resulted in RORγt phosphorylation ( Figure 1H). These data collectively indicate that Pak2 phosphorylates RORγt at S316 in Th17 cells.

Inhibition of Pak2 enhances IL-17 expression and colitis
To gain insights into the functional consequence of Pak2-mediated phosphorylation of RORγt, we analyzed the expression of Il17a and Il17f mRNA in in vitro-generated Th17 cells from wild-type and Pak2 f/f CD4 Cre mice. Results of real-time PCR experiments showed that Pak2 deficiency enhances the expression of Il17a and Il17f ( Figures S2A and S2B). However, no significant difference in Rorc mRNA level was observed ( Figure S2C). To gain in vivo evidence for Pak2-mediated phosphorylation of RORγt in colonic inflammation, we sorted CD4 + T cells from wild-type and Pak2 f/f CD4 Cre mice, and the cells were differentiated under Th17 polarizing conditions. The cells were then adoptively transferred into Rag1 −/− mice as described before (Lee et al., 2009). The Rag1 −/− mice that received Pak2 −/− Th17 cells showed more body weight loss, an increased fecal occult blood (FOB) score, increased diarrhea, splenomegaly, reduced colon length, and a higher weight-to-length ratio of the colon compared with the mice that received wild-type Th17 cells ( Figures  2A-2E). Colonoscopic examination showed increased inflammation in mice that received Pak2 −/− Th17 cells compared with those that received wild-type Th17 cells (Figures 2F and 2G). Similarly, histo-logical analysis of H&E-stained sections showed greater infiltration of inflammatory cells, more crypt damage, and higher clinical scores in mice that received Pak2 −/− Th17 cells than wild-type cells ( Figures 2H and 2I). Real-time PCR analysis showed higher expression of ll17a and Il17f mRNA in the colonic mucosa of mice that received Pak2 −/− Th17 cells ( Figures 2M and 2N). Further, flow cytometric analysis ( Figure S2D) showed an increased IL-17 expression by the adoptively transferred Pak2 −/− Th17 cells in Rag1 −/− mice compared with wild-type Th17 cells ( Figure 2J). However, no significant changes in interferon gamma (IFN-γ) production ( Figures 2K and S2E) was observed. Also, as expected, no change in IL-17 expression by innate lymphoid cells (ILCs) of host Rag1 −/− mice was observed ( Figure 2L). These data suggest that Pak2-mediated phosphorylation in Th17 cells plays a crucial role in colonic inflammation.
To test if increased IL-17 expression by the adoptively transferred Pak2 −/− Th17 cells is potentially due to elevated Rorc mRNA expression, we performed real-time PCR analysis. No change in Rorc mRNA level ( Figure 2O) was noted. However, immunoblotting the lysate of cLPLs showed an increased level of RORγt protein in mice that received Pak2 −/− Th17 cells compared with wild-type Th17 cells ( Figure 2P). These data suggest that Pak2 regulates RORγt protein turnover without affecting RORγt transcription.

Pak2 enhances the ubiquitination of RORγt
Next, we sought to investigate how Pak2 regulates RORγt protein turnover. We have previously shown that Itch targets RORγt for ubiquitination (Kathania et al., 2016). Since phosphorylation can affect protein turnover via ubiquitination (Hunter, 2007), we hypothesized that Pak2-mediated phosphorylation promotes RORγt ubiquitination. To test this hypothesis, we performed a ubiquitination assay by co-expressing either wild-type RORγt, RORγt-ΔS316A kinase mutant, or RORγt-ΔS316D phospho-mimetic mutants along with the Itch. Our results showed an increased polyubiquitination of RORγt-ΔS316D ( Figure 3A), suggesting that phosphorylation regulates ubiquitination of RORγt. To further confirm that phosphorylation promotes RORγt ubiquitination, we performed in vitro ubiquitin assays using recombinant wild-type RORγt or RORγt mutants (RORγt-ΔS316A or RORγt-ΔS316D) along with recombinant Itch. Increased ubiquitination of RORγt-ΔS316D phospho-mimetic mutant was observed compared with the RORγt-DS-A phospho-null mutant ( Figure 3B). Finally, we analyzed ubiquitination of RORγt in in vitro-generated Th17 cells from Pak2 f/f CD4 Cre mice lentivirally transduced with wild-type Pak2 or the Pak2-ΔK278R kinase-dead mutant. We found an increased ubiquitination in the cells transduced with wild-type Pak2 compared with the Pak2-ΔK278R kinase-dead mutant ( Figure 3C).
To investigate if phosphorylation-regulated ubiquitination affected RORγt protein turnover, we performed a cycloheximide (CHX) chase experiment. The primary CD4 + T cells from Rorc −/− mice were lentivirally transduced with wild-type RORγt and RORγt-ΔS316D phospho-mimetic mutants. The cells were then treated with CHX, and the level of RORγt was analyzed by immunoblotting. Our results showed a reduced abundance of RORγt protein in RORγt-ΔS316D compared with wild-type RORγt transduced cells ( Figure 3D). Similarly, the CHX chase experiment in in vitro-generated Th17 cells from Pak2 f/f CD4 Cre mice reconstituted with wild-type Pak2 or the Pak2-ΔK278R kinase-dead mutant also showed a reduced abundance of RORγt protein in wild-type Pak2 transduced cells compared with the Pak2-ΔK278R kinase-dead mutant ( Figure 3E). These results strongly suggested that phosphorylation increases RORγt protein turnover. Additionally, the mRNA-to-protein ratio of RORγt in Rorc −/− CD4 + T cells reconstituted with wild-type RORγt and RORγt-ΔS316D phospho-mimetic mutant was lower compared with wild-type-RORγt ( Figure 3F).
Itch is a HECT type E3 ligase that contains four WW domains (each of which contains two conserved tryptophan residues), which recognizes proline-rich PPXY (P, proline; X, any amino acid; Y, tyrosine) motifs in its substrate proteins (Venuprasad et al., 2015). We showed earlier that Itch binds to the conserved PPLY motif on RORγt via its WW domain (Kathania et al., 2016). To gain molecular insights into the mechanism by which phosphorylation at S316 promotes RORγt ubiquitination, we performed homology modeling of mouse RORγt. In silico analysis of the modeled structure showed that S316 made an H-bond(3.6 Å) with a side-chain amino group of asparagine (N) 253 of the neighboring α-helix and stabilized the ligand-binding domain (LBD) of RORγt, hence there was less accessibility of the PPLY motif ( Figure 3G). We then substituted the S316 with phosphomimetic aspartic acid residues (D) 316, and the energy of the modeled structure was minimized after the substitution mutation. The modeled structure revealed that the H-bond interaction between S316 to N253 is abolished after phosphorylation of serine, suggesting that phosphorylation may provide increased accessibility of the PPLY motif of RORγt to Itch ( Figure 3G). To validate the computational analysis, we performed a GST pull-down assay using purified wild-type Itch, GST-RORγt, GST-RORγt-ΔS316A, and GST-RORγt-ΔS316D mutants. Our results showed that Itch association with RORγt-ΔS316D mutant was more prominent compared with GST-RORγt or GST-RORγt-ΔS316A mutant ( Figure 3H). These results suggest that phosphorylation enhances the binding affinity of Itch to RORγt.

Phosphorylation of RORγt inhibits IL-17 expression and colonic inflammation
To confirm that Pak2-mediated phosphorylation prevents excessive IL-17-mediated inflammation, we reconstituted Rorc −/− CD4 + T cells with wild-type RORγt or RORγt-ΔS316A or RORγt-ΔS316D mutants. Cells were cultured under Th17 polarizing conditions, and expression of IL-17 was measured by real-time PCR. Our results showed an increased Il17a expression in Rorc −/− cells expressing RORγt-ΔS316A mutant compared with wild-type RORγt, whereas significantly reduced Il17a expression was observed in cells expressing RORγt-ΔS316D mutant ( Figure 3I). However, no significant difference in Rorc mRNA was observed ( Figure S2F).
To investigate the physiological impact of RORγt phosphorylation on the regulation of IL-17-mediated inflammation in vivo, we utilized an adoptive transfer colitis model (Lee et al., 2009). We sorted CD4 + CD25 − cells from Rorc −/− mice and reconstituted them with either empty vector, wild-type RORγt, or RORγt-ΔS316A or RORγt-ΔS316D mutants. The cells were differentiated under Th17 polarizing conditions for 5 days, and 5 × 10 5 cells were adoptively transferred into Rag1 −/− mice ( Figure 4A). The mice were monitored for signs of disease, including weight loss, FOB, and diarrhea. Rag1 −/− mice that received Th17 cells expressing a phosphorylation-deficient mutant, RORγt-ΔS316A, exhibited greater loss of body weight, higher FOB, diarrhea scores, splenomegaly, reduced colon length, and a higher weight-to-length ratio of the colon compared with host mice that received Th17 cells expressing RORγt-ΔS316D mutant and wild-type RORγt ( Figures 4B-4F). Colonoscopic examination revealed enhanced inflammation in Rag1 −/− mice that received Th17 cells expressing RORγt-ΔS316A mutant compared with RORγt-ΔS316D mutant or wild-type RORγt (Figures 4G and 4H). Histological analysis of H&E-stained sections showed greater infiltration of inflammatory cells, more crypt damage, and higher clinical scores for Rag1 −/− mice that received RORγt-ΔS-A mutant expressing cells compared with RORγt-ΔS-D mutant or wild-type RORγt cells (Figures 4I and 4J). Rag1 −/− mice that received Th17 cells expressing RORγt-ΔS316A mutant had higher expression of ll17a and Il17f mRNA in the colonic mucosa ( Figure 4K). Similarly, flow cytometric analysis of cLPLs showed increased IL-17 expression in Rag1 −/− mice that received Th17 cells expressing the RORγt-ΔS316A mutant compared with cells expressing the RORγt-ΔS316D mutant or wild-type RORγt. However, there was no change in IFN-γ production by adoptively transferred cells ( Figure  4L and 4M). Further expression of IL-17-induced chemokines and MMPs Cxcl1, Ccl2, Ccl7, Ccl20, Mmp1, Mmp2, Mmp3, and Mmp13 were significantly upregulated in Rag1 −/− mice that received Th17 cells expressing RORγt-ΔS316A mutation ( Figures 3A and 3B). To test the potential difference in Rorc gene expression in adoptively transferred Th17 cells expressing wild-type RORγt and RORγt mutants, we performed real-time PCR experiments using RNA isolated from cLPLs of Rag1 −/− mice. As shown in Figure 4K, no significant changes in Rorc mRNA expression were observed. However, immunoblotting the lysates with anti-V5 antibody showed increased RORγt protein expression of the RORγt-ΔS316A mutant, suggesting a defect in turnover of RORγt protein ( Figure 4N). Together, these findings suggest that Pak2-mediated phosphorylation of RORγt negatively regulates IL-17 expression and controls colonic inflammation.

DISCUSSION
Genome-wide association studies have highlighted the central role of the RORγt:IL-17 pathway in several human inflammatory diseases (Duerr et al., 2006;Lock et al., 2002;Parkes et al., 2013). Accordingly, the IL-17-inducing transcription factor RORγt has emerged as an attractive target for pharmacological interventions (Beringer et al., 2016). Our results show that Pak2-mediated phosphorylation of RORγt at S316 inhibits IL-17 expression by promoting ubiquitination of RORγt. Pak2 −/− Th17 cells are highly colitogenic due to elevated IL-17 expression. Similarly, transfer of Th17 cells expressing RORγt-ΔS316A phosphor mutant resulted in severe colitis, whereas the RORγt-ΔS316D mutation that mimics the constitutively phosphorylated form of the RORγt mutant resulted in attenuated colitis in Rag1 −/− mice. Mechanistically, we demonstrated that S316 phosphorylation of RORγt enhances its interaction with Itch due to increased exposure of the E3 ligase Itch interacting PPLY motif. Our results could be exploited therapeutically to inhibit IL-17-mediated inflammation.
Pak2 was shown to be essential for maintaining regulatory T cell (Treg) stability and their suppressive function, and loss of Pak2, specifically in Tregs, resulted in defective Treg function (O'Hagan et al., 2017). Our data show that Pak2 plays a distinct role in Th17 cells by facilitating RORγt degradation to curb excessive IL-17-mediated inflammation. IL-17 is produced by a variety of immune cells, including Th17 cells, ILCs, Tc17 cells, natural killer (NK) cells, and neutrophils (Kumar et al., 2021b). The cell-surface receptors involved and the intracellular signaling pathways leading to the secretion of cytokines significantly differ in these cells. However, it remains poorly understood if different immune cells' regulation of IL-17 expression varies or adopts the same pathways. The data presented here identify a previously unknown mechanism of regulation of the RORγt-IL-17 pathway by Pak2 in Th17 cells. Our assay systems exclude the regulation of other IL-17-producing cells such as NK cells, CD8 + T cells, and ILCs. If Pak2 regulates IL-17 expression in these cells by a similar mechanism remains unknown, which requires further detailed studies, including cell-type-specific knockout mice (e.g., NK cell or ILC-specific Pak2 −/− mice) and additional adoptive transfer experiments.
The upstream mechanisms that activate the Pak2-Itch pathway to trigger RORγt degradation in Th17 cells remain un clear. Pak2 is shown to be activated by Cdc42/Rac downstream of Vav following T cell receptor (TCR) stimulation (Ha et al., 2015;Taglieri et al., 2014). Therefore, it is possible that in Th17 cells, Pak2 acts as a feedback mechanism and inhibits IL-17 expression by phosphorylation of RORγt. It is also possible that IL-17R signaling activates Pak2 in Th17 cells to impede the pathogenicity of Th17 cells. Such a phenomenon has been reported recently via the induction of IL-24 (Chong et al., 2020). Additionally, Pak2 is involved in TGF-β signaling (Sato et al., 2013;Wilkes and Leof, 2006;Wilkes et al., 2003Wilkes et al., , 2005Wilkes et al., , 2009Yan et al., 2012), which regulates Th17 development (Korn et al., 2009;Patel and Kuchroo, 2015). Therefore, it is also possible that Pak2 activated via the transforming growth factor β (TGF-β) pathway may trigger RORγt phosphorylation. Further, a detailed investigation of these pathways is essential to fully understand how RORγt protein turnover is regulated during an inflammatory response to prevent chronic inflammation.
The current strategies of targeting IL-17 using antagonistic antibodies (against IL-17 or its receptor) have shown some success in treating psoriasis and ankylosing spondylitis (Beringer et al., 2016); however, clinical trials using this approach led to mixed results in rheumatoid arthritis (RA) (Fragoulis et al., 2016;Genovese et al., 2013;Hueber et al., 2010) and failure in IBDs (Fitzpatrick, 2013;Hueber et al., 2012;Targan et al., 2016). Several recent studies have shown that RORγt function and protein stability are modified by posttranslational mechanisms, including ubiquitination, acetylation, and SUMOylation (Kumar et al., 2021b). A defect in RORγt protein degradation has been reported in tumorinfiltrated Th17 cells of patients with colorectal cancer (Sun et al., 2021). CRNDE-h protein was shown to form a complex with the PPLY region of RORγt, and binding of CRNDE-h to RORγt in patients' Th17 cells prevented ubiquitination and proteasomal degradation of RORγt by impeding its association with the Itch protein (Sun et al., 2021). On the contrary, our results show that Pak2 facilitates the binding of Itch to RORγt and promotes RORγt degradation. A clear understanding of the mechanism that regulates RORγt degradation could lead to alternative avenues to promote RORγt degradation at the site of inflammation. Such a strategy could transiently inhibit IL-17, resulting in therapeutic inhibition of Th17mediated inflammation without deleterious effects observed in antibody-mediated systemic blocking of IL-17.

Limitations of the study
Although our study comprehensively shows that phosphorylation promotes ubiquitination of RORγt by enhancing its interaction with Itch, additional biophysical and cryoelectron microscopy evidence is necessary for the phosphorylation-dependent conformational changes in RORγt. In our in vivo experiments, we have lentivirally expressed RORγt-ΔS316A in CD4 + T cells; studies using RORγt-ΔS316A point mutation knockin mice will be necessary to determine the broader physiological relevance of our findings. Finally, in this study, we utilized only the Th17-adoptive transfer colitis model to demonstrate that phosphorylation of RORγt regulates Th17 cell-induced colon inflammation. If the Pak2-RORγt-Itch pathway operates in other Th17-dependent disease models needs to be tested.

STAR★METHODS RESOURCE AVAILABILITY
Lead contact-Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact: K Venuprasad (venuprasad.poojary@utsouthwestern.edu).

Materials availability-This work did not generate any unique reagents.
Data and code availability-This paper does not report the original code. All software utilized is freely or commercially available and is listed in the key resources table. All data reported in this paper will be shared by the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice-C57BL/6 mice, Rag1 −/− mice, Rorc tm1Litt/J mice, and CD4 cre mice were purchased from the Jackson Laboratory. Pak2 f/f mice were described before (Kosoff et al., 2013). Equal number of male and female mice were used. All mice were housed in micro isolator cages in the barrier facility of the University of Texas Southwestern Medical Center. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.

METHOD DETAILS
Plasmid construction and cell transfection-Myc-Itch, HA-Ub (wild-type), and Flag-mRORγt have been described (Kathania et al., 2016). Transient transfection of 293T cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Myc-Pak2 and Myc-Pak2-ΔK278R were created and cloned into pCDNA3.1 between NotI and BamHI sites. The sequences of all clones were verified. Lentiviral transduction-Expression clones were created by Gateway cloning technology according to the manufacturer's instructions (Invitrogen). Lentiviral expression clones of mouse mRORγt-ΔS316A, mRORγt-ΔS316D, Pak2, and Pak2-ΔK278R were constructed by first subcloning into pENTR-3C entry vector (Invitrogen), and then subcloned to pLenti6.2/N-Lumio/V5-DEST (Invitrogen) via Gateway cloning. The ViraPower lentiviral expression system (Invitrogen) was used for packaging and producing lentiviruses in 293FT cells. The supernatant was harvested after 72 h. CD4 + T cells were transduced using lentiviruses in the presence of 5 μg/mL polybrene (Santa Cruz), and the media was changed after 12 h.

Protein identification by liquid chromatography (LC)-tandem MS-
The protein samples were processed and analyzed at the Mass Spectrometry Facility of the Department of Pathology at the University of Michigan. Gel slices (10 slices/lane) were destained with 30% methanol for 4 h. Upon reduction (10 mM DTT) and alklylation (65 mM 2-Chloroacetamide) of the cysteines, proteins were digested overnight with sequencing grade, modified trypsin (Promega). The resulting peptides were resolved on a nano-capillary reverse phase column (Acclaim PepMap C18, 2 micron, 25 cm, ThermoScientific) using a 1% acetic acid/acetonitrile gradient at 300 nL/min and directly introduced in to Orbitrap Fusion tribrid mass spectrometer (Thermo Scientific, San Jose CA). MS1 scans were acquired at 120K resolution. Data-dependent high-energy C-trap dissociation MS/MS spectra were acquired with top speed option (3 sec) following each MS1 scan (relative CE ~32%). Proteins were identified by searching the data against the Mus musculus database (24861 entries) using Proteome Discoverer (v1.4, Thermo Scientific). Search parameters included MS1 mass tolerance of 10 ppm and fragment tolerance of 0.2 Da; two missed cleavages were allowed; carbamidimethylation of cysteine was considered fixed modification and oxidation of methionine were considered a potential modification. Percolator algorithm was used for discriminating between correct and incorrect spectrum identification. The false discovery rate (FDR) was calculated at the peptide level and peptides with <1% FDR were retained. Immunoprecipitated proteins were separated by SDS-PAGE. In-gel digestion with trypsin, followed by protein identification using LCtandem MS, was performed as described elsewhere.43 Briefly, tryptic peptides were resolved on a nano-LC column (Magic AQ C18; Michrom Bioresources, Auburn, CA) and introduced into an Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA). The Orbitrap was set to collect a high-resolution MS1 (FWHM 30,000@400 m/z), followed by the data-dependent collision-induced dissociation spectra on the "top 9" ions in the linear ion trap. Spectra were searched against a human protein database (UniProt release 2011_05) using the X!Tandem/TPP software suite.44 Proteins identified with a Protein Prophet probability Z0.9 false discovery rate o2% were considered for further analysis.
Real-time PCR analysis-Total RNA was prepared using RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using Verso cDNA kit (Thermo Scientific). Quantitative realtime PCR was performed on a Mastercycler Realplex2 (Eppendorf). Light Cycler 480 SYBR Green I master reaction mix (Roche) was used in a 20 μL reaction volume. The expression of individual genes was normalized to the expression of actin. Cycling conditions were: 95°C for 2 min, followed by 50 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 20 sec.
Th17 cell differentiation-For in vitro experiments, naïve CD4 + T cells from the spleen and lymph nodes were differentiated into Th17 cells in plates coated with anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL). The cells were cultured in the presence of TGF-β (5 ng/mL), IL-6 (20 ng/mL), anti-IL-4 (5 μg/mL) and anti-IFN-γ (5 μg/mL) antibodies for 5 days. Immunoprecipitation and immunoblot analysis-Cell lysates were prepared from 293T cells transfected with Myc-Pak2 and Flag-RORγt plasmids after 36 h by homogenizing in NP-40 lysis buffer. Protein estimations were done using the Pierce BCA protein assay kit according to the manufacturer's protocol. Whole-cell lysates were precleared with 20 μL of Protein A/G plus agarose beads (Santa Cruz) for 1 h at 4°C. Lysates were then incubated with 1 μg of the desired antibody overnight at 4°C followed by a further 1 h of incubation at 4°C with 25 μL of Protein A/G beads. The immunocomplexes were washed five times with lysis buffer and denatured using 4X Laemmli buffer. Further, they were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Blots were visualized using Amersham ECL plus immunoblot analysis detection system (GE, Chicago, IL) on a Biorad ChemiDoc. For reprobing, membranes were stripped by incubation in a stripping buffer (62.5 mM Tris-HCl, pH 6.7; 100 mM 2-mercaptoethanol and 2% SDS) at 55°C for 45 min and washed thoroughly before reprobing.
In vitro kinase assays-Pak2 kinase activity was assessed using ADP-Glo Kinase Assay Kit (V9101, Promega), following the manufacturer's instructions. Recombinant RORγt protein (1 μg) was incubated with 100 ng of recombinant Pak2 kinase. The kinase reaction with a final ATP concentration of 50 μM was incubated at room temperature for 2 h. The kinase reaction was then terminated by adding ADP-Glo reagent for 40 min, followed by the addition of kinase detection reagent for 30 min incubation before reading the luminescence on a multimode microplate reader.
Ubiquitination assay-293T cells were transfected with Flag-RORγt, Myc-Itch, and various other constructs as indicated. MG132 was added 6 h before cell lysis. Cells were washed three times with PBS and lysed in NP-40 lysis buffer. Immunoprecipitation was performed using anti-Flag antibody. RORγt-associated ubiquitin was analyzed by immunoblot using antibody against HA. An in vitro ubiquitination assay was performed

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Pak2 directly binds and phosphorylates RORγt at S316  (E) Pak2 was knocked down in CD4 + T cells using shRNA and cultured under Th17inducing conditions. The cells were restimulated with anti-CD3 and anti-CD28 antibodies, and the lysate was subjected to immunoprecipitation (IP) with anti-RORγt antibody and analyzed by p-Ser specific antibody. (F) ADP-glow kinase assay result is represented as relative luciferase units (RLUs). (G) CD4 + T cells from Pak2 f/f CD4 Cre were reconstituted with either wild-type or kinasedead mutant of Pak2 and cultured under Th17 condition. Cell lysates were subjected to IP with anti-RORγt antibody and analyzed by p-Ser specific antibody. (H) CD4 + T cells were isolated from Rorc −/− mice, reconstituted with either wild-type or RORγt-ΔS316A mutant, and cultured under Th17 condition. Cell lysates were subjected to IP with anti-V5 antibody and analyzed by p-Ser specific antibody. Data are representative of two independent experiments. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


s2fieldsofstudy: Biology Medicine

text:
PRR34-AS1 promotes exosome secretion of VEGF and TGF-β via recruiting DDX3X to stabilize Rab27a mRNA in hepatocellular carcinoma

Background Exosomes are deemed to be an important tool of intercellular communicators in cancer cells. Our study investigated the role of PRR34 long non-coding RNA antisense RNA 1 (PRR34-AS1) in regulating exosome secretion in hepatocellular carcinoma (HCC) cells. Methods Quantitative real-time polymerase chain reaction (RT-qPCR) analyzed the expression of PRR34-AS1. We assessed the function of PRR34-AS1 on the biological changes of THLE-3 cells and HCC cells. The downstream interaction between RNAS was assessed by mechanistic experiments. Results PRR34-AS1 expression was upregulated in HCC cells in comparison to THLE-3 cells. PRR34-AS1 depletion repressed HCC cell proliferation, migration and invasion as well as EMT phenotype, while PRR34-AS1 up-regulation accelerated the malignant phenotypes of THLE-3 cells. PRR34-AS1 recruited DDX3X to stabilize the mRNA level of exosomal protein Rab27a. Moreover, PRR34-AS1 facilitated the malignant phenotypes of THLE-3 cells by elevating Rab27a expression to promote the exosome secretion of VEGF and TGF-β in HCC cells. Conclusions The current study revealed a novel function of PRR34-AS1 in accelerating exosome secretion in HCC cells and offered an insight into lncRNA function in the regulation of tumor cell biology. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12967-022-03628-9.

Background
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, with a high morbidity and mortality rate [1,2]. Surgical resection is identified as the most effective therapy for HCC treatment, but high recurrence and distant metastasis after surgery result in poor prognosis of HCC [3,4]. Exosomes play important roles in multiple aspects of HCC, which include angiogenesis, chemoresistance and metastasis [5]. Moreover, some exosome biomarkers are used for the early diagnosis and prognosis of HCC patients [6,7]. Cancer cell-secreted exosomes were considered as important messengers in intercellular communication, thus many studies have unveiled the function of exosome in tumor microenvironment [8]. Nevertheless, few studies have addressed the underlying molecular mechanisms of tumor cell secretion of exosomes.
Exosomes are 30-150 nm nano-sized vesicles containing various types of nucleic acids, such as proteins, RNAs and lipids [9]. Exosome biogenesis is formed by the budding of multivesicular body (MVB) membranes inwards to shape intra-luminal vesicles, ultimately maturing and contained within MVBs [10]. Multiple effectors and molecular mechanisms are involved in regulating the intracellular trafficking steps such as MVB movement, docking, and integration of exosomes with the plasma membrane before release [11]. Rab GTPases are responsible for regulating MVB motility and plasma membrane docking [12]. The Rab family contains many subtypes, and some Rab GTPases are crucial mediators in modulating exosome secretion, such as Rab27A and Rab27B. Rab27A and Rab27B have been reported to be the primary components of vesicle trafficking in exosome secretion and play critical roles in tumor progression and metastasis [13]. Nevertheless, the mechanisms by which Rab GTPases control the secretion of exosome in HCC cells remain to be further investigated.
According to global gene expression data in mammalian species, the majority of the genome is transcribed into non-coding RNAs (ncRNAs) [14]. Long non-coding RNA (lncRNA) is a class of ncRNAs greater than 200 nucleotides in length, and has been emerged as pivotal regulators in cancer progression [15]. LncRNAs can regulate gene expression through transcriptional regulation, post-transcriptional regulation, chromatin modification as well as genomic imprinting [16]. Aberrantly expressed lncRNAs can participate in HCC progression as oncogenes or tumor suppressors [17]. MCM3AP-AS1 exerts an oncogenic role in HCC progression via interacting with miR-194-5p to elevate FOXA1 expression [18]. MAGI2-AS3 impedes cell proliferation and migration in HCC via the miR-374b-5p/SMG1 axis [19]. LncRNA PRR34 long non-coding RNA antisense RNA 1 (PRR34-AS1) regulates HCC cells malignant phenotypes through miR-296-5p/SOX12/E2F2 axis [20] or miR-498/ TOMM20/ITGA6 axis [21]. In this study, to further reveal the effect of PRR34-AS1 in HHC, we probed into the role of PRR34-AS1 in HCC, and investigated the mechanism between HCC cells and THLE-3 cell by exosome secretion. Our study might offer a novel sight for HCC treatment.

Quantitative real-time PCR (RT-qPCR)
Total RNAs were extracted with the application of Trizol reagent (Invitrogen, USA). For the evaluation of gene expression, cDNA was synthesized with the application of PrimeScript RT master mix (Takara, Japan). Next, SYBR Green PCR Master Mix (Applied biosystems) was utilized to conduct qPCR with 2 −△△CT calculation. GAPDH served as control.

5-Ethynyl-2′-deoxyuridine (EdU)
Cell proliferation was assessed with the application of EdU kit (RiboBio, Guangzhou, China). Cells in 96-well plates were incubated to 90% confluence, and then cultured with 100 μL of 50 μM EdU diluent for 2 h. After fixation with 4% paraformaldehyde and 0.5% Triton X-100 treatment, cells were incubated in the dark with 100 μL of 1× Apollo ® staining reaction. DAPI was applied for nuclear redyeing. Images were acquired by a laser confocal microscope.

Colony formation
Cells were planted into 6-well plates (600 cells/ well) for 2 weeks, followed by the treatment of 4% paraformaldehyde and 0.5% crystal violet. Finally, the number of colonies (> 50 cells) was counted.

Wound healing
Cells were planted into 6-well plates until completely adhered to the wall, and then cells were scratched for cell culture. Images of wound healing were obtained at 0 h and 24 h after scratching, and the relative wound width was calculated.

Transwell
Transwell chambers (8-μm pores, Corning, USA) precoated with Matrigel (BD, USA) were used for invasion assay. Cells were planted into the upper chamber, and the bottom chamber was filled with medium containing 10% FBS. After 24 h of incubation, cells were removed from the upper surface, and the invaded cells were subjected to the fixation with 4% paraformaldehyde for 10 min at room temperature, and stained in 0.5% crystal violet. The average number of invaded cells per field was assessed using a microscope.

Western blot
Cells were cultured in RIPA buffer (Beyotime) for the extraction of proteins. BCA protein assay kit II (Beyotime) was performed for determining protein concentrations. The extracted proteins were subjected to the separation on 10% SDS-PAGE and then transferred onto PVDF membrane (Invitrogen), followed by being (1/1000) and all these antibodies were procured from Abcam. Next, blots were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies. The blot signals were measured through enhanced chemiluminescence reagents.

Immunofluorescence staining
Cells were treated with 4% paraformaldehyde and 0.5% Triton X-100 for fixation and permeabilization, and then blocked in 3% bovine serum albumin. Subsequently, cells were cultured overnight at 4 °C with primary antibodies and cultured for 1 h at 37 °C with specific secondary antibodies. After washing, DAPI was utilized for nuclear redyeing. The images of Immunofluorescence staining were obtained under a confocal microscope.

Exosome isolation
Exosomes were separated from cells using Ribo exosome isolation reagent (RiboBio, Guangzhou, China) based on the user manual. Cells were centrifuged at 2000×g for 30 min, and mixed with Ribo exosome solution reagent. The mixtures were refrigerated at 4 °C overnight, followed by centrifugation at 1500×g for 30 min. Exosomes were obtained after removing the supernatants.

Exosome labeling
PKH67 (1 μM, Sigma-Aldrich) was commercially obtained to label the exosomes according to the supplier's protocol. Cell nuclear was double-stained by DAPI. Images were observed under a laser scanning microscope.

Nanoparticle tracking analysis (NTA)
The concentration and distribution of exosome size was measured using ZetaView PMX 10 (Particle Metrix, Germany). Exosomes were re-suspended in granular PBS and added to the instrument. Each sample was recorded in 5 videos to confirm the numbers and size distribution of exosomes.

Transmission electron microscopy (TEM)
Cells were centrifuged and treated with 2.5% glutaraldehyde and then treated by osmium tetroxide. Next, cells were embedded with epoxy resin and cut into sections at a thickness of 100 nm, followed by the treatment of uranyl acetate and lead citrate. The images of microscopic samples were captured using a transmission electron microscope.

Subcellular fractionation
Subcellular isolation of RNAs was performed with the application of Cytoplasmic and Nuclear RNA Purification Kit which was procured from Norgen (Thorold, ON, Canada). Nuclear and cytoplasmic fractions were measured via RT-qPCR.

Luciferase reporter assay
For Rab27a promoter analyses, the sequence of Rab27a promoter was firstly sub-cloned into pGL3-basic reporter vectors (Promega, Madison, WI, USA). After the co-transfection of downregulated PRR34-AS1 and plasmids, the activity of Rab27a promoter was evaluated using a luciferase assay kit (Promega).

RNA-binding protein immunoprecipitation (RIP)
Magna RNA-binding protein immunoprecipitation kit (Millipore, USA) was utilized to assess the binding between RNAs. Cell lysates were cultured with RIP buffer and antibodies (anti-Ago2, anti-SNRNP70 and anti-DDX3X) conjugated with magnetic beads for 1 h. The enrichment of RNAs was evaluated by RT-qPCR.

RNA pull down assay
Pierce Magnetic RNA-Protein Pull-down kit (Thermo fisher scientific) was used in this assay in the light of supplier's instructions. PRR34-AS1 sense and PRR34-AS1 antisense were in vitro transcripted and labeled. Biotin-labeled PRR34-AS1 (50 pmol) was cultured with streptavidin magnetic beads (50 μL) with rotation, and then hatched in cell lysates. The eluted proteins were measured by western blot after mass spectrometry.

Actinomycin D assay
After reaching 80% confluence, cells were treated with actinomycin D (4 µg/mL, Sigma-Aldrich, USA) to inhibit transcription. The relative mRNA levels were analyzed by RT-qPCR at the indicated time points.

Statistical analysis
Data were processed by GraphPad Prism 6.0 Software and displayed as means ± standard deviation of three replicates. The Student's t-test and two-way ANOVA were used to analyze differences between two groups or more than two groups. Data were defined as statistical significance if P < 0.05.

PRR34-AS1 overexpression enhances the capacities of proliferation, migration, invasion and EMT of THLE-3 cells
First, GEPIA 2 (http:// gepia2. cancer-pku. cn) database was applied to assess PRR34-AS1 expression in liver hepatocellular carcinoma (LIHC) tissues. PRR34-AS1 exhibited a markedly high expression level in LIHC tissues (Additional file 1: Fig. S1A). Consistently, RT-qPCR analysis indicated that PRR34-AS1 was highly expressed in HCC cells including HLF, Huh-7, SNU-449, HepG2 and LM3 in comparison to THLE-3 cells (Fig. 1A). Since HepG2 and LM3 cells presented the highest PRR34-AS1 expression among the selected cells, both HepG2 and LM3 cells were selected for subsequent assays. We stably silenced PRR34-AS1  Fig. S1B). As shown in EdU and colony formation assays, PRR34-AS1 depletion reduced the percent of EdU positive cells and the number of colonies (Additional file 1: Fig. S1C, D). Meanwhile, the relative wound width was obviously increased when PRR34-AS1 was knocked down, suggesting that PRR34-AS1 silencing inhibited cell migratory ability in HCC (Additional file 1: Fig. S1E). Additionally, the number of invaded cells was also decreased in PRR34-AS1-silenced HepG2 and LM3 cells (Additional file 1: Fig. S1F). Moreover, we also used western blot to evaluate the role of PRR34-AS1 depletion on EMT in HCC cells. It turned out that PRR34-AS1 absence caused an upregulation in the level of epithelium marker (E-cadherin) whereas diminished the levels of mesenchymal markers (N-cadherin and Vimentin) and EMT transcription factors (Slug and Twist), implying that PRR34-AS1 silencing suppressed the EMT process in HCC cells (Additional file 1: Fig.  S1G). Collectively, PRR34-AS1 enhances cell proliferative, migratory, invasive and EMT phenotypes in HCC. We next up-regulated PRR34-AS1 expression in THLE-3 cells (Fig. 1B) and found that the EdU positive stained cells were sharply increased in THLE-3 cells after PRR34-AS1 overexpression (Fig. 1C). Likewise, PRR34-AS1 overexpression led to an augment on the number of colonies (Fig. 1D), suggesting that PRR34-AS1 up-regulation facilitated the proliferation ability of THLE-3 cells. Besides, we found that the relative wound width was shorter in PRR34-AS1 groups compared to NC group, implying that up-regulated PRR34-AS1 promoted the migratory capacity of THLE-3 cells (Fig. 1E). Simultaneously, transwell assays disclosed that PRR34-AS1 overexpression enhanced the invasive capacity of THLE-3 cells (Fig. 1F). Additionally, we also assessed the influence of PRR34-AS1 up-regulation on EMT in THLE-3 cells. E-cadherin level was obviously reduced whereas the levels of N-cadherin, Vimentin, Slug and Twist were enhanced in PRR34-AS1-up-regulated THLE-3 cells (Fig. 1G). Similarly, the intensity of E-cadherin was weakened whereas N-cadherin intensity was strengthened in THLE-3 cells when PRR34-AS1 was overexpressed, implying that PRR34-AS1 overexpression accelerated EMT in THLE-3 cells (Fig. 1H). Taken together, PRR34-AS1 up-regulation facilitated the malignant capacities of THLE-3 cells.

Enhanced cell proliferative, migratory, invasion and EMT abilities are observed in co-cultured HCC and THLE-3 cells
Then, we wondered whether HCC cells could secrete exosomal PRR34-AS1 and transmit them to THLE-3 cells, leading to the promotion of cell proliferation, migration, invasion and EMT in THLE-3 cells. After the co-culture of HepG2 or LM3 cells with THLE-3 cells, we performed functional assays in co-cultured cells. Cell proliferation ability was enhanced in co-cultured cells relative to THLE-3 cells ( Fig. 2A, B). Consistently, the potential of cell migration and invasion was markedly promoted in co-cultured cells (Fig. 2C, D). Moreover, we found that E-cadherin level was reduced while the level of N-cadherin, Vimentin, Slug and Twist were elevated in co-cultured cells (Fig. 2E). Meanwhile, the data from immunofluorescence staining also uncovered that the cocultured cells had a stronger EMT capacity than THLE-3 cells (Fig. 2F). However, PRR34-AS1 expression had no marked change in co-cultured cells (Fig. 2G), which excluded the hypothesis of exosomal PRR34-AS1 in HCC cells. Collectively, the increased cell malignant phenotypes were observed in co-cultured HCC and THLE-3 cells.

HCC cell-secreted exosomes promote cell malignant phenotypes
Then, we speculated the existences of other exosome transmitted from HCC cells to THLE-3 cells. Exosomes from HCC cells were isolated and labeled with PKH67 dye (green). The result showed that PKH67-labeled exosomes dispersed in HCC cell cytoplasm (Fig. 3A). Besides, we found that exosomes ranged from 50 to 150 nm in diameter (Fig. 3B). Moreover, we found that the exosomes under TEM had the typically round or cupshaped morphology (Fig. 3C). Additionally, western blot analysis further confirmed that HCC cells had abundant exosomal proteins (CD9, CD63, HSP70 and TSG101) and extracellular vesicle proteins (CD81, ERBB2 and HLA-A) (Fig. 3D). All above results validated the existence of exosomes in HCC cells. Subsequently, we treated HCC cell-secreted exosomes into THLE-3 cells to observe the changes of their cellular phenotypes. As shown in Fig. 3E, F, the proliferation ability was obviously increased in THLE-3 cells (Fig. 3E, F). Meanwhile, the capacities of migration and invasion were apparently enhanced in THLE-3 cells treated with HCC cell-secreted exosomes (Fig. 3G, H). Moreover, we found that the EMT process was also accelerated in THLE-3 cells treated with HCC cell-secreted exosomes (Fig. 3I). Expectedly, the expression of PRR34-AS1 had no significance change in THLE-3 cells treated with HCC cell-secreted exosomes (Fig. 3J). Collectively, HCC cell-secreted exosomes enhanced cell proliferation, migration, invasion and EMT.

PRR34-AS1 regulates exosomal protein Rab27a in HCC cells
We extracted the exosomes secreted from HCC cells and found that the level of total exosomal protein, exosome markers (CD9, CD63, HSP70 and TSG101) and extracellular vesicle proteins (CD81, ERBB2 and HLA-A) was obviously decreased when PRR34-AS1 was downregulated ( Fig. 4A-D). Therefore, we speculated that the level of HCC cell-secreted exosomal proteins might be regulated by PRR34-AS1. We then analyzed the level of exosomal proteins in HCC cells treated with downregulated PRR34-AS1. The level of Rab27a was substantially inhibited in HCC cells after PRR34-AS1 silencing and other exosomal proteins had no marked changes (Fig. 4E). Taken together, PRR34-AS1 regulated exosomal protein Rab27a in HCC cells.

PRR34-AS1 interacts with DDX3X to regulate Rab27a mRNA stability
To uncover the regulatory mechanism of PRR34-AS1 in HCC cells, we firstly assessed the cellular distribution of PRR34-AS1 in HCC cells. As shown in Fig. 5A and Additional file 2: Fig. S2A, PRR34-AS1 was primarily distributed in cell cytoplasm, indicating that PRR34-AS1 might post-transcriptionally regulate Rab27a in HCC cells. Meanwhile, luciferase reporter assays further confirmed this point, as indicated that PRR34-AS1 could not affect the luciferase activity of Rab27a promoter (Fig. 5B). It is well-acknowledged that lncRNA acts as a ceRNA to regulate cancer progression at post-transcriptional levels. We wondered whether PRR34-AS1 existed in the RNA-induced silencing complex (RISC) to bind to miR-NAs and thereby regulated Rab27a expression. However, the outcome from RIP assays dispelled this hypothesis, as indicated that the enrichment of PRR34-AS1 had no significant change in Anti-Ago2 groups (Fig. 5C). Thus we further treated protein synthesis inhibitor (actinomycin D) into HCC cells to assess Rab27a mRNA level.
The half-life of Rab27a mRNA was obviously declined after PRR34-AS1 silencing, indicating the regulatory effect of PRR34-AS1 on the stability of Rab27a mRNA (Fig. 5D). Then, the electrophoretic gel indicated that one specific band appeared at approximately 73 kDa and we finally identified it as DDX3X protein (Fig. 5E). Furthermore, the combination between PRR34-AS1 and DDX3X was confirmed by western blot and RIP analyses (Fig. 5F, G). Intriguingly, we also found the combination between Rab27a and DDX3X in HCC cells via RNA pull down assays (Fig. 5H). Moreover, we found that the combination of Rab27a and DDX3X was lessened in HCC cells after the downregulation of PRR34-AS1 (Fig. 5I). Thus we conjectured that PRR34-AS1 might interact with DDX3X to regulate Rab27a mRNA stability. We knocked down the levels of DDX3X and revealed that the levels of Rab27a were markedly decreased in HCC cells after DDX3X depletion (Additional file 2: S2B, C). Meanwhile, the stability of Rab27a mRNA was also reduced when DDX3X was silenced in HCC cells treated with actinomycin D (Fig. 5J). Collectively, PRR34-AS1 interacted with DDX3X to mediate Rab27a mRNA stability.

PRR34-AS1 facilitates THLE-3 cells proliferation, migration, invasion and EMT via elevating Rab27a expression to increase the exosome secretion of VEGF and TGF-β
Next, we investigated the role of Rab27a in THLE-3 cells.
We overexpressed Rab27a expression in THLE-3 cells and observed that the proliferation ability was obviously enhanced when Rab27a was up-regulated in THLE-3 cells (Fig. 6A-C). In parallel, the capacities of migration and invasion were promoted in THLE-3 cells after Rab27a up-regulation (Fig. 6D, E). Moreover, we found that Rab27a overexpression elevated the EMT process in THLE-3 cells (Fig. 6F, G). It has been reported that Rab27a promotes tumor growth via increasing VEGF and TGF-β secretion in vitro [22]. Herein, we also found that the protein concentration of VEGF and TGF-β in THLE-3 cells as well as their protein levels in HCC cell-secreted exosomes was increased after Rab27a upregulation (Fig. 6H, I). All above results suggested that Rab27a increased VEGF and TGF-β secretion in HCC cells and transmitted them into THLE-3 cells to elevate cell proliferative, migratory and invasive phenotypes as well as EMT.
To explore the effect of PRR34-AS1 and Rab27a on the malignant phenotypes of THLE-3 cells, rescue experiments were arranged after the verification of the knockdown efficiency of Rab27a (Additional file 3: Fig. S3A). As indicated in Additional file 4: Fig. S4A, B, Rab27a silencing reversed the enhanced proliferation ability in THLE-3 cells after PRR34-AS1 overexpression. Besides, it was found that the enhanced migration and invasion capacities induced by PRR34-AS1 up-regulation were abolished by co-transfection of sh/Rab27a#a (Additional file 4: Fig.  S4C, D). Moreover, the decreased protein level of E-cadherin and the enhanced levels of N-cadherin, Vimentin, Slug and Twist in THLE-3 cells transfected with PRR34-AS1 overexpression was overturned by co-transfection of sh/Rab27a#a (Additional file 4: Fig. S4E). Meanwhile, the data of immunofluorescence assays further confirmed that Rab27a depletion abrogated the increased EMT process in THLE-3 cells after PRR34-AS1 augment (Additional file 4: Fig. S4F). Taken together, PRR34-AS1 facilitated THLE-3 cells proliferation, migration, invasion and EMT via elevating Rab27a expression.

Discussion
In this research, we verified that lncRNA PRR34-AS1 recruited DDX3X to stabilize Rab27a mRNA and thereby promoted cell proliferative, migratory, invasive and EMT phenotypes in HCC. Meanwhile, PRR34-AS1 up-regulated Rab27a expression to increase the exosome secretion of VEGF and TGF-β in HCC cells and transmitted them into THLE-3 cells to accelerate the malignant phenotypes of THLE-3 cells. Our study revealed a novel function of PRR34-AS1 in regulating exosome secretion from HCC cells to THLE-3 cells, which provides a promising method for HCC treatment.
In recent years, lncRNAs have emerged as a pivot in the field of cancer research. LncRNA-D16366 is lowly expressed in HCC and might be an independent diagnostic and prognostic indicator for HCC [23]. RGMB-AS1 plays as a tumor suppressing role in HCC and is an independent favorable prognostic factor for patients with this disease [24]. All above studies demonstrated the anticancer effects of lncRNAs in HCC. In our research, we proved the oncogenic effect of lncRNA PRR34-AS1 in HCC. Furthermore, PRR34-AS1 exhibited a high expression level in HCC. PRR34-AS1 knockdown inhibited HCC cell proliferative, migratory, invasive and EMT abilities. Taken together, lncRNAs play an important role in the regulation of HCC progression.
Our study also found that PRR34-AS1 overexpression facilitated THLE-3 cell malignant phenotypes, suggesting the possibility of exosomes in HCC cells. Exosome-mediated communication in the tumor microenvironment contributes to HCC progression [5]. Exosome-transmitted lncRNA SENP3-EIF4A1 inhibits cell migration and invasion in HCC [25]. High expression of exosomal H19 enhances the proliferation and motility of HCC cells [26]. All these evidence suggested the regulation of exosomal lncRNA in HCC progression. In our study, we confirmed that the capacities of proliferation, migration, invasion and EMT were increased in THLE-3 cells co-cultured with HCC cells as well as in THLE-3 cells treated with HCC-secreted exosomes. However, PRR34-AS1 expression was not affected in THLE-3 cells co-cultured with HCC cells as well as in THLE-3 cells treated with HCCsecreted exosomes, which ruled out the existence of exosomal PRR34-AS1 in HCC.
As we all know, exosomes are a type of secretory membrane vesicle with the structural and biochemical characteristics of multivesicular endosomes [27]. Several Rab GTPases, including Rab27a, Rad27b, and Rab35, were previously found to play a crucial role in regulating exosome secretion and influencing cellular process [28]. It has been reported that lncRNA HOTAIR motivates exosome secretion via mediating RAB35 and SNAP23 in HCC [29]. In our study, we demonstrated that PRR34-AS1 silencing obviously reduced the expression of Rab27a. It has been reported that KIBRA modulates exosome secretion via repressing the proteasomal degradation of Rab27a [30]. All above results uncovered that PRR34-AS1 regulated Rab27a to affect exosome secretion in HCC cells.
Moreover, our study found that PRR34-AS1 interacted with DDX3X to regulate the stability of Rab27a mRNA. In line with our study, HHIP-AS1 inhibits HCC progression through recruiting HUR to stabilize HHIP mRNA [31]. FAM83A-AS1 expedites HCC progression by interacting with NOP58 to increase the mRNA stability of FAM83A [32]. After confirming the regulatory mechanism between PRR34-AS1 and Rab27a, we revealed that Rab27a accelerated cell malignant phenotypes in THLE-3 cells. More importantly, Rab27a upregulation also promoted exosome secretion of VEGF and TGF-β, suggesting that PRR34-AS1 regulated Rab27a to promote the exosome secretion of VEGF and TGF-β and thereby transmitted them into THLE-3 cells to accelerate cell malignant phenotypes.
However, our study had some limitations. We didn't unravel the detailed mechanism of Rab27a on regulating exosome secretion of VEGF and TGF-β, which will be addressed in the future.


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Malassezia in Inflammatory Bowel Disease: Accomplice of Evoking Tumorigenesis

Accumulating evidence indicates that patients with inflammatory bowel disease (IBD) have a significantly higher risk of developing different cancers, while the exact mechanism involved is not yet fully understood. Malassezia is a lipid-dependent opportunistic yeast, which colonizes on mammalian skin and internal organs. Also, dysbiosis in fungal communities accompanied by high level of Malassezia are fairly common in inflammatory diseases such as IBD and various cancers. In cancer patients, higher levels of Malassezia are associated with worse prognosis. Once it is ablated in tumor-bearing mice, their prognostic conditions will be improved. Moreover, Malassezia manifests multiple proinflammatory biological properties, such as destruction of epithelial barrier, enrichment of inflammatory factors, and degradation of extracellular matrix (ECM), all of which have been reported to contribute to tumor initiation and malignant progression. Based on these facts, we hypothesize that high levels of Malassezia together with mycobiome dysbiosis in patients with IBD, would aggravate the microecological imbalance, worsen the inflammatory response, and further promote tumorigenesis and deterioration. Herein, we will discuss the detrimental properties of Malassezia and explore the key role of this fungus in the correlation between IBD and cancer, in order to take early surveillance and intervention to minimize the cancer risk in individuals with IBD.

INTRODUCTION
Inflammatory bowel disease (IBD) belongs to chronic idiopathic gastrointestinal (GI) inflammatory diseases, characterized by imbalance of the intestinal microbiome (1). Typically, IBD contains Crohn's disease (CD) and ulcerative colitis (UC), of which the common characteristic is the inflammation in the GI wall. Their main distinction is the site and depth of lesions. UC is generally limited to the colon, while CD may include the whole intestine ranging from the mouth to the rectum (2). The rapid pace of IBD expansion over only several decades is disturbing, which has led to serious socioeconomic burden (3).
Emerging evidence suggests that the damaged intestine barrier and gut microbiome dysbiosis are closely linked with the genesis of IBD (4)(5)(6). Generally, the batches of bacteria, viruses, archaea, fungi, and eukaryotic microbes inhabiting in the GI tract are referred as gut microbiota, which have already formed a mutually beneficial correlation with the host (7,8). Among the collection of bacteria, persistent enteric pathogens including adhesion-invasive Escherichia coli (AIEC) and Clostridium difficile, may act as a trigger for IBD (6). Besides bacterial contribution, there is increasing awareness regarding the impact of mycobiome as immunologically reactive components. On the whole, IBD and ordinary population differ in mycobiome. Alterations in gut mycobiome as well as their communications with other intestine microbiota are essential to maintain the intestinal barrier, such as in IBD (9)(10)(11)(12)(13)(14). Reports showed that higher abundance of Candida albicans was observed in the intestinal tract of UC patients, when compared to controls, conversely, the decreased amount of C. albicans indicated the improvement and recovery of UC (12). Studies also confirmed that when compared with healthy people, one specific commensal fungi Malassezia yeast has been identified particularly abundant in people with IBD, which may scale up inflammatory cytokine production and aggravate inflammation in IBD (10,11,13,14). In addition, when the microflora balance is disturbed or the host immune defense is impaired, fungi may spread or transfer from the original symbiotic habitat to other important organs such as the gut, thus becoming a susceptible factor for lifethreatening infection (15). The mouse model of psoriasis showed that pre-exposure of symbiotic fungus may significantly worsen tissue inflammation through enhancing T helper type 17 (Th17)dependent immune responses and phagocytosis of neutrophils (16).
Recently, it has been noticed that malignancies are prevalent in people with IBD (17)(18)(19)(20). Reports showed that standardized incidence ratios (SIRs) for pancreatic cancer (PC) in IBD cases were 7.6-fold higher than the ordinary persons (21). For colorectal cancer (CRC), when compared with the reference individuals, the cancer risk in patients with IBD increased considerably, up to around 10 times (22,23). Studies indicated a strong association between IBD and bile duct cancer, and the SIR could rise remarkably in patients with UC for intrahepatic cholangiocarcinoma (24,25). Extraintestinal analyses also revealed that the IBD group had a significantly elevated risk for skin cancer (e.g., melanoma), particularly among CD patients or elder population (26)(27)(28). Furthermore, evidence has demonstrated that inflammation acts a substantial role in oncogenesis (29,30).
However, the underlying mechanism linking IBD to cancer remains to be further clarified. Malassezia, as representative fungal commensals, may be the key to push aside this dense fog. Based on this, this work is intended to discuss and elaborate the interplay between Malassezia, IBD, and cancer.

HYPOTHESIS
Considering current evidence, we hypothesize that the particular enrichment of Malassezia genius in the gut microbiome could promote inflammatory responses in IBD patients through the following microbiological characteristics: disrupting the integrity of epithelial barrier; increasing the release of proinflammatory molecules; and degrading the extracellular matrix (ECM). In this manner, Malassezia can further induce neoplasia and raise cancer incidence in the IBD population under inflammatory conditions ( Figure 1).

General Properties of Malassezia
Malassezia pertains to the category of lipid-dependent yeast and is an essential symbiotic organism resident on the mammalian skin, hair, and GI tract (31). Under certain scenario, Malassezia spp. may switch to opportunistic pathogens.
When referring to microbial variability in different body sites, the skin microbiome is known for its relatively high fungal presence (32). Malassezia is the dominated eukaryotic component of microbial communities identified in human and is most abundant in craniofacial sebaceous glands and relatively low in arms or trunk, due to the enrichment of the lipid nutritional sources (33,34). So far, as many as 20 species of Malassezia strains have been identified (35,36). Malassezia restricta and Malassezia globosa are the top 2 most abundant in human skin (37). However, the distribution of Malassezia is not confined to the skin, high-throughput sequencing revealed that they are also detected at a relatively high frequency in the GI and respiratory tract sample sets, with positive rates of 88% and 86%, respectively (38,39).
Shaping on fungal community is changing with age. The skin microbiota of neonates delivered vaginally resembled their mother's vaginal mycobiome, while that of neonates born through cesarean section was similar to the skin surface of mothers (40,41). Malassezia colonized once after birth and was more resembling adult microbiome assemblage, as driven by maternal hormones (42,43). The relative abundance of Malassezia in infant skin was only 2%, while cesarean-born infants owned lower (44). The sebaceous gland entered a dormant state within 6 months after birth, thus Malassezia returned to low enrichment. M. globosa predominately colonized on prepubertal skin. During adolescence, the increase of lipid levels in the sebaceous glands can lead to a simultaneously increased percentage with Malassezia (45). Skin fungal community analysis showed that Malassezia fungus was absolutely dominant in adults, with predominance up to over 90%. In contrast, Malassezia was relatively lower in children under 14, but the fungal community was more diverse (46).

Pathogenic Properties of Malassezia
Studies showed that Malassezia spp. were associated with numerous inflammatory diseases, such as dermal inflammation (16,47), IBD (13,14), CRC (48), pancreatic carcinoma (31), and severe infections (49). In general, Malassezia is mainly colonized in the skin (50). However, the availability of lipid nutrition within the GI tract may facilitate the localization and survival of Malassezia in the gut, and its nutrition can be accessed from host diet or intestinal fungal synthesis (51,52). It is speculated that Malassezia might immigrate to the GI tract along with the diet; however, solid evidence is lacking. Manifold factors can influence the pathogenicity of Malassezia, which involves the virulence and quantity of Malassezia, environmental conditions including humidity, temperature, oxygen, and fatty acid nutrients, as well as the susceptibility of the host (53). So far, several viewpoints have been proposed to reveal the pathogenic behaviors of Malassezia, which may bring more dawn for understanding the inflammatory or infectious pathogenesis.

Epithelial Barrier Defects
The epithelial barrier is organized as a protective and complicated system that allows nutrient exchange while preventing the displacement of microorganisms and their metabolites. This tight barrier is obviously a main hurdle which must be broken through for microbial antigens to enter the human body, for example, in the lumen of the gut (54). Malassezia could produce increased irritating free fatty acids through metabolism, typified by oleic acid (OA), which may damage the permeability of the skin barrier and lead to skin itching or even exfoliation (55,56). Because of this, when applying OA to the scalp, it would induce scalp flaking in patients with seborrheic dermatitis (55). Malassezia species were kind of lacking synthase genes for fatty acid, which may be supplemented by enhancing the expression of genes encoding secretory hydrolases in Malassezia genome to generate fatty acid (57). Among them, the extracellular lipases and phospholipases secreted by Malassezia could severally influence its virulence factors on the release of distinct metabolites and the cell wall characteristics itself, in order to facilitate epithelium targeting, lesion aggravation, and barrier disruption (57,58). It is reported that the lipase activity of Malassezia is linked to the pathogenesis of inflammatory skin diseases in vitro (59). Malassezia phospholipase activity has also been reported to be related to its virulence in dogs (60). What is more, other environmental determinants such as increased epidermal water loss, loss of tight junction proteins, decrease in both cholesterol and free fatty acid, and high pH value, could exhibit catalytic effects in the pathogenesis of Malassezia, thus inducing and exacerbating cutaneous inflammation (61,62). Defective cutaneous barriers failed to provide adequate protection against microbes or allergens, instead they may assist Malassezia to enter the blood circulation system, resulting in immune activation and inflammatory process (63).
We own innate immunity in our genomes which provides a defense against Malassezia infection. As a positive regulator, mast cells (MC) can detect and control the fungi Malassezia at the infected site, which may be activated by Toll-like receptor 2 (TLR2) (64,65). Macrophages can effectively defend the host against the attack of opportunistic fungal pathogens through phagocytosis and collection of phagocytic contents, affected by TLR9 (66,67). As innate immune receptors, Dectin-2 and Mincle were mainly involved in the immune recognition to Malassezia, arousing the production of pro-and anti-inflammatory factors (68). Moreover, Malassezia may induce a reciprocal activation between natural killer (NK) cells and dendritic cells (DCs), in which NK cells would promote the maturation and costimulatory capacity of DCs, as well as accelerate the release of interleukin-8 (IL-8) in DCs (69,70). These innate immune responses will play an important role in the subsequent adaptive immunity including activation of T cells. Malassezia may also promote the progression of inflammation directly or indirectly by mediating host receptor recognition, promoting the release of proinflammatory cytokines and secreting chemicals or vesicles. Within the host, C-type lectin receptor (CLR) family could specifically recognize fungal microorganisms and initiate adaptive immune responses (68,71). CLRs were specialized in sensing the carbohydrates in the cell wall of Malassezia, mediating the signaling adaptor caspase recruitment domaincontaining protein (CARD)-9 to drive the polarization of CD4+ T lymphocytes into IL-17-producing immune cells, such as gd T cells or innate lymphoid cells (ILCs) (71,72). Malassezia could generate indoles, the ligands for the aryl hydrocarbon receptor (AhR), which may also promote Th17 differentiation and IL-17 secretion through activation of AhR signaling (73,74). By eliciting the production of inflammatory cytokine IL-17 strongly, Malassezia spp. has been reported to stimulate tissue inflammation, destroy skin integrity, and further contribute to or augment epicutaneous infections such as atopic dermatitis (AD) in murine models (72). Other proinflammatory cytokines including IL-18, IL-8, and IL-6 and Th22 chemokines including C-C Motif Chemokine Ligand 17 (CCL17) were significantly increased after exposure to Malassezia as well, so as to worsen local inflammation (75,76). It has been confirmed that, compared with the negative group, Malassezia colonization can induce CARD9-S12N polymorphism and strongly enhance the release of cytokines such as IL-10 or tumor necrosis factor alpha (TNF-a) in either wild-type (WT) or Card9 −/− colitis mice (13). Moreover, Malassezia was able to activate NLRP3 inflammasome through Dectin2/CARD9 signal, accelerating the generation of IL-1b to aggravate inflammation (77). Zhang et al. also demonstrated that Malassezia can produce nanovesicles rich in allergens or proteins, which may initiate and maintain inflammation by activating the nuclear factor-kB (NF-kB) pathway and upregulate IL-6 production in the immune microenvironment (78).

ECM Degradation
ECM is a highly dynamic acellular network composed of collagen, fibronectin, and several other proteins (79). It is of great importance in the inflammatory process. Reports showed that ECM was involved in the signal transmission to recruit inflammatory cells, stimulate cell migration, and restore inner homeostasis for coping with external stimulus (80). Variation or degradation of ECM components has been confirmed to have a close relationship with the progression of various inflammatory diseases, such as AD (81,82). Some virulence factors including acid sphingomyelinase and aspartate protease, which were secreted by Malassezia, could mediate ECM degradation and participate in the pathogenesis (83). MgSAP1, one unique secreted M. globosa protease, has been implicated in hydrolyzing host proteins to provide nutrition and destroy ECM elements, so as to facilitate pathogen adhesion in the inflamed areas (84). Adhesion is a decisive step in the pathogenesis of microbial infection (85). One close homolog of secretory MgSAP1 protease produced by Malassezia furfur is MfSAP1, which owned high catalytic efficiency in extracellular proteins of human skin, particularly when substrate collagen was thermally denatured (86,87). It has been proved that MfSAP1 was likely to modify the epidermal and dermal environment through degrading key components of skin-correlated ECM, such as vitronectin, fibronectin, and thrombospondin, even at low proportions of enzyme to the substratum. Accordingly, high concentrations of MfSAP1 could rapidly and sensitively cleave these ECM proteins and inhibit cell migration and attachment to the fundamental ECM, thus attenuating re-epithelization process and retarding cutaneous wound healing in an acute traumatic cell model (86).
Apart from proinflammatory motivation, Malassezia has manifested some other biological features, including unique cell-defense characteristics. Malassezia owns quite thick and unique multilayered cell walls, which could protect themselves from complex environmental stress and help to escape phagocytosis (88). In particular, Malassezia was able to form biofilms on their surface, which was correlated to the emergence of drug resistance and the maintenance of virulence (89). Beyond these described circumstances, Malassezia may promote disease progression by modulating the pathogenicity of other microorganisms, for instance Staphylococcus aureus through microbial communications (90).

INCREASED MALASSEZIA AND ITS RELEVANCE WITH INFLAMMATION IN IBD AND OTHER INFLAMMATORY DISEASES
In recent years, the detection of Malassezia in the digestive tract has attracted public attention. Microbial diversity analysis showed that Basidiomycota phyla ranked as second major dominators, following Ascomycota phyla, within gut fungal microbiota in both healthy controls and IBD patients, although there were some variations among different disease phenotypes (14). As contrasted to healthy persons, Malassezia genera were observed with a high prevalence in people with IBD, which was one primary reason to the increase of Basidiomycetes in IBD (11,14). Sokol and his team revealed that the higher abundance of Malassezia was noticed in the acute stage of IBD population (14). In one retrospective cohort, when compared with healthy volunteers, increased abundance of Malassezia was also detected in patients with IBD (50). Limon and his colleagues reported that Malassezia species, represented by M. restricta, were more enriched in the sigmoid colon mucosa of CD patients, where massive monocyte-derived DCs were involved in the following pathogenesis (13). Statistic data suggested that M. sympodialis and its extract were able to initiate MC to secret cysteinyl leukotrienes and intensify IgE-dependent immunoreactions in vitro, which might possibly lead to deteriorative inflammation in IBD as well (91,92). The polymorphism of CARD-9 in CD would encourage the colonization of M. restricta yeast in the intestine, which may worsen or even exacerbate the intestinal inflammation by strongly evoking proinflammatory responses of macrophages and monocyte-derived DCs in murine models (13). Beyond this, Liguori et al. also demonstrated that the overall load of Malassezia in CD patients was significantly increased in contrast to ordinary ones (p < 0.05), suggesting that Malassezia may play a role in the pathogenesis of mucositis (11). Similar results have been obtained from the clinical analysis on UC. Regarding fecal fungal composition, there was a significant difference in only several fungal genera between UC patients and non-IBD controls, among which Malassezia owned a relatively higher abundance in patients with UC, particularly when at the active phase (93).
Aside from IBD, evidence suggests that the enrichment of Malassezia is also obviously increased in other chronic or inflammatory diseases, such as psoriasis, AD, and cystic fibrosis (CF). Scholars found out that the average quantity and species diversity of Malassezia in psoriatic patients were higher than those in healthy individuals, particularly in skin lesions (94). Malassezia-positive culture rate of scalp skin samples was 85% and 50% in patients with psoriasis and healthy subjects, respectively (95). The more serious the lesion, the higher is the positive rate. Studies also indicated that Malassezia allergens could induce immunoglobulin E (IgE)-mediated sensitization in AD subjects (96). There was a significant positive correlation between Malassezia-specific IgE levels and AD severity (97). Mittermann and colleagues reported that compared with healthy controls, Malassezia was more abundantly detected in sputum specimens of asthmatic patients, particularly in children (98). High detection rates of Malassezia were observed in respiratory tract of CF patients as well (99,100). Beyond this, higher Malassezia colonization was detected in subjects with neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (101)(102)(103). In most cases, the severity of the mentioned diseases is positively correlated with the abundance of Malassezia detected.
It seems ubiquitous that the appearance frequency of Malassezia yeast differs in distinct human body site. As Malassezia strains are resident members of cutaneous mycobiome, it is not so esoteric to understand the relationship between the high prevalence of Malassezia and psoriasis or AD. Apart from this, Malassezia may be an overlooked fungal pathogen that is neglected to participate in inflammatory splanchnic diseases, particularly in IBD.

Malassezia Is Associated With Cancer
Host-microflora interactions in the tumor microenvironment play an important driving role in tumorigenesis and progression. In various noninfectious diseases such as cancer, the emphasis of most research is concentrated in bacteriome. Recently, scholars have recognized the significance of mycobiome and microbial dysbiosis in genesis and development of neoplasms. Since its powerful potential in cancer development, the role of one cardinal fungus genus Malassezia has just begun to come under scrutiny.
Epidemiological data suggested that abundant yeast niches of Malassezia were overlapping with cancerous regions of basal cell carcinoma in humans and animals such as dogs or cats (96,104). This yeast may promote dermal carcinogenesis through synthesis and activation of AhR ligands and further inhibition of cell caducity (104). Moreover, Aykut and his team reported that Malassezia was remarkably elevated in the pancreas of PC patients and mice models than that in healthy volunteers. Furthermore, they have demonstrated that repopulation with Malassezia could promote PC through mannose-binding lectin (MBL) signal pathway, thus accordingly its ablation with amphotericin B in murine models was found to slow oncogenic progression (31,105). Malassezia also showed higher relative abundance in patients with oral squamous cell carcinoma (OSCC), compared with control volunteers (106). In addition, Gao and collaborators discovered that although there was no significant difference in stool mycobiota diversity between CRC patients and healthy controls, the fungal subgroup Malassezia genus was more enriched in people with CRC, which was positively correlated with tumor progression (48). Similar results were obtained in another analysis performed by Coker and coworkers. They found that when compared with control subjects, Malassezia strains were elevated significantly and the mycobiome diversity was specifically modified in CRC (107). This may provide a potentially predictive diagnostic marker for CRC. However, so far, there is no exact hypothesis about the underlying mechanism of Malassezia involved in cancer, and the relationship between Malassezia and CRC still awaits further study.

Inflammation and Cancer
There are many hypotheses about the pathogenesis of tumors, among which the theory of inflammatory mechanism is the widely accepted one. Usually, inflammation is fundamental to fight against harmful or pathogenic stimuli, hasten the wound to restore and maintain normal function of tissues, which involves in endothelial cells, immune cells, and inflammatory agents (108). Self-limited acute inflammation is beneficial in the healing process (109). However, when it is out of control, it may develop into chronic inflammation, induce tissue lesions and predispose to cancer (110), including tumorigenesis, progression, and metastasis (111). Only a small portion of cancers are ascribed to germ line mutations, while 90% of cancers are associated with somatic mutations and environmental hazards, and the latter is always linked to chronic inflammation or infections (112). Epidemiological investigations showed that inflammation was closely related to the occurrence of about 20% of all cancers (113). Triggers of chronic inflammation for enhanced risk or progression of cancer include microbial infections, such as Helicobacter pylori in gastric adenocarcinoma and chronic hepatitis B virus (HBV) in hepatocellular carcinoma (HCC), and inflammatory diseases (e.g., UC in CRC), which were closely bound up with genetic instability (114)(115)(116). Accordingly, with H. pylori being eradicated, the conditions of atrophic gastritis and intestinal metaplasia would be improved or eliminated, thus potentially suppressing the generation of gastric cancer (117). Moreover, existing evidence has shown that hypoxia-associated inflammatory cytokines or chemokines were significantly elevated in the tumor microenvironment, for example, IL-6, IL-1, and TNF (29,118). Anti-inflammatory drugs may benefit cancer patients, such as TNF blockade and nonsteroidal antiinflammatory drugs (NSAIDs) (119,120).

Malassezia-Related Inflammation Contribute to Tumorigenesis
As mentioned above, Malassezia has exhibited profound proinflammatory effects and been linked to oncogenesis. On the other hand, inflammation can influence all the stages of tumorigenesis and drive tumor development and metastasis. Based on prior elaboration, Malassezia-associated inflammation may promote tumor initiation and malignant progression through multiple approaches: (1) Barrier impairment-epithelial barrier deterioration, including aberrant production of mucin and defective expression or organization of tight junctional proteins, induced by oncogene activation, was an early malignant behavior in intestinal tumorigenesis (121). Correspondingly, loss of mucus would result in increased penetration of epithelial barriers and enhanced microbial translocation through the colon, leading to colorectal neoplasms in mouse models (122). (2) Proinflammatory molecules-many inflammatory cytokines and growth factors have been reported to facilitate tumor development or antitumor immunity. In mice bearing breast cancer, IL-1 was upregulated and its related signaling was enhanced, which could promote angiogenesis, endothelial cell adhesion, lymphocyte polarization, and recruitment of myeloid cells, thus contributing to cancer progression and bone metastasis (123)(124)(125). Suppressed action of IL-1 dramatically inhibited tumor growth in ovarian cancer mouse models (126). IL-17 can promote GI tumorigenesis by binding to its receptor, and this signaling could induce the activation of the mitogen-activated protein kinase (MAPK) and NF-kB pathways and boost colonic epithelial cell proliferation and further support malignant transformation in mice (121,127). TNF-a exhibited its protumorigenic features through activation of representative c-Jun Nterminal kinase (JNK) and NF-kB signaling pathways, resulting in enhanced epithelial to mesenchymal transition (EMT) and accelerated tumor cell invasion (128, 129). (3) ECM remodeling-ECM remodeling such as degradation or stiffening was tumorigenic (130). It could contribute to tumor invasion and metastasis, in which integrin clustering could encourage focal adhesions, intensify ERK and PI3K pathways, and thus promote cell proliferation and invasion (131,132).
Moreover, other mechanisms may also participate in the process of Malassezia-related inflammation-cancer transformation, such as DNA lesion accumulation and imbalance of oncogenes and antioncogenes. Inflammatory cells may release cytotoxic chemicals such as reactive oxygen species (RONS) to induce DNA damage (133). Continuous inflammatory conditions may result in aggravation and accumulation of DNA damage in cells, which may promote genetic mutations, generate genomic instability, and eventually cause carcinogenesis (134). Another powerful toxic polycyclic aromatic hydrocarbon, 7,12dimethylbenz[a] anthracene (DMBA), has been reported to induce inflammation-dependent dermal tumorigenesis in mice through the cGAS-STING signaling pathway (135) and even distant metastasis in mouse models of breast cancer (136). Accumulated mutations in oncogenes such as c-Myc, which may be induced by inflammatory cytokines or DNA damage, could show synergism with inflammatory stimulus to enhance oncogenous process including enhanced cell proliferation, differentiation, and malignant transformation (137,138). In addition, chronic inflammation may contribute to tumor protein 53 (TP53) mutations in the epithelium, and this accumulation could lead to deep loss of tumor suppressor functionality in cells. As a result, chromosomal instability increased and eventually cancer occurred (139,140). In turn, the tumor microenvironment may exacerbate tumorigenic inflammation, leading to a persistent vicious circle between inflammation and cancer.
High-abundance Malassezia has already been detected in multiple cancers, implying that Malassezia may be a key in initiating and accelerating cancer development. Furthermore, the presence of Malassezia was inextricably linked with its induced inflammation. Based on this, during the process of Malassezia in cancer promotion, inflammation may be the biggest contributor.

CONCLUSION
In general, patients with IBD exhibit a high incidence in a series of cancers. For the IBD population, increased level of Malassezia is connected to the imbalance of microflora, fungal translocation, and inflammatory deterioration. In addition, the abundance of Malassezia is positively correlated with the pathogenesis and progression of various cancers, suggesting that Malassezia may be a key component to relate IBD with cancer. Accordingly, broad-spectrum antifungal drugs, aimed to reduce the production of Malassezia strains or their metabolites, can inhibit tumorigenesis and slow down tumor progression, by restoring internal fungal homeostasis and reducing inflammatory responses. This may provide new thoughts for cancer monitoring and novel therapeutic approaches. However, more studies are still required to verify the clinical benefits of Malassezia genus inhibition in IBD or cancer group. Although Malassezia is considered to be a pathogenic fungus which may participate in the pathogenesis of IBD and promote this disease to further develop into cancer, more informative pathogenesis still needs to be addressed. Multidisciplinary cooperation has become an inevitable trend, so the next arduous task is to deeply explore and fully utilize Malassezia-relevant mycology, and then combine it with metabolomics and immunology. This future research will reveal the potential of Malassezia strains as therapeutic targets, aiming at relieving inflammatory reaction, improving patient outcome in IBD, and further reducing the incidence of cancer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
QY and JO wrote the first draft edition of the manuscript. DP provided critical revisions of this manuscript. LF and JY conceived and designed the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


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Two distinct stem cell‐like subtypes of hepatocellular carcinoma with clinical significance and their therapeutic potentials

Dear Editor Hepatocellular carcinoma (HCC) is among the most common cancers worldwide, causing about 600,000 deaths annually [1]. In HCC, stem cell-like characteristics, which drive early recurrence and therapy resistance, are major contributors to poor prognosis [2]. In this current study, we integrated and analyzed gene expression data from human fetal liver cells and primary HCC tumors (n = 1231) and uncovered two clinically and biologically distinct hepatic stem cell (HS) subtypes, potential biomarkers associated with these subtypes, and a potential new therapeutic intervention for these subtypes. By analyzing gene expression data from human fetal liver cells [3], we identified 609-, 2538-, and 1139-gene signatures for gestational 10-week fetal liver cells, 17-week fetal liver cells, andmature hepatocytes, respectively (Supplementary Fig. S1A). Because 10and 17-week fetal liver cells reflect different degrees of stemness of hepatic lineage, as reflected by α-fetoprotein expression (Supplementary Fig. S1B), we renamed the gene signatures specific to the 10and 17-week cells hepatic stem cell type 1 (HS1) and hepatic stem cell type 2 (HS2), respectively. To estimate the clinical relevance of stem cell-like characteristics in primary HCC, we trichotomized HCC tumors as HS1, HS2, or differentiated HCC (dHCC) subtypes according to their degree of stemness by applying the prediction algorithm to the gene expression data from HCC tumors (n = 1226) (Supplementary Fig. S1C). The HS1 subtype was associated with worst overall survival, the HS2 subtype exhibited moderate overall survival, and the dHCC subtype exhibited the best overall survival (P < 0.001, Figure 1A). When patients with Barcelona Clinic Liver Cancer stage A or B

disease were stratified by HS subtype, the subtypes successfully stratified high-risk patients at both stages (Supplementary Fig. S2). In addition to clinical stage and resection status, the HS subtypes were significant predictors of overall survival, except HS2 for recurrence-free survival, in multivariate Cox proportional hazards regression analyses (Supplementary Table S1 and S2). The HS subtypes were also significantly associated with some clinicopathological variables ( Supplementary Fig. S3).
Interestingly, while there was no significant difference in the subtypes' mutation rates, HS1 subtype showed significantly different copy number alteration score when compared with other subtypes ( Figure 1B). The HS1 subtype showed higher rates of TP53 and RB1 mutations, while the HS2 subtype showed frequent IL6ST and CDKN2A mutations ( Supplementary Fig. S4). Since the HS1 subtype showed the highest microRNA expression ( Supplementary  Fig. S5), it will be interesting to test in future studies if they play important roles in maintaining the stemness of cancer cells.
As a combination of targeted treatment and immune checkpoint blockade has been reported to have encouraging results in HCC [4], we assessed each subtype's potential response to immunotherapy using tumor immune dysfunction and exclusion (TIDE) scores which reflects their degrees for immunotherapy resistance [5]. Both HS subtypes showed high TIDE scores ( Figure 1C), suggesting that patients with either HS subtype would not benefit substantially from immunotherapy. In agreement with this, the HS subtype probability and TIDE scores were positively correlated ( Supplementary Fig. S6A). In an analysis using the CIBERSORT algorithm [6], which estimates the relative fractions of immune cells ( Supplementary  Fig. S6B), the non-activated naïve M0 macrophage fraction was significantly higher in both HS subtypes (Supplementary Fig. S6C, D), suggesting that a lack of active macrophages could be contributing to the HS subtypes' low immunotherapy response. Furthermore, the fraction  Fig. S7A). Likewise, the expression of major immune checkpoints (PD-1, PD-L1, and CTLA4) was significantly different in the HS subtypes ( Supplementary Fig. S7B, C), further supporting the notion that the HS subtypes are immune-suppressive.
Interestingly, the HS1 subtype was associated with potential response to sorafenib, while the HS2 subtype was not ( Supplementary Fig. S8) (see extended discussion in Supplementary File). We next applied the HS signatures to the gene expression data of 75 patient-derived xenograft (PDX) HCC tumors. The HS signatures of the PDX tumors are highly similar to primary HCC ( Supplementary Fig. S9A), indicating that stem cell features are well-maintained in PDX tumors. Most PDX tumors appeared to be stable as the passage number of the PDX models differed only slightly among subtypes (Supplementary Fig. S9B). Using targeted ELISA for 122 serum proteins (Oncology MAP screening), we performed a proteomic analysis with serum samples from HCC patients (n = 45, a subset of our cohort) and identified the top five serum markers for the HS1 and HS2 subtypes ( Supplementary Fig. S10).
Gene network analysis revealed that oncogenes were activated in the HS1 and HS2 subtypes (Supplementary Table S3 and S4). In particular, YAP1 was highly activated in the HS1 subtype. Since YAP1 regulates HS and is associated with poor prognosis in HCC [7], we next examined the potential interaction of other transcription regulators with YAP1 by integrating the downstream target genes of each of the transcription regulators. BRD4 exhibited the highest interaction with YAP1 as it shared more target genes with YAP1 than any of the other regulators (Supplementary Fig. S11A). Of BRD4's 37 target genes, 15 (41%) were shared with YAP1 ( Supplementary Fig. S11B), suggesting that BRD4 interacts with or regulates YAP1 in HCC. We also evaluated the expression of HCC-associated stem cell markers and found that many of these stem cell markers, including SALL4, were highly expressed in the HS1 subtype ( Supplementary Fig. S12).
BRD4 is a member of the bromodomain and extraterminal domain (BET) family and a key transcriptional regulator for many oncogenes, including MYC [8]. Next, we tested if BET members were involved in the regulation of YAP1 expression. HCC cells treated with the BET family inhibitor JQ1 had reduced expression of YAP1 but not MYC, a de facto target of BET proteins ( Figure 1D, Supplementary Fig. S13A) [8], suggesting that the oncogenic activity of the BET family is mediated by the regulation of YAP1 in HCC cells. Furthermore, JQ1 significantly reduced the viability and migration of HCC cells ( Figure 1E, Supplementary Fig. S13B, C), suggesting that JQ1 can inhibit the growth and invasion of HCC cells by suppressing YAP1.
Since JQ1 could inhibit most BET family members, we silenced the expression of BET members to identify the key members regulating YAP1. Surprisingly, complete YAP1 suppression required the depletion of all BET members ( Figure 1F), suggesting that all BET members are active and functional in HCC cells. Most HCC cells consistently expressed all BET members (Supplementary Fig. S13D). In agreement with this, BET family member gene expression was highly correlated with YAP1 expression in human HCC tumors (Supplementary Fig. S13E), which supports the notion that BET genes regulate YAP1 in HCC.
Previous studies identified HCC subtypes with stem cell features [9,10] but provided only general descriptions of those features while also listing potential therapeutic targets without providing functional validation or guidance for treatment. In the current study, we found that HCCs with stem cell features were not clinically or genomically homogeneous. They differed not only in their degree of stemness but also in their clinical outcomes and underlying biology. Importantly, the HS1 subtype, which has a poor prognosis, appears to be sensitive to BET inhibitors. The newly identified serum markers associated with these subtypes may provide opportunities to develop marker-based clinical trials. Furthermore, the potential marker genes we identified are well-preserved in PDX models, which shows promise for the development of accurate disease models for preclinical study.

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE
Serum samples were obtained from HCC patients who provided informed consent under an IRB approved protocol at Mayo Clinic, Rochester, Minnesota, USA.

C O M P E T I N G I N T E R E S T S
The authors report no conflicts of interest.