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Inhibition of tamoxifen's therapeutic effects by emodin in estrogen receptor-positive breast cancer cell lines

Purpose This study was aimed to investigate the combination effect of endoxifen and emodin on estrogen receptor (ER) positive breast cancer cell lines and to explain the mechanism of the combination effect. Methods We conducted this study on MCF-7 (ER+/human epidermal growth factor receptor-2 [HER2]−), T47D (ER+/HER2−), ZR-75-1 (ER+/HER2+), and BT474 (ER+/HER2+) cell lines, which confirmed combination effect of endoxifen and emodin. Optimal concentrations for combination were determined to study the effects on proliferation of MCF-7 and ZR-75-1 cells. Analysis of the combination effect was carried out in the CompuSyn software. The combination of downstream mechanisms, and combined effects of other similar compounds were tested on the MCF-7 and ZR 75-1 cell lines. Protein expression was confirmed by western blot. Results The combination of endoxifen and emodin had antagonistic effects on MCF-7 and ZR-75-1cell lines (combination index > 1). We validated the antagonistic effect in T47D and BT474 cell lines. During the combined treatment, the results showed elevated amounts of cyclin D1 and phosphorylated extracellular signal-regulated kinase (pERK). Analysis of drug interactions showed antagonistic effect between endoxifen and chemical compounds similar to emodin, such as chrysophanol or rhein, in MCF-7 and ZR-75-1 cells. Conclusion Addition of emodin attenuated tamoxifen's treatment effect via cyclin D1 and pERK up-regulation in ER-positive breast cancer cell lines.

INTRODUCTION
The proportion of patients with estrogen receptor (ER)-positive breast cancer increased from 49.6% in 1996 to 70.0% in 2010 [1]. In patients with hormone receptor-positive (ER-positive and/or progesterone receptor; [PR] positive) breast cancer, antiestrogen therapy is the main treatment. The mechanism of action of tamoxifen is mediated by competitive binding to ER. Tamoxifen inhibits estrogen-related gene expression and blocks G1 phase of the cell cycle [2]. There was a randomized trial of tamoxifen for the treatment of early breast cancer. The proportion of recurrence for women with ER-positive breast cancer who used tamoxifen for 5 years was reduced by 50% (P < 0.01), and the mortality rate was reduced by 28%. On the other hand, in women with ER-negative breast cancer, the benefits of tamoxifen are unclear [3]. The antiestrogen therapy such as tamoxifen or an aromatase inhibitor lasting for 5 years reduces breast cancer recurrence and increases overall survival. Thus, tamoxifen has become the global standard of care for premenopausal patients with breast cancer. The recommendation of treatment takes into consideration that after 5 years, patients should receive additional antiestrogen therapy for 10 years [4]. However, adjuvant hormone therapy was discontinued for various reasons. Aiello Bowles et al. [5] reported discontinuation of hormone therapy and related adverse effects among patients with early breast cancer. The researchers reported that 18.2% of the patients had discontinued hormone therapy. Patients who discontinued treatment experienced adverse effects in many cases, with 41.8% reporting hormone/menopause-related symptoms.
The aims of the present study were to investigate the combination effect of tamoxifen and emodin on ER-positive breast cancer cell lines and to explain the mechanism of the combination effect. In this study, we looked at patients with tamoxifen for passible material use to improve their hormone/ menopause-related symptoms.

Measurement of cell viability
Cell viability was measured using the EZ-Cytox cell viability assay kit (Itsbio, Seoul, Korea). The cells were incubated for 1 hour at 37℃ in a serum-free medium diluted with 1 kit reagent. Next, harvested cells resuspended in the media were carefully moved to empty 96-well plate and absorbance was measured using a microplate reader at 450 nm.

Isobologram analysis
Cell viability data were analyzed using in the CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). This software determines the combined effects of drugs: whether they are synergistic, antagonistic, or additive. The software was developed by Dr. Dorothy Chou in 2005. This software is based on the median-effect principle of the mass-action law [15]. In the isobologram graph, an antagonistic effect is above the oblique line (combination index [CI] > 1), a synergistic effect is below the oblique line (CI < 1), and an additive effect is on the line (CI = 1) [15,16].

Western blot analysis
Cell lines were harvested and dissolved in radio immunoprecipitation assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS], supplemented with a protease inhibitor cocktail) (Gendepot, Katy, TX, USA). Equal amounts of protein (20-50 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred on to a nitrocellulose membrane. Membranes were blocked by incubating them with 2.5% skim milk for 1 hour and then incubated overnight with the appropriate primary antibodies (diluted 1:1,000), followed by a 1.5-hour reaction with the secondary antibody (diluted 1:10,000). Immunoreactive proteins were visualized by means of the enhanced-chemiluminescence reagent (Amersham Bio-

Statistical analysis
All the results were compiled from a minimum of 3 independent experiments. Student t-test was performed to compare results of untreated (control) and treated group. Data were analyzed in the IBM SPSS ver. 18.0 (IBM Co., Armonk, NY, USA). Statistical significance was set to P < 0.05.
This study was exempted from review by the Institutional Review Board of the Konkuk University (study number: 7001355-201507-E-036).
The effects of the drug combinations were calculated in the CompuSyn software (Fig. 3A, B). In the MCF-7 breast cancer cell line, the CI value was 1.06586 between endoxifen (4 µM) and emodin (60 µM). In ZR-75-1 cells, the CI value was 1.20447 under the same conditions. Both CI values were >1, indicating an antagonistic effect between emodin and endoxifen in MCF-7 and ZR-75-1 breast cancer cells.
Subsequently, we validated the antagonistic effect in other breast cancer cell lines. The ER-positive/HER2 negative breast cancer cell line, T47D, showed similar antagonistic effects (

Analysis of the downstream mechanism of the antagonistic effect
To confirm the mechanism of the antagonistic effect, we performed western blotting assays. The MCF-7 and ZR-75-1 breast cancer cell lines were treated with emodin and endoxifen for 48 hours, and the harvested proteins were analyzed by western blotting. Treatment with the endoxifen and emodin combination; increased cyclin D1 and p-ERK amounts (Fig. 5).

Combination effects for similar chemical compounds on breast cancer cell lines
Subsequently, we studied the drug interactions between chemical compounds that are similar to emodin and endoxifen. Emodin, chrysophanol, and rhein are categorized as  The CI value was calculated and found to be 3.09017. Chrysophanol and rhein had antagonistic effects on ZR-75-1 breast cancer cell line. The cell viability of ZR75-1 cells was 86.7% when treated with endoxifen (4 µM) and chrysophanol (60 µM) for 48 hours (as compared with 60 µM chrysophanol alone: 97.7%, or 4 µM endoxifen alone: 87.5%). The CI value was found to be 1.63508. The combination of endoxifen (4 µM) and rhein (60 µM) yielded a cell viability of 97.2% of MCF-7 cells after 48 hours (as compared with 60 µM rhein alone: 96.5%, or 4 µM endoxifen alone: 88.6%). The CI value was calculated and found to be 3.72066.

DISCUSSION
The results of this study reveal antagonistic effects of emodin and endoxifen when used in combination against breast cancer cell lines. These antagonistic effects are associated with up-regulation of cyclin D1 and p-ERK. These results suggest that emodin, one of phytoestrogens, may interfere with the therapeutic effect of tamoxifen against ER-positive breast cancer cell lines.
In this study, we tested the drug combination on MCF-7 and ZR-75-1 breast cancer cell lines and validated the results in T47D and BT-474 breast cancer cell lines. The MCF-7 breast cancer cell line originated from the pleural effusion of a 69-year-old patient with metastatic breast cancer [17]. MCF-7 cells are widely used as an ER-positive cell line [13]. The T47D breast cancer cell line originated from the pleural effusion of a 54-year-old patient with metastatic breast cancer [18]. The T47D breast cancer cell line expresses high levels of PR. Thus, T47D cells are considered a major model for the research one progesterone in human cells [19]. The MCF-7 and T47D breast cancer cell lines are classified as luminal A (ER+, PR+/-, HER2-) [13]. The ZR-75-1 breast cancer cell line originated in the ascites of a 47-year-old patient with metastatic breast cancer [18] and is regarded as a luminal B (ER+/HER2+) subtype [13]. The BT-474 breast cancer cell line originated from a primary invasive ductal carcinoma of a 60-year-old patient with breast cancer [20] and is also a luminal B subtype (ER+/HER2+) [13]. In our study, MCF-7, T47D, ZR-75-1, and BT474 breast cancer cell lines all showed the antagonistic effects of our drug combination. According to these results, the combination of endoxifen and emodin has an antagonistic effect regardless of HER2 status. Cyclin D1 forms a complex with cyclin-dependent kinase 4 (CDK4). The cyclin D1-CDK4 complex promotes S-phase entry by triggering E2F-dependent gene transcription [21]. In the western blotting analysis an increased cyclin D1 amount was observed in the cells treated with the drug combination. This result suggests that the drug combination promotes entry into the S-phase of the cell cycle. Cyclin D1 overexpression has been noted in breast carcinoma [21]. In the ZR-75-1 breast cancer cell line, EGFR expression increased in response to the drug combination. EGFR is composed of an extracellular region, a transmembrane segment, a juxtamembrane segment, a kinase domain, and sites of tyrosine phosphorylation. The extracellular region of EGFR undergoes dimerization, resulting in transmission of downstream signals [22]. The Ras-Raf-mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, phospholipase Cγ pathway, the signal transducer and activator of transcription pathway, and the Src kinase pathway are all downstream effectors of EGFR [12]. EGFR plays an important role in many cancers, where it stimulates tumor growth, metastasis, and angiogenesis via these signaling cascades [23]. The Ras-Raf-MAPK pathway controls cell proliferation and survival. After phosphorylation of EGFR, EGFR forms a complex with proteins Grb2 and Sos, or interacts with them through the adaptor molecule Shc. This interaction recruits Ras-GDP and activates Ras-GTP. Ras-GTP activates Raf-1, and then stimulates Erk-1 and Erk-2. These 2 acts as transcription factors in the nucleus and ultimately promote cell proliferation and survival. The PI3K-Akt-mTOR pathway is associated with cell growth, apoptosis resistance, cell invasion, and cell migration [12]. PI3K phosphorylates phosphatidylinositol diphosphate to phosphatidylinositol triphosphate (PIP3). PIP3 activates the oncogene Akt. Akt inhibits tuberous sclerosis complex-2. As a result, Akt activates mTOR, which has effects on cell survival, cell proliferation, and metabolism [24]. In our study, increased p-ERK levels could explain the antagonistic effects of endoxifen and emodin on cell proliferation.
We performed drug interaction analyses between endoxifen and anthraquinone compounds similar to emodin (chrysophanol and rhein). Anthraquinones are benzoquinone derivatives that are synthesized by diverse pathways in plants and fungi. Anthraquinones have antimutagenic and anticarcinogenic activities. In addition, emodin, chrysophanol, and rhein have inhibitory effects on benzo[α]pyrenemediated DNA damage in the HepG2 hepatoma cell line [25]. Nevertheless, in this study, emodin, chrysophanol, and rhein showed effects that are antagonistic to endoxifen's action on MCF-7 and ZR-75-1 breast cancer cell lines. Although we did not perform a mechanistic analysis of this finding, we hypothesized that the mechanism is similar to that of emodin.
Some investigators reported inhibitory effects of a combination of tamoxifen and genistein [26,27]. Genistein is a phytoestrogen from soy that has been demonstrated to potentially inhibit the growth of breast cancer cell lines in vitro and to provide a relief of postmenopausal symptoms. Jones et al. showed that genistein inhibits tamoxifen's effects on the T47D breast cancer cell line. Tamoxifen (1.0 µM) decreased cell proliferation (92.8%) compared with the control (100%). By contrast, the combined treatment with genistein and tamoxifen improved proliferation (96.7%) of the T47D breast cancer cell line. This result was interpreted as a reversal of cell cycle arrest: Tamoxifen induces G1 arrest; however, the combined treatment with genistein and tamoxifen reverses the G1 arrest, thereby normalizing the cell cycle [26]. Ju et al. [27] determined whether genistein negates the inhibitory effect of tamoxifen on the growth of MCF-7 breast cancer cells in nude mice. They studied the effect of the drug under several conditions and showed antagonistic effects. The combination treatment with genistein and tamoxifen resulted in a significantly larger tumor surface area compared with tamoxifen alone (2.5 TEG, 75.1 mm 2 ; 5TEG, 50.9 mm 2 ; 2.5TE, 14.4 mm 2 ; 5TE, 13.7 mm 2 ). This study revealed that the combination treatment recovered pS2 expression via cyclin D1 overexpression as compared to the treatment with tamoxifen alone. Thus, these 2 studies verified the antagonistic effect between tamoxifen and other phytoestrogens, in agreement with our results.
In conclusion, our data indicate that the combination treatment with endoxifen and emodin has an antagonistic effect via cyclin D1 and pERK overexpression in ER-positive breast cancer cell lines. These results suggest that emodin may attenuate tamoxifen's therapeutic effects. Therefore, patients with ERpositive breast cancer should be careful when consuming phytoestrogens.

CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.


s2fieldsofstudy: Biology Medicine

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Human-Biting Ixodes Ticks and Pathogen Prevalence from California, Oregon, and Washington

Abstract From July 2006 through August 2017, a passive surveillance study of Ixodes ticks submitted from California, Oregon, and Washington was conducted by the TickReport program at the University of Massachusetts, Amherst. In total, 549 human-biting Ixodes ticks were submitted comprising both endemic and nonendemic species. We found that 430 endemic ticks were from 3 Ixodes species: Ixodes pacificus, Ixodes spinipalpis, and Ixodes angustus, whereas Ixodes scapularis (n = 111) was the most common species among the 119 nonendemic ticks. The submission peak for nymphal I. pacificus and I. spinipalpis was June, while submission peak for adult I. pacificus and nymphal I. angustus was April and September, respectively. Endemic ticks commonly attached to the lower extremities of their victims, and individuals younger than 9 years old were frequently bitten. The infection prevalence of Borrelia burgdorferi sensu lato, Borrelia miyamotoi, and Anaplasma phagocytophilum in I. pacificus ticks was 1.31%, 1.05%, and 0.52%, respectively, and the prevalence of B. burgdorferi s. l. and A. phagocytophilum in I. spinipalpis ticks was 14.29% and 10.71%, respectively. Furthermore, two species within the B. burgdorferi s. l. complex were detected in West Coast ticks: B. burgdorferi sensu stricto and Borrelia lanei. I. spinipalpis had the highest Borrelia prevalence among endemic ticks, and it was caused exclusively by B. lanei. Borrelia mayonii, Babesia microti, and Ehrlichia muris-like agent were not detected in these endemic ticks. In this study, we show that many nonendemic Ixodes ticks (119/549) are most likely acquired from travel to a different geographic region. We report cases of conventionally recognized nonhuman feeders (I. spinipalpis and I. angustus) parasitizing humans. The highest pathogen prevalence in I. spinipalpis may indicate a larger public health threat than previously thought, and the enzootic life cycle and pathogenicity of B. lanei warrant further study.

Introduction
T he single prostriate genus Ixodes comprises medically important ticks throughout the world Clifford 1978, Piesman andEisen 2008). Several members within the Ixodes ricinus complex are the primary tick vectors that are involved in transmission of human pathogens (Xu et al. 2003). For example, in the eastern and north-central United States, Ixodes scapularis is the primary vector of several pathogens, including Borrelia burgdorferi (Lyme disease), Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti (Piesman and Eisen 2008). In the western United States, Ixodes pacificus is the primary vector of these pathogens (Burgdorfer et al. 1985, Lane et al. 1994, Eisen et al. 2016.
The prevalence and ecology of Lyme disease in the western United States differs significantly from that in the northeastern United States. The overall incidence of Lyme disease in the western United States is 0.2 cases compared to 30-80 cases (per 100,000 persons per year) in the northeastern United States (Schwartz et al. 2017). There is also greater vector and spirochete diversity in the western United States than in the northeastern United States (Brown and Lane 1992). In the Northeast, Lyme spirochetes are primarily maintained in white-footed mice (Peromyscus leucopus) and other small mammals by I. scapularis (Spielman et al. 1985). However, in the western United States, Peromyscus mice have low tick load and low prevalence of B. burgdorferi sensu lato (s. l.) (Brown and Lane 1992), whereas, the western gray squirrel (Sciurus griseus) and dusky-footed woodrat (Neotoma fuscipes) appear to be the predominant reservoir hosts of B. burgdorferi (Salkeld et al. 2008, 2014a, Salkeld and Lane 2010. I. pacificus mainly serves as a ''bridge'' vector of spirochetes to humans (Burgdorfer et al. 1985, Lane and Lavoie 1988, Clover and Lane 1995. In California, Borrelia species are also maintained among dusky-footed woodrats, California kangaroo rats (Dipodomys californicus), and other small mammals by Ixodes spinipalpis, Ixodes angustus, and Ixodes jellisoni, although they rarely bite humans (Burgdorfer et al. 1985, Brown and Lane 1992, Lane et al. 1994. In addition to differences in hosts and tick vectors, the species and population of Lyme spirochetes are more diverse in California (Girard et al. 2009). Up to now, only four Borrelia species within B. burgdorferi s. l. have been found in the northeastern and upper midwestern United States: Borrelia andersonii, B. burgdorferi sensu stricto (s. s.), Borrelia kurtenbachii, and Borrelia mayonii, with B. burgdorferi s. s. representing the dominant species (Margos et al. 2010(Margos et al. , 2014. Conversely, in addition to B. burgdorferi s. s., several species within the B. burgdorferi s. l. complex, including Borrelia americana (Rudenko et al. 2009), Borrelia bissettiae (Postic et al. 1998), Borrelia californensis (Postic et al. 2007), and Borrelia lanei (Margos et al. 2017) have been found in the western United States. B. burgdorferi s. s. is the only species associated with Lyme borreliosis: it contains two genetically distinct, but phylogenetically related populations in the Northeast and Midwest (Qiu et al. 2002, Hoen et al. 2009). However, the population structure of California strains of B. burgdorferi s. s. is more heterogeneous than the Northeast strains (Girard et al. 2009).
Several tick surveillance studies have been conducted to connect tick data with the risk of tick-borne disease in the western United States, including research by traditional tick flagging (Lane et al. 2004, Salkeld et al. 2014a, 2014b and host trapping (Castro and Wright 2007). However, these studies generally do not link information about ticks and tick-borne diseases directly to human-tick encounters. A more detailed analysis relating the pathogen prevalence in different species of ticks parasitizing humans in the western United States is needed to better assess risk. To address this gap in knowledge, we report results of passive surveillance involving ticks that were submitted from the western United States and tested for pathogens by TickReport at the University of Massachusetts Amherst.

Ticks and morphological identification
TickReport is a public outreach service at the University of Massachusetts Amherst providing individuals with information about potential pathogen exposures associated with their tick bites and provides information about risk vis-a-vis the biting tick's species, its infection status with respect to several pathogen species, and an assessment of the tick's feeding status. Ticks analyzed in the present study were submitted to TickReport from July 2006 through August 2017. Orders were placed via an online form where submitters are asked to provide the location and date of tick collection; age, gender, and species of the host; and attachment site of the tick on the host's body (Xu et al. 2016).
The Ixodes genus-level identification of each tick was determined by morphological characterization of the anal groove, which is distinctively located anterior to the anus in all Ixodes ticks (Keirans and Clifford 1978).

DNA extraction and molecular identification
DNA was extracted from each tick using Epicenter Master Complete DNA and RNA Purification Kits (Epicenter Technologies, Madison, WI) following the manufacturer's protocols. Differentiation of I. scapularis and I. pacificus was performed by a species-specific TaqMan PCR assay (Table 1). Other Ixodes species were identified by amplifying and sequencing a fragment of the tick 16S rRNA gene (Table 1) (Krause et al. 2016, Xu et al. 2016. B. burgdorferi s. l., B. miyamotoi, B. mayonii, B. microti, A. phagocytophilum, and Ehrlichia muris-like agent (EMLA) were detected by a multiplex TaqMan PCR assay targeting different genes (Table 1). Borrelia detection was performed by first applying a genus-specific detection assay for a conserved target, followed by specific qPCR assays for each of the three species (B. burgdorferi s. l., B. miyamotoi, B. mayonii). All B. burgdorferi s. l. isolates were further differentiated by multilocus sequence analysis (MLSA) using eight housekeeping loci (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) (Margos et al. 2017).

Results
Tick species, geographical distributions, and seasonal activity From July 2006 to August 2017, we received and identified 601 Ixodes ticks (58 from 2006 to 2013 and 543 from 2014 to 2017) from California, Oregon, and Washington, of which 549 (91.3%) had bitten humans, 43 were from dogs, 3 were from cats, and 6 were from lawns or unknown sources. The majority of submissions were received from 2014 to 2017. Analyses of ticks and associated pathogens were restricted to the 549 Ixodes ticks found on human subjects submitted from these three states (Table 2).
Surprisingly, we also found that 21.67% (119/549) of the ticks from California, Oregon, and Washington residents are not endemic to the western United States. These four tick species are as follows: I. scapularis (N = 111: 8 larvae, 47 nymphs, 56 adults), I. ricinus (N = 6: 5 nymphs, 1 adult), Ixodes cookei (N = 1 nymph), and Ixodes holocyclus (N = 1 adult). Among the 119 nonendemic ticks submitted, 107 (89.9%) bit people who reported in the TickReport order questionnaire, a travel history to the eastern or north-central United States or foreign countries. No travel history was volunteered for the remaining 12 individuals, but in all likelihood, these nonendemic ticks were also acquired during travel. Based on the travel history, it is clear that the 5    I. ricinus ticks and the single I. holocyclus tick were brought to the United States from Europe and Australia, respectively.
Over 94.2% of total ticks were submitted from Oregon, Northern California, and Washington. Only a small proportion, 5.8% (N = 25), of ticks were submitted from Southern California. For the three endemic tick species: 85.7% of I. angustus ticks were submitted from Washington; 79.3% of I. pacificus ticks were submitted from California; and 46.4% and 42.9% of I. spinipalpis ticks were submitted from California and Washington, respectively (Table 2).
We received I. pacificus in every month of the year except September and October. The nymphal and adult I. pacificus exhibited different seasonal activity patterns (Fig. 1). We received 96.5% of nymphs between March and August, with a clear peak in June. Adult I. pacificus were abundant from November to July and then absent from September and October, with a peak in April (Fig. 1). We only found nymphal human-biting I. spinipalpis in this study. They were submitted between February and July, with a peak in June. Nymphal I. angustus were submitted from May to October, with a peak in September (Fig. 1).

Age distribution and tick attachment sites of tick bite victims
We received age data for 413 of the tick bite victims (Fig. 2). The age pattern was quite different for humans bit by I. pacificus nymphs compared to adults: 22% of attached adults and 57% of attached nymphs were found in children younger than 9 years. Adult ticks (70%) were commonly found among the age group of 25-64 years, however, only 30% of nymphs were found among this age. The individuals aged 0-9 years also had the largest proportion of I. spinipalpis and I. angustus submitted: 56% of I. spinipalpis nymphs and 47% of I. angustus nymphs (Fig. 2). The number of I. angustus adults was too small to compare age groups.
B. burgdorferi s. l. prevalence in I. pacificus nymphs was higher than in adults (3.5% vs. 0.9%). On the contrary, B. burgdorferi s. l. prevalence in I. scapularis nymphs was lower than that in adults (25.5% vs. 32.1%). B. burgdorferi s. l. prevalence was 14.29% (4/28) in I. spinipalpis nymphs, but no I. spinipalpis adults were submitted in this study.
Based upon alignment from MLSA (GenBank accession nos. MH378169-MH378227), we concluded that two members of the B. burgdorferi s.l. complex were found in the present study: B. burgdorferi s. s. and the recently named B. lanei (genomospecies 2) (Schwan et al. 1993, Postic et al. 1994, Margos et al. 2017 (Fig. 3). Of the five Borreliainfected I. pacificus ticks, four were infected with B. burgdorferi s. s., and one was infected with B. lanei. B. lanei was the only Borrelia species found in I. spinipalpis ticks.

Discussion
The levels of tick infestation, reservoir competence, and Borrelia infection prevalence vary widely among hosts in the western United States (Brown and Lane 1992, Eisen et al. 2004, Salkeld et al. 2008, Salkeld and Lane 2010. Passive tick surveillance studies have shown that the nymphal stage of I. pacificus is the primary vector of B. burgdorferi to humans in California (Clover and Lane 1995). Certain human behaviors, such as sitting on logs, can pose considerable risk of multiple exposures to nymphal ticks in this region (Lane et al. 2004). So too, travel history to the northeastern or upper midwestern United States impacts Lyme disease reporting from California patients diagnosed by an erythema migrans rash. Onset of the rash in patients exposed in California peaked in May-July, while the onset in patients (N = 219) with a travel history peaked in June-August (N = 177); reflecting differences in tick seasonal activity in each region (Salkeld et al. 2014a). Some tick species, such as I. spinipalpis and I. angustus are considered ''rare'' human biters (Damrow et al. 1989, Brown and Lane 1992, Merten and Durden 2000, Peavey et al. 2000, however, their encounter frequency and public health impact are yet to be determined. In this study, we found that 21.67% of Ixodes ticks from California, Oregon, and Washington residents are not endemic to the western United States. These data indicate that tick species identification and travel history are critical factors for human-based tick surveillance in the western United States. Although I. pacificus and I. scapularis are closely related species, the acarologic risk of exposure to pathogens in these ticks is very different (Eisen et al. 2004, Hahn et al. 2018. Accuracy of species identification is challenging for several reasons. First, I. pacificus is very similar to I. scapularis morphologically. Second, most human-biting ticks are in poor condition, often with missing mouth parts, further complicating proper identification. Finally, the geographic distributions of these two species do not overlap (Eisen et al. 2016). Therefore, it is often assumed that a human-biting tick in a certain area is representative of the local tick species. In this study, we used real-time PCR and DNA sequencing to overcome these complications and accurately identify each sample to the species level. With proper species identification, we were able to conclude that nonendemic Ixodes ticks may influence local tick-borne disease due to human travel between nonoverlapping tick ranges.
Our data indicate that West Coast residents traveling to the northeast United States have a much higher risk of being bitten by an infected tick compared to nontraveling residents who are exposed to local tick populations. We found that 20.22% (111/549) of the total human-tick encounters were cases of I. scapularis ticks biting travelers. Second, B. burgdorferi s. l., B. miyamotoi, and A. phagocytophilum prevalence in I. scapularis is 20.6, 2.6, and 13.7 times higher, respectively, compared to I. pacificus (Table 2). We do not know how many tick-borne diseases were acquired from traveling for the West Coast residents. However, the 10-year data for humans with erythema migrans in California (2001California ( -2011 showed two different frequency peaks: one is likely local exposure in May-July and another peak is likely travel-associated exposure in June-August (Salkeld et al. 2014). Assessing risk for tickborne diseases in the western United States without accurate species identification and travel information would likely overestimate the number of local cases.
I. angustus and I. spinipalpis are normally considered nonhuman feeders but were experimentally demonstrated to be competent vectors of B. burgdorferi (Dolan et al. 1997, FIG. 3. Dendrogram showing only Borrelia burgdorferi s.s. and Borrelia lanei were detected within B. burgdorferi sensu lato in the present study. Reference Borrelia strains from GenBank are indicated by species (and strain where applicable). Isolates from the present study are represented by the tick species, in which they were detected (starting with TR#, red color). The dendrogram was constructed by minimum evolution method of MEGA 6 software with Borrelia relapsing fever group as outgroup. Numbers on the branches represent bootstrap support with 1000 bootstrap replicates. Merten andDurden 2000, Peavey et al. 2000). In this study, 11.40% (49/430) of the endemic ticks recovered from humans in the western United States were I. angustus and I. spinipalpis. We found 4.88% (21/430) of human-biting ticks were I. angustus. Both nymphal and adult I. angustus occasionally quest openly in moist and cool habitats, especially in coastal areas (Eisen et al. 2006). In this study, most I. angustus (14/21) were nymphs from Washington.
I. spinipalpis is more important than the human-biting ''bridge'' vector, I. pacificus, in maintaining the enzootic spirochete cycle in the western United States (Brown and Lane 1992, Oliver et al. 2003, Eisen et al. 2009). However, we found the human risk for exposure to I. spinipalpis and the pathogens it transmits may be underestimated. First, 6.52% (28/430) of the endemic ticks recovered from humans in the western United States were I. spinipalpis in this study. This species may use a primarily nidicolous host-seeking strategy in hot and dry climates, but commonly quest for hosts openly in moister habitats (Eisen et al. 2006). In some redwoodassociated woodland sites in California, a larger number of openly host-seeking I. spinipalpis nymphs were collected by the dragging flag method, compared to I. pacificus (Eisen et al. 2006). All human-biting I. spinipalpis were nymphal ticks in this study. Second, the pathogen infection rate in I. spinipalpis was higher than that in I. pacificus: 14.29% (4/28) versus 1.31% (5/381) and 10.71% (3/28) versus 0.52% (2/381) for B. burgdorferi s. l. and A. phagocytophilum, respectively.
Although only B. burgdorferi s. s. and B. mayonii in B. burgdorferi s. l. complex have been culture confirmed as human pathogens in the United States (Steere et al. 1983, Pritt et al. 2016, several other species, including B. americana, B. bissettiae, B. burgdorferi s. s., Borrelia californiensis, and B. lanei, have been found in I. pacificus or I. spinipalpis ticks (Postic et al. 2007, Rudenko et al. 2009, Fedorova et al. 2014, Margos et al. 2017. Based upon results of MLSA in the present study, human-biting I. pacificus and I. spinipalpis are associated with only two species within the B. burgdorferi s. l. complex: B. burgdorferi s. s. and the recently named B. lanei (genomospecies 2) (Schwan et al. 1993, Postic et al. 1994, Margos et al. 2017 (Fig. 3). Although the Borrelia prevalence was 14.29% (4/28) in I. spinipalpis ticks, B. lanei was the only Borrelia species found in this tick species. B. lanei was recently found in I. spinipalpis ticks collected from rabbits in California and Canada, suggesting that rabbits may play an important role in the transmission cycle of B. lanei (Margos et al. 2017, Scott et al. 2017. It is well known that B. burgdorferi s. s. is pathogenic to humans in North America; however, human cases of B. lanei infection have not been found in the western United States (Margos et al. 2017). The vector role of I. pacificus and I. spinipalpis in the enzootic cycle of B. lanei and the pathogenicity of B. lanei to humans warrants further study.
The role of adult I. pacificus in disease transmission is not clear, however, we found adult ticks carry B. burgdorferi, B. miyamotoi, and A. phagocytophilum in California and Oregon. The nymphal stage of I. pacificus is the primary vector of Lyme disease in California (Clover and Lane 1995), but nymphs are difficult to detect and remove before repletion due to the tick's small body size (Xu et al. 2016). We found that a high rate (57%) of nymph bite-related encounters was in children aged 9 years and younger. Of these nymphal at-tacks, 96.5% of them were found between March and August, with a clear peak in June. We also received 56% of I. spinipalpis nymphs and 47% of I. angustus nymphs in victims aged 9 years and younger. In addition, we found that 48% of I. pacificus adults were attached to the lower extremities and back, whereas 30%, 31%, and 38% of I. pacificus, I. spinipalpis, and I. angustus nymphs were attached to the lower extremities. To help prevent tick-borne disease, it is critical to educate parents of young children to check for nymphal ticks during March-August in the West Coast, paying close attention to the lower extremities of young children.

Conclusions
We show that many nonendemic Ixodes ticks (119/549) submitted to the TickReport public testing program from the West Coast are most likely acquired from travel to a different geographic region. Traveling to an area where the tick population has a relatively higher pathogen infection rate increases the risk of exposure to infected ticks: as in the case of West Coast residents traveling to the northeast United States. For tick species that are endemic to the West Coast, we report cases of conventionally recognized nonhuman feeders (I. spinipalpis and I. angustus) parasitizing humans. Importantly, I. spinipalpis had the highest pathogen prevalence of the endemic species, indicating that it may pose a larger public health threat than previously thought. Furthermore, two species within the B. burgdorferi s. l. complex were detected in West Coast ticks; B. burgdorferi s. s. and B. lanei. B. lanei was detected in I. spinipalpis and I. pacificus ticks, corroborating previous reports. The vector role of I. pacificus and I. spinipalpis in the enzootic cycle of B. lanei and the pathogenicity of B. lanei to humans warrants further study. Seasonal activity, age distribution, and site of attachment data have yielded valuable public health information: for instance, young children are at an increased risk of tick bites, likely around their lower extremities, especially during the month of June when seasonal activity peaks for both nymphal I. pacificus and I. spinipalpis ticks. Public health professionals and physicians are encouraged to use this information to limit tick bites and tick-borne pathogens.
These results highlight the importance of passive surveillance of human-biting ticks and pathogens. Epidemiological case reports and/or entomological infection rate of questing ticks provide only proxies of human risk of tick-borne disease. Testing human-biting ticks allows assessment of the biological correlates of risk (tick species, degree of engorgement, and infection status) and the most reliable geographic distribution of that risk.


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Intergrated analysis of ELMO1, serves as a link between tumour mutation burden and epithelial-mesenchymal transition in hepatocellular carcinoma

Background Epithelial-mesenchymal transition (EMT) is critical for cancer cell metastasis. Recently, EMT was reported to be associated with the inflammatory tumour microenvironment and, therefore, might be a predictive biomarker for immune checkpoint blockade agents. However, the underlying mechanism is still unclear. Methods Patient survival data for our HCC cohort, TCGA and GEO datasets were determined by Kaplan-Meier analysis. The functional roles of ELMO1 in HCC were demonstrated by a series of in vitro and in vivo experiments. Gene microarray analysis was used to demonstrate potential mechanisms of ELMO1. Data retrieved from the TCGA datasets were used to determine the relationships of ELMO1, EMT and TMB. Findings Here, we report an indispensable role for ELMO1 in linking EMT with tumour mutation burden (TMB), which is a promising biomarker for the immune checkpoint blockade agent response. Upregulated ELMO1 expression is associated with a poor prognosis in hepatocellular carcinoma (HCC), as well as increased cell growth, invasion, migration, angiogenesis and EMT in vitro and in vivo. Mechanistically, we provide evidence that ELMO1 regulates SOX10 expression and induces EMT through PI3K/Akt signalling. Moreover, ELMO1 is negatively associated with TMB, indicating a negative relationship between EMT and TMB. Interpretation ELMO1 serves as a link between EMT and TMB, providing a mechanistic basis for the further development of ELMO1 as a therapeutic target against HCC and potentially a promising biomarker of the immune checkpoint blockade agent response. Fund National Natural Science Foundation of China; Natural Science Foundation of Guangdong Province; Young Teacher Training Program of Sun Yat-sen University; Science and Technology Plan of Guangdong Province; Special Support Program of Guangdong Province, Science and Technology Innovation Youth Talent Support Program; the Pearl River Science and Technology New Talent of Guangzhou City; Medical Scientific Research Foundation of Guangdong Province.

Introduction
Metastasis accounts for major cancer-related mortality, including HCC, which remains a leading cause of cancer death worldwide [1,2]. Metastasis is a complex process that requires tumour cells with the ability to transit from the epithelial to mesenchymal state, which is considered the primary attribute driving tumour progression from initiation to metastasis [3]. Although epithelial to mesenchymal transition (EMT) has been reported to endow tumour cells to become highly malignant, with considerable invasiveness and an ability to form distant metastases in various cancers [4,5], the underlying regulatory mechanism is still unclear. In particular, it has also been recently reported that EMT is significantly associated with an inflammatory tumour microenvironment in lung adenocarcinoma [6]. A further study has revealed a link between the EMT signature and PD-L1 blockade non-response [7]. However, how EMT impacts the efficiency of immune checkpoint blockade remains unknown.
The majority of studies exploring the role of EMT in cancer focus on metastasis and alteration of susceptibilities to treatment [8][9][10]. Recent studies indicate that EMT is highly associated with the tumour environment [11] and has the potential to predict PD-1/PDL-1 blockade benefit [12]. Furthermore, tumour mutation burden (TMB) can predict responses to anti-PD-1 or anti-PD-L1 treatments [13][14][15]. The absence of a response to anti-PD-L1 treatment with low TMB is associated with the transforming growth factor β(TGF-β) signalling signature [7], which is a classical regulatory mechanism of EMT [16][17][18]. However, whether TMB is associated with EMT and the underlying molecular mechanism are largely unknown.
Engulfment and cell motility protein 1 (ELMO1) is a critical regulator of Rac, a small GTPase that regulates cell motility through cytoskeletal interactions [19,20]. ELMO1 can also interact with dedicator of cytokinesis 180 (Dock180) to control cellular processes, including cell migration and phagocytosis [21]. Increased expression of ELMO1 promotes cell invasion and is an independent predictor of OS for patients with acute myeloid leukaemia (AML) [22]. In addition, ELMO1 is elevated in HCC and promotes a poor prognosis, but the regulatory mechanism remains unclear. Moreover, ELMO1 has been linked to the KRAS mutation [23], resulting in a higher mutation burden via the alteration of genes involved in cell-cycle regulation, DNA replication and damage repair [24], suggesting a potential role for ELMO1 in chromosome instability.
In the present study, we sought to determine the role of ELMO1 in HCC metastasis and how ELMO1 links TMB to EMT, which may supports its use as a predictive biomarker for immune checkpoint blockade treatment.

Patients and follow-up
Two hundred sixty-three patients diagnosed with HCC were enrolled from The First Affiliated Hospital, Sun Yat-Sen University, Guangdong, China between 2006 and 2009. All patients underwent hepatectomy, and surgical specimens were identified histologically as HCC. Patients were regularly followed up at the outpatient clinics every 3 months for the first 2 years, every 6 months for the next 3 years, and once annually thereafter. Patients under aged 18 years, lacking clinical or laboratory data, or lacking follow-up were excluded. The last follow-up ended in December 2012. The disease-free survival (DFS) and overall survival (OS) were calculated from date of surgery to the date of recurrence or HCC-associated death, respectively. Informed consent was obtained for each enrolled patient, and the research was carried out with approval from the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangdong, China).

Haematoxylin and eosin (H&E) and immunohistochemistry (IHC)
For routine histological study, intrahepatic tumours were fixed in 4% paraformaldehyde and embedded in paraffin. Haematoxylin and eosin (H&E) staining was performed according to the manufacturer's instructions.
Tissue microarrays containing tumour samples, as well as the matched adjacent noncancerous tissue, were constructed as previously described [25]. A Dako Real Envision Kit (K5007, Dako Denmark A/S, Denmark) was used for IHC staining. Primary antibodies against ELMO1 (1:100 dilution, Santa Cruz), E-cadherin (1:200 dilution, Cell Signal), N-cadherin (1:200 dilution, Cell Signal), Snail (1:200 dilution, Abcam) and SOX10 (1:100 dilution, Santa Cruz) were used for this study. The degree of immunostaining was reviewed and scored independently by two observers based on the proportion of positively stained tumour cells and intensity of staining as previously reported [26].

Cell lines, cell culture, signal inhibitor
The liver cancer cell lines HepG2, BEL7402, MHCC97L, MHCC97H, and SMMC7721 and the human normal liver cell line LO2 were purchased from the Cell Bank of the Typical Culture Preservation Committee of Chinese Academy of Science (Shanghai, China). MHCC97L and MHCC97H cell lines have different metastatic potentials but share the same genetic background, and they were used for stable cell line establishment. Cell lines were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone, Logan, UT, USA) in a humidified atmosphere of 5% CO2 at 37°C. LY294002, a classic inhibitor of PI3K/Akt signalling, was purchased from Cell Signal (9901; 50 μM).

Establishment of stable overexpression and knockdown cell lines
ELMO1 lentiviral activation particles and control lentiviral activation particles and ELMO1 shRNA pGFP-C-shLenti vector and scrambled shRNA pGFP-C-shLenti vector were purchased from GeneChem

Research in context
Evidence before this study Recent research as reported that EMT regulates metastasis and affects susceptibility to treatment. EMT is highly associated with the tumour environment and has the potential to predict PD-1/PDL-1 blockade benefit. Furthermore, the tumour mutation burden (TMB) can predict the response to anti-PD-1 or anti-PD-L1 treatments. A lack of a response to anti-PD-L1 treatment with low TMB is associated with the transforming growth factor β(TGF-β) signalling signature, which is a classical regulatory mechanism of EMT. However, whether TMB is associated with EMT and the underlying molecular mechanism are largely unknown.

Added value of this study
In the present study, we report an indispensable role for ELMO1 in linking EMT with the tumour mutation burden (TMB), which is a promising biomarker for immune checkpoint blockade agent response. Upregulated ELMO1 expression is associated with a poor prognosis in hepatocellular carcinoma (HCC), as well as increased cell growth, invasion, migration, angiogenesis and EMT in vitro and in vivo. Mechanistically, we provide evidence that ELMO1 regulates SOX10 expression and induces EMT through PI3K/Akt signalling. Moreover, ELMO1 is negatively associated with TMB, indicating a negative relationship between EMT and TMB.

Implications of all the available evidence
Our study demonstrates that ELMO1 can serve as a link between EMT and TMB, providing a mechanistic basis for further development of ELMO1 as a therapeutic target against HCC as well as a promising biomarker of the immune checkpoint blockade agent response.
For cell growth measurements, cells (1 × 10 3 ) were seeded in 96well plates in complete DMEM. Cell density was determined with the Cell Proliferation Kit I (MTT) (#11465007001, Roche) according to the supplier's protocol starting 24 h post-seeding of the cells. The optical density was measured at 490 nm. All experimental points comprised three replicates, and all experiments were repeated at least three times. The data were graphically displayed using GraphPad Prism 5. Each point (mean ± standard deviation) represents the growth of treated compared with untreated cells.

Western blot analysis
The indicated tissues and cells were suspended in protein lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40) with a cocktail of protease and phosphatase inhibitors (cOmplete Mini, Roche; Phosphatase Inhibitor Cocktail 2 and 3, Sigma). The Bradford (Bio-Rad) method was used to quantify the total amount of protein. Equal volumes of total lysate were processed for protein analysis, which were separated by SDS-PAGE and transferred to PVDF (Roche Life Sciences, Switzerland) membrane. Then, the membranes were blocked with 5% milk in TBST (TBS + 0.1% Tween-20) for at least 1 h at room temperature and incubated with primary antibodies diluted in 5% milk in TBST overnight at 4°C. The membranes were then washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Primary antibodies were detected with anti-mouse IgG HRP-linked (7076S) or anti-rabbit IgG HRP-linked (7074S) antibody, which were purchased from Cell Signalling and used at a 1:5000 dilution. The antigen-antibody complex was detected with enhanced chemiluminescence reagents (Merck Millipore, Massachusetts, USA) and visualized with the Bio-Rad imaging system. The antibody information is listed in Supplementary Table 2.

Co-immunoprecipitation (Co-IP)
The co-immunoprecipitation assay was conducted using a coimmunoprecipitation kit (#26149; Pierce; Rockford, IL, USA) according to the kit's protocols. In brief, 500 μg of MHCC97L or MHCC97H cell lysates for each sample was pre-cleared using a control agarose resin and then incubated with the column containing 5 μg antibody against ELMO1 or IgG at 4°C overnight. After washing to remove non-specific binding, the co-immunoprecipitated proteins were then eluted and analysed by western blotting with the antibody for ELMO1 or SOX10. The input was used as positive control, while IgG was used as negative control.
2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) Briefly, total RNA was isolated with TRIzol Reagent (Life Technologies, Carlsbad, CA) from frozen tissue samples or HCC cell lines according to the manufacturer's protocol. cDNA was obtained from RNA using a universal cDNA synthesis kit (TAKARA, Shiga, Japan). The resultant products were amplified using a SYBR Green PCR kit (Toyobo, Tokyo, Japan) for qRT-PCR analysis. Amplification plots were analysed using Bio-Rad IQ5 software (Bio-Rad Laboratories Inc., California, USA). All quantifications were normalized to the level of endogenous GAPDH as a control. All primers were purchased from GeneCopia (Rockville, MD, USA), and a representative experiment of at least three independent experiments is shown.

Gene expression profiling using RNA microarrays
For mRNA expression profiling analysis, tumour tissues in HCC patients and their counterpart non-cancerous tissues (five pairs) were constructed as described above, and total RNA was extracted with TRIzol® Reagent (Thermo Fisher Scientific), respectively. The Agilent Whole Human Genome Oligo Microarray was performed by Kangchen Biotech Inc. (Shanghai, China). Agilent Feature Extraction software (version 11.0.1.1) was performed to analyse the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes with at least 1 out of all samples containing flags ("All Targets Value") were chosen for further data analysis. Differentially expressed genes between the two samples were identified through fold change filtering.

Human epithelial to mesenchymal transition (EMT) RT 2 profiler™ PCR array
Changes in expression of EMT-related genes in MHCC97L-ELMO1 versus MHCC97L-control, MHCC97H-shELMO1 versus MHCC97Hcontrol cells were investigated using the EMT specific RT 2 Profiler PCR Array (Kangchen Biotech Inc. Shanghai, China) according to the manufacturer's protocol.

Mutation data analysis
For the discovery set, the somatic mutation data (level 2) of liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD), lung squamous cell carcinoma (LUSC), diffuse large B cell lymphoma (DLBC), prostate adenocarcinoma (PRAD), colon adenocarcinoma (COAD), and oesophageal carcinoma (ESCA) were retrieved from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/findArchives. htm). The total number of somatic mutations was adopted to assess the mutation burden, which is convenient and significantly correlated with the number of non-synonymous mutations. Missense, nonsense, nonstop, silent, and frameshift/in-frame insertions and deletions were counted and summed, and germline mutations without somatic mutations were excluded [27]. The frequencies of 6 classes of mutations were calculated by dividing the total number of mutations in each category by the total number of mutations in the 6 categories combined.

Cell migration and invasion assays
Briefly, 1 × 105 cells in 300 μL of DMEM (with 1% FBS) were seeded in the upper chambers of transwell membranes (Corning, New York, USA). The bottom wells of the chambers were filled with 500 μL DMEM (with 10% FBS) and cultured under hypoxic conditions. After 20 h of incubation, the transwell membranes were fixed with 95% ethanol and then stained with 1% crystal violet. Images of five different fields were captured from each membrane, and the number of migrated cells was counted. A similar procedure was adopted for the cell invasion assay, except that the applied inserts were precoated with Matrigel.

Immunofluorescence
Cells were seeded on coverslips, then fixed with 4% paraformaldehyde in PBS for 15 min and washed twice with PBS. Primary antibodies against E-cadherin (Proteintech Group) and N-cadherin (Cell Signalling Technology), F-actin (Genecopia, Guangzhou, China) were incubated overnight at 4°C. E-cadherin and N-cadherin, F-actin were visualized using FITC-conjugated goat anti-mouse or anti-rabbit IgG (Genecopia, Guangzhou, China). DAPI was used as a nuclear counterstain (Genecopia, Guangzhou, China). A Leica DMRA fluorescence microscope (Leica, Wetzlar, German) was used to obtain the images.

Xenograft studies
All experimental procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication nos. 80-23, revised 1996) and were performed according to the institutional ethical guidelines for mouse experiments. All studies involving mice were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Male BALB/C nude mice (5 weeks old) were raised in an SPF environment in the animal facility during the experimental procedures with free access to diet or water. Briefly, 4 × 106 cells suspended in 100 μl PBS were injected subcutaneously into the flanks of nude mice. Tumour volumes and mouse weights were monitored once per week. Tumour size was measured using a calliper twice weekly, and weights were determined at the same time. Tumour volume was calculated as follows: length × width 2 × 0.51. After 6 weeks, tumours established in this step were collected, and subcutaneous tumours were resected, cut into 1 mm3 pieces, and then implanted into the left liver to mimic primary HCC. Twelve mice were randomly divided into each study group. Eight weeks after implantation, in vivo imaging system (IVIS, PerkinElmer) was used to detect GFP fluorescence in tumour-bearing mice. The mice were then sacrificed, and the liver was analysed by standard histological examination.

Gene set enrichment analysis (GSEA)
For gene set enrichment analysis (GSEA), the javaGSEA Desktop Application was downloaded from http://software.broadinstitute.org/ gsea/index.jsp. GSEA was used to determine statistically significant molecular signatures with the indicated data. The input data for GSEA were a complete table of genes ranked by the test statistics from the cuffdiff results and a catalogue of functional gene sets from the HALLMARK gene sets, GO gene sets (Molecular Signatures Database v6.0, http:// software.broadinstitute.org/gsea/msigdb/index.jsp). Default parameters were used. The normalized enrichment score (NES) is the primary statistic for examining gene set enrichment results. The nominal P value estimates the statistical significance of the enrichment score. A gene set with a nominal P ≤ 0.05 was considered to be significantly enriched in genes.

Statistical analysis
Statistical analyses were performed using SPSS 17.0. Data are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Quantitative data were compared between groups using the Student's t-test. Categorical data were analysed by the c2 or Fisher's exact test. Spearman's rank analysis was used to evaluate correlations between protein expression levels. The Kaplan-Meier and log-rank tests were used to analyse differences in OS and DFS. Regression analysis of survival and recurrence was determined using a Cox proportional hazards model. P values b0.05 were considered significant. Correlations between gene expression levels were conducted using a linear regression test.

ELMO1 is upregulated and can confer risk of a poor prognosis in HCC
To identify gene targets in HCC, we performed a gene microarray assay between tumour and adjacent non-tumour tissues. Of five tumour-non-tumour tissues pairs, ELMO1 was upregulated in three of five tumour tissues (patient No. 3, No. 4 and No. 5) ( Supplementary  Fig. 1a). The Gene Expression Profiling Interactive Analysis (GEPIA) platform revealed that ELMO1 mRNA was particularly overexpressed in ACC, DLBCL, ESCA, HNSC, KIRC, LIHC, PAAD, PCPG, SARC, STAD, and TGCT ( Supplementary Fig. 1b). Indeed, the data showed that TCGA, ELMO1 mRNA expression was significantly upregulated in LIHC (Supplementary Fig. 1c). Moreover, similar results were found in the Gene Expression Omnibus (GEO) databases of HCC (GSE84598, GSE76427, GSE57957 and GSE62232) ( Supplementary Fig. 1d). Consistently, western blot analysis revealed a higher level of ELMO1 protein in tumour tissues in 5/6 tumour-non-tumour patient tissue pairs. (Fig. 1a). Together, these results suggest that ELMO1 is upregulated in HCC.
To characterize clinical ELMO1, we collected 263 patient-matched tumour-non-tumour liver tissues. Interestingly, IHC staining demonstrated that ELMO1 expression was dramatically enhanced in 41.4% HCC samples (109/263), compared with adjacent nontumourous tissues (Fig. 1b). ELMO1 overexpression was correlated with vascular invasion (Cox regression analysis, p = 0.017), whereas it was associated with other clinical factors such as gender, age, hepatitis B surface antigen level, alpha-fetoprotein level, capsulation, Edmondson grading, tumour size, liver cirrhosis, or number of nodules (Supplementary  Table 3). Notably, high expression of ELMO1 was associated with a poor prognosis in our cohort, which demonstrated a shorter DFS and OS (Fig. 1c). Consistently, data from TCGA also revealed a correlation of high ELMO1 expression with shorter DFS in LIHC (Fig. 1d). Furthermore, both univariate and multivariate analyses revealed that the expression of ELMO1, tumour size, capsulation, number of tumours and vascular invasion were independent risk factors of lower overall survival and early tumour recurrence (Supplementary Table 4). These data together show that high ELMO1 expression is negatively associated with OS and DFS in HCC patients, suggesting that ELMO1 may play a predictive role in HCC prognosis.

ELMO1 is required for the growth, migration and invasion of HCC cells
Little is known regarding the regulation or function of ELMO1 in human cancer cells. We first analysed ELMO1 expression in cancer cell lines. To date, the Cancer Cell Line Encyclopedia (CCLE) project has demonstrated ELMO1 expression across a diversity of cancer types, including liver cancer (Supplementary Fig. 1e). Furthermore, ELMO1 mRNA and protein expression were higher in HepG2, SMMC7721 and MHCC97H but lower in BEL7402, with no significance in MHCC97L, as compared to the human normal liver cell line LO2 (Fig. 1e, f). Based on the previously reported increased metastatic potential of MHCC97H compared with MHCC97L, we identified MHCC97L and MHCC97H as the most appropriate model for our screen. We then overexpressed ELMO1 in MHCC97L and knocked down ELMO1 in MHCC97H as an initial step to investigate the effect of ELMO1 in HCC.
To understand how ELMO1 affects HCC cells, we investigated ELMO1 in the gene expression profiling using mRNA microarray. Gene ontology (GO) analysis showed that ELMO1 could affects cell cycle, cell proliferation, cell migration and vascular angiogenesis (Supplementary Fig. 1f). Then we further explored ELMO1 effects in the TCGA database. Interestingly, ELMO1 was positively correlated with MKI67, MMP3, VEGFA and VEGFB in the TCGA LIHC data ( Supplementary Fig. 1g, h, i, j), which are known markers of cell proliferation (MKI67), cell metastasis (MMP3) and angiogenesis (VEGFA and VEGFB). In addition, we monitored cell growth, migration and invasion abilities by generating ELMO1  overexpressed or knocked down HCC cells. Indeed, upregulated ELMO1 expression promoted cell growth (Fig. 1g), migration and invasion (Fig. 1h) capabilities, whereas knocked down ELMO1 resulted in the opposite. Together, these results strongly support a critical role of ELMO1, at least partially, in promoting cell growth, migration and invasion.

ELMO1 is negatively associated with tumour mutation burden
Cell cycle, DNA replication, DNA damage response (DDR) and mismatch repair (MMR) mainly accounted for TMB [28,29], a promising positive predictive biomarker for immune checkpoint blockade. Mutation rates of ELMO1 ranged from 0 to 18.69% in provisional TCGA datasets across various cancer types ( Supplementary Fig. 2a), and rates of amplification and deletion ranged from 0 to 5.21%. The majority of the mutations were related to ELMO1, which was overexpressed at the mRNA level across cancer types ( Supplementary Fig. 2a), which confirmed ELMO1 upregulated in HCC. Moreover, TMB was significantly decreased in high ELMO1 mRNA expression tumours compared with low ELMO1 mRNA expression tumours in the TCGA datasets (Fig. 2a). To further elucidate the molecular mechanism by which ELMO1 regulates TMB, we examined the mutator phenotype in ELMO1 high or low tumours. Notably, the C N T mutation frequency, which is most commonly seen in MMR-deficient tumours [30], was markedly higher in low ELMO1 tumours (Fig. 2b, c, d), and it was confirmed to be the major type of mutator phenotype in cancer, suggesting that MMR serves as a key node between ELMO1 and TMB. In addition, in the TCGA datasets, the DDR, MMR signature was enriched in ELMO1-high tumours ( Supplementary Fig. 2b), which further demonstrated a positive association between ELMO1, DDR and MMR regulators, including ERCC4, PALB2, RPA3 and MSH3, in CCLE ( Supplementary Fig. 2c, d, e, f). These findings suggested that ELMO1 might be regulated TMB through DDR or MMR.

ELMO1 links TMB with EMT in HCC
Recent studies have shown that TMB renders tumours more susceptible to PD-L1 blockade [15], whereas TGFβ exhibit the opposite trend, and further research has revealed that cancer cell lines with high TGFβ-EMT signature tend to carry a lower mutation rate [7], suggesting a negative relationship between TMB and EMT. However, whether TMB is correlated with EMT and the underlying mechanism are unclear. First, we applied GSEA to drive enrichment scores from high or low TMB tumours in the TCGA LIHC datasets. Notably, positive enrichment of EMT was observed in low TMB tumours but was irrelevant in high TMB tumours (Fig. 2e). Moreover, EMT was enriched in high ELMO1 tumours with low TMB (Fig. 2f). Thus, our data support a negative correlation of TMB with EMT, and ELMO1 may participate in the crosstalk.

ELMO1 induces EMT
Given that ELMO1 promoted HCC cell invasion and migration, we wondered whether ELMO1 could directly induce EMT. Strikingly, in both the TCGA LUAD and TCGA STAD datasets, a positive correlation between overexpressed ELMO1 and enriched EMT genes was observed (Fig. 3a), suggesting a regulator role of ELMO1 on the EMT signature of cancer. To genetically dissect the contribution of ELMO1 in HCC, we analysed the relationship between ELMO1 protein expression and EMT-related genes in our HCC cohorts. Of note, ELMO1 protein expression level was positively correlated with N-cadherin and Snail, both of which are established essential players in promoting EMT, whereas it showed a negative association with E-cadherin, a canonical marker of suppressed EMT (Fig. 3b, c, d). Furthermore, upregulated N-cadherin and snail mRNA expression in HCC cell lines was observed with overexpressed ELMO1 and downregulated E-cadherin with knocked down ELMO1; in contrast, cell lines with knocked down ELMO1 exhibited the opposite trend (Fig. 3e). Interestingly, comparable observations were observed western blot analyses of HCC cell lines expressing different levels of ELMO1, which demonstrated a significant increase in Ncadherin, snail and decrease in E-cadherin in ELMO1 overexpressed cells. Conversely, decreased ELMO1 expression displayed the opposite trend (Fig. 3f). Moreover, Immunofluorescence assay showed consistent results (Fig. 3g), suggesting that ELMO1 could regulate EMT.

ELMO1 activates PI3K/Akt signalling
The molecular mechanism linking ELMO1 with EMT is unclear. Notably, gene set enrichment analysis identified PI3K/Akt signalling as the feature associated with ELMO1 from the HCC GEO datasets (Fig. 4a), and genes involved in the PI3K/Akt signalling pathway were significantly overexpressed in ELMO1 overexpressed tumours. Consistently, using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations by STRING, the PI3K/Akt signalling pathway was activated, as well as focal adhesion, VEGF signalling, and the MAPK signalling pathway, among others (Fig. 4b), which have been previously reported to be associated with EMT initiation. Of Note, although PI3K/Akt signalling was not the most activated pathway regulated by ELMO1, upregulated PIK3CA, a key member of PI3K/Akt [31], was involved in various signals, such as VEGF signalling, viral carcinogenesis, pancreatic cancer, and non-small cell lung cancer (Fig. 4b). Notably, the PI3K/Akt gene signature was significantly enriched in ELMO1 overexpressed tumours in TCGA LIHC patients (Fig. 4c). We further revealed a positive relationship between ELMO1 and PIK3CA, PIK3CB, PIK3CD, and PIK3CG in TCGA ( Supplementary Fig. 3a). Moreover, PIK3CA and PIK3CB were further validated as positively correlated with ELMO1 in CCLE datasets across 760 cancer cell lines ( Supplementary Fig. 3b). Importantly, ELMO1 could upregulate PI3K (Fig. 4d, e), and Akt and GSK3β were phosphorylated by overexpressed ELMO1, whereas phosphorylated Akt and pGSK3β were decreased with downregulated ELMO1 in HCC (Fig. 4e). Together, these findings suggest that ELMO1 can activate the PI3K/ AKT signalling pathway.
To further validate that the PI3K/Akt pathway is downstream of ELMO1, we applied a PI3K/Akt signalling pathway inhibitor, LY294002, which is widely used to attenuate PI3K/Akt pathway expression. Indeed, ELMO1 enhanced PI3K/Akt signalling, as demonstrated by a significant increase in the levels of p-Akt in MHCC97L-ELMO1 cells, and LY294002 could antagonize this effect (Fig. 4f). Furthermore, ELMO1 knocked down the inhibitory Akt signal with a marked decrease in p-Akt levels in HCC cells, and the inhibitory effect was enhanced in  the presence of LY294002, as evidenced by an attenuation of the level of p-Akt (Fig. 4f). Altogether, these findings provide evidence that ELMO1 acts as positive regulator of the PI3K/Akt signalling pathway, potentially providing an explanation for the pro-metastatic effect of ELMO1 in HCC.

ELMO1 induces EMT through the PI3K/Akt signalling pathway
Recent studies have revealed a regulator role of the PI3K/Akt signalling pathway in EMT [32]. Therefore, we considered the possibility that ELMO1 could regulate EMT through the PI3K/Akt signalling pathway. Indeed, ELMO1 protein interacts with the protein of PIK3CA, CDH1 (Ecadherin) and VEGFA derived from STRING, a functional protein association networks database ( Supplementary Fig. 3c), suggesting a potential link between ELMO1, PI3K/Akt signalling and EMT. Moreover, overexpression of ELMO1 induced a dramatic rearrangement of F-actin in MHCC97L cells, and inhibition of PI3K/Akt signalling by addition of LY294002 could abolish the inducing effect of ELMO1 on F-actin (Supplementary Fig. 3d). Furthermore, LY294002 could reduce the positive effect of ELMO1 on MHCC97L cell invasion and migration (Fig. 4g). In addition, overexpressed ELMO1 induced a dramatic enhancement of N-cadherin and Snail in MHCC97L cells, whereas inhibition of PI3K/ Akt signalling by the addition of LY294002 could abolish the inducing effect of ELMO1 (Fig. 4h). Together, these results suggest that ELMO1 is critically involved in mediating EMT through the Akt signalling pathway.

ELMO1 promotes cell growth, metastasis and EMT in vivo
We next evaluated ELMO1 overexpression or knocked down cell lines injected subcutaneously in mice (Fig. 5a). Notably, the tumour volume and weight of MHCC97L-ELMO1-derived tumours was significantly increased compared with MHCC97L-control. In contrast, ELMO1 knockdown achieved the opposite effect (Fig. 5b, c). Moreover, we transplanted the subcutaneous tumour into liver (Fig. 5e), although no obvious metastasis was observed in the lung by the end of the experiments (Fig. 5e). ELMO1 enhanced the rate of intrahepatic metastasis in tumours derived from MHCC97L-ELMO1 cells compared with control tumours from parental MHCC97L-control cells (Fig. 5d). Conversely, there was a lower incidence of intrahepatic metastasis in tumours derived from MHCC97H-shELMO1 cells compared with control tumours from parental MHCC97H-shcontrol cells (Fig. 5d). In addition, ELMO1 increased the intrahepatic tumour volume, whereas ELMO1 knockdown resulted in a decreased intrahepatic tumour volume (Fig. 5e, f). Together, these results suggest that ELMO1 promotes cell growth and cell metastasis in vivo.
We further examined the relationship between ELMO1 and EMTassociated markers in intrahepatic tumours by IHC. Similar to the in vitro results, increases in N-cadherin, Snail and SOX10 protein expression were detected in tumours from ELMO1 overexpression cells, and decreased E-cadherin protein levels were observed in tumours from ELMO1 knockdown cells. In contrast, tumours derived from MHCC97H-shELMO1 HCC cells exhibited the opposite effects (Fig. 5g). These findings suggest that, in vivo, the availability of EMT-associated factors can be affected by ELMO1, thus revealing a critical role of ELMO1 in the induction of EMT of HCC cells.
3.9. SOX10 is critical for ELMO1-enhanced cell invasion, metastasis and PI3K/Akt signalling Our data support ELMO1-activated Akt signalling as a cause of HCC metastasis, but the full mechanism was still unclear. We performed EMT-associated regulator analyses using human the Epithelial to Mesenchymal Transition (EMT) RT 2 Profiler™ PCR Array, and we identified various EMT-associated regulators that could be affected by ELMO1 (Supplementary Fig. 4a, b). Notably, sex determining region Y (SRY) and the related HMG box-containing factor 10 (SOX10), a member of the SOX family transcription factors, were upregulated in the ELMO1 overexpression HCC cells and suppressed in ELMO1 knockdown HCC cells, suggesting that SOX10 expression is likely responsible for the increased or decreased ELMO1 expression levels. This result was further confirmed by western blot analysis in ELMO1 overexpression or knockdown HCC cells (Fig. 6a). Interestingly, we found that ELMO1 could directly bind to SOX10 (Fig. 6b). Additionally, SOX10 protein expression was higher than in adjacent non-tumour tissues by IHC (Fig. 6c). Furthermore, a positive relationship between ELMO1 and SOX10 was observed in orthotopic HCC (Fig. 5g). Therefore, it seemed likely that ELMO1 mediated SOX10 expression in HCC. These data support the conclusion that ELMO1 can increase SOX10 expression in HCC.
To evaluate the clinical relevance of SOX10 in cancer, we explored the TCGA datasets. Notably, although SOX10 had no significant effect on OS in LIHC patients, LIHC patients with high SOX10 expression showed a shorter DFS (Fig. 6d). Furthermore, in human SKCM patient cohorts of TCGA, patients with high SOX10 displayed a poorer OS when compared with patients with low SOX10 (Fig. 6d). Thus, we concluded that SOX10 could predict the prognosis of HCC patients, at least partially, indicating DFS in HCC.
Recent studies have indicated that SOX10 regulates EMT in cancer [33,34]. This finding was further supported in our GSEA of GEO databases using HALLMARK gene collection (Fig. 6e), which revealed an enhancement of the EMT signature in ELMO1 overexpression tumours, suggesting that SOX10 is involved in initializing EMT in HCC.
Triggered by our initial finding of ELMO1 mediating PI3K/Akt signalling and subsequently inducing EMT, we wondered whether SOX10 was involved in this axis. However, to the best of our knowledge, the relationship between SOX10 and PI3K/Akt signalling has not yet been explored, and recent studies have reported that SOX9, another member of SOX family, can activate PI3K/Akt signalling [35]. Therefore, we hypothesized that SOX10 might mediate PI3K/Akt activation by ELMO1. As expected, KEGG analysis from platform STRING revealed an enrichment of PI3K/Akt signalling in the presence of SOX10, confirming the involvement of SOX10 in PI3K/Akt signalling activation, although PI3K/ Akt was not the most enriched signalling pathway ( Supplementary  Fig. 4c). Furthermore, we evaluated whether SOX10 was essential for ELMO1 mediation of the PI3K/Akt/Snail axis. SOX10 silencing resulted in reduced mRNA and protein levels of PI3K and snail, as well as Akt phosphorylation. However, overexpression of SOX10 showed the opposite effect (Fig. 6f, g), suggesting that SOX10 functions as a downstream target of ELMO1 that leads to PI3K/Akt signalling activation. Additionally, upregulated SOX10 in the ELMO1-low HCC cell line led to enhanced cytoskeletal staining, whereas downregulated SOX10 in the ELMO1high HCC cell line demonstrated an opposite effect (Fig. 6h), supporting a role for ELMO1 in promoting cell metastasis through regulating SOX10. Together, these results suggest that ELMO1 regulates cell metastasis and PI3K/Akt signalling in a manner dependent on ELMO1 regulatory activity towards SOX10.

Discussion
In the present study, we demonstrated that increased ELMO1 expression is associated with HCC metastasis and a poor prognosis. ELMO1 induces EMT via the SOX10/PI3K/Akt axis. Moreover, increased ELMO1 is correlated with a lower TMB and positively with DDR (ERCC4, PALB2), MMR (RPA3), MSI (MSH3). In addition, ELMO1 can serve as a link between EMT and TMB. These results provide insight into the regulatory mechanism underlying EMT and how EMT is linked to TMB in HCC, which may improve our understanding of predictive biomarkers in immune checkpoint blockade.
This study indicated the importance of ELMO1 in HCC prognosis, which is in agreement with previous research implicated in AML [22]. Furthermore, it should be emphasized that the current findings are consistent with prior studies [36], suggesting that ELMO1 expression is upregulated in HCC and associated with microscopic vein invasion in HCC. Together, the results imply the potential of ELMO1 as a predictive prognostic biomarker in HCC and its association with the HCC vascular invasion phenotype. Furthermore, we extended these findings in a larger LIHC patient cohort, in TCGA, combined with the notion that plasma markers of methylated ELMO1 detect 86% of gastric adenocarcinoma with 95% specificity [37], supporting ELMO1 as a promising noninvasive predictive biomarker of HCC screening, surveillance and prognostic prediction. However, further studies corroborated in a larger cohort in HCC are still warranted, and whether mRNA-or protein-based quantification of ELMO1 for HCC prognosis is a better predictor is unclear.
Notably, we further revealed a link between ELMO1 and EMT. Although it has been previously reported that ELMO1 can stabilize snail via NF-kB signalling, this phenomenon only occurred in the context of IL-8 binding to CXCR1 [38]. Therefore, whether ELMO1 upregulated in the absence of the indicated background can induce EMT remains unclear. Furthermore, a recently published article has challenged the classic function of EMT in cancer metastasis [39,40]. In our study, we found that ELMO1 could upregulate SOX10 and induce EMT via PI3K/Akt signalling in HCC. SOX10 is a member of the SOX transcription factor family. In this study, SOX10 was found to be overexpressed in HCC and was a predictor of DFS, although it failed to predict OS in the TCGA LIHC cohort. Although the importance of SOX10 in cancer initiation and progression is well-recognized in several cancer types [41], the regulation of SOX10 remains poorly characterized. Our data suggested ELMO1 could directly regulate SOX10, which means that ELMO could act upstream of SOX10. SOX10 has been reported to play a pivotal role in driving EMT and to link Stem-and EMT-like characteristics in breast cancer [42]. Consistently, we demonstrated SOX10 associated with the EMT phenotype with PI3K/Akt signalling as a key downstream target of ELMO1/SOX10. However, whether the combination of ELMO1 and SOX10 inhibition could increase the antitumour effect in cancer requires further investigation.
TMB is an important biomarker of the immune checkpoint blockade response in tumour therapy [13][14][15], based on increased expression of tumour neoantigens that are recognized by immune cells [43]. Interestingly, higher ELMO1 expression resulted in lower TMB. Furthermore, ELMO was associated with the DDR, MMR phenotype, a pivotal regulatory mechanism in tumour mutability. These results implied that ELMO1 might decrease TMB through DDR and MMR. In brief, ELMO1 has the potential to predict the response to immune checkpoint blockade therapy, lending insight into personalized cancer therapy strategies. However, the mechanism by which ELMO1 regulates tumour mutability through DDR or MMR remains to be explored.
Although most studies have focused on the effect of EMT in cancer metastasis and drug resistance, recent studies have explored the role of EMT in reprogramming the tumour immunity microenvironment. A strong association between EMT status and an inflammatory tumour microenvironment with elevation of PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA and CTLA-4 was identified in NSCLC [44]. Despite a failure to identify an association between EMT and tumour mutation burden in NSCLC, Harbhajanka et al. revealed a higher frequency of STK11 or KEAP1 mutations in "epithelial" than "mesenchymal" lung adenocarcinomas [45], suggesting that mesenchymal tumours have fewer neoantigen mutations. Moreover, one recent report has demonstrated that low TMB is correlated with TGFβ signalling, which is known as a main driver of EMT [12]. Notably, this study provides evidence that EMT is inversely correlated with TMB in HCC and that ELMO1 links the two phenotypes. However, it is noteworthy that EMT was correlated with elevated PD-L1 expression, which is a potential biomarker for anti-PD-1/PD-L1 therapy [7]. Therefore, this finding seems contradictory to our results showing that EMT was negatively correlated with TMB, which was recently reported as a biomarker of better clinical outcomes for immune checkpoint blockade therapy. Notably, some patients with high PD-L1 expression benefit little from immune checkpoint blockade therapy, while some patients with low PD-L1 expression show a greater benefit [46], suggesting that the PD-L1 expression level is not an exclusive biomarker for the response to immune checkpoint blockade therapy. Therefore, our study provides insights into ELMO1, which links EMT and TMB. The use of TMB detection is restricted due to its high cost [47]. Therefore, examination of ELMO1 expression levels could represent a substitute solution with a much lower cost.
In conclusion, ELMO1 is an independent prognostic factor of HCC and some other cancer types. ELMO1 overexpression promotes HCC cell proliferation, invasion, and migration via EMT in vitro and in vivo by regulating SOX10/PI3K/Akt signalling, suggesting a tumouractivator role of ELMO1 in HCC. Furthermore, ELMO1 is inversely correlated with TMB by regulating MMR or DDR. In addition, ELMO1 links EMT with TMB. Close monitoring of ELMO1 levels in patients has the potential to predict the response to immune checkpoint blockade therapy in HCC either alone or combined with TMB. This study highlights the role of ELMO1 as a biomarker for HCC prognosis and the immune checkpoint blockade therapeutic response and as a potential therapeutic target for HCC therapy.

Declaration of Competing Interest
The authors declare that they have no conflicts of interest.


s2fieldsofstudy: Biology Medicine Chemistry

text:
Pd-Ia exerts anti-inflammatory effects by activating PPAR-α in human umbilical vein endothelial cells

Pd-Ia (dl-praeruptorin A), isolated from Chinese traditional herbal medicine Qian-Hu, has been linked to cardiovascular diseases because of its effective role on left ventricular dysfunction, exhibit anti-inflammatory properties through reducing the level of pro-inflammatory factors in ischemia/reperfusion myocardium. However, the potential roles and possible mechanisms in endothelial cells inflammatory response have yet to be fully clarified. In the present study, human umbilical vein endothelial cells (HUVECs) were selected to perform in vitro assays. Pd-Ia significantly reduced the mRNA expression of pro inflammatory cytokines and chemokines, including tumor necrosis factor α (TNF-α), interleukin (IL) 1β, IL 6, IL 8, monocyte chemoattractant protein 2 (MCP-2) and vascular adhesion molecule 1(VCAM 1) that were upregulated by LPS stimulation. Pd-Ia selectively increased peroxisome proliferator-activated receptor-α (PPAR-α) expression and enhanced the phosphorylation of PPAR-α that were downregulated by LPS. Furthermore, knockdown of PPAR-α by siRNA abrogated the effects of Pd-Ia on pro inflammatory cytokines TNF-α and IL 1β expression in LPS stimulated HUVECs. Additionally, Pd-Ia decreased the LPS upregulated NF-κBp65 phosphorylation at tested timepoints, which was abolished by PPAR-α antagonist MK886. These results demonstrated that Pd-Ia inhibited the pro inflammatory genes expression through activating PPAR-α and suppressing NF-κB activity. Thus, Pd-Ia may have the potential of being developed as an anti-inflammatory agent.

Introduction
The inflammatory response of endothelial cells is critical for the pathogenesis of various diseases, such as endotoxic shock, cancer, thrombosis, atherosclerosis and diabetes mellitus. Inflammatory activation of the endothelium has been shown to decrease its vasomodulatory ability and antithrombotic properties, contributing to the pathogenesis of atherosclerosis [1]. Chronic inflammation in endothelial cells produces a variety of inflammatory mediators to exacerbate endothelial dysfunction, which plays a dominant role in atherosclerosis, hypertension and diabetes-induced vasculopathy and vascular remodelling [2][3][4]. Endothelial cells are activated to release inflammatory mediators such as TNF-α and IL-1β, chemokines (MCP-1 and IL-8), adhesion molecules (ICAM-1 and VCAM-1) [5,6]. In turn, the release of inflammatory factors, chemokines and adhesion molecules as well as coagulation factors activates the vascular endothelial cells and subsequently promotes the vascular endothelial injury [7]. Lipopolysaccharide (LPS) has been identified as one of the strongest stimulators that target the endothelium, LPS induce the morphological damage, reduce cell viability of HUVECs and increase TNF-α, IL-8, IL-1β and MCP-1 expression [6,8,9]. Therefore, endothelial cells are one of the active participants in regulation of inflammatory reactions.
Pd-Ia (dl-praeruptorin A), isolated from Chinese traditional herbal medicine Qian-Hu, has a long history of application for the treatment of cough with thick sputum and dyspnea, non-productive cough and upper air-way infections in traditional medicinal practice [10,11]. Our previous study had revealed that Pd-Ia had cardioprotective action and relieved inflammatory reaction and apoptosis in rat isolated ischemia-reperfusion myocardium through inhibiting the activity of However, whether Pd-Ia may act at PPAR-α to play the antiinflammatory role is not well understood. In this study, we investigated the effects of Pd-Ia on LPS-induced inflammatory responses. We found that PPAR-α plays a critical role in mediating the anti-inflammatory effects of Pd-Ia in LPS-stimulated HUVECs.

Cell culture
Human umbilical vein endothelial cells (HUVECs) were harvested and isolated from fresh healthy volunteer maternal newborn umbilical cords. This study was performed in accordance with the Helsinki Declaration and approval of the Ethical Committees of School of Medicine, Xiamen University and Xiamen Cardiovascular Hospital. All subjects have consented to this study. HUVECs were digested by trypsogen and kept in 0.2% gelatin-coated cell culture dishes in DMEM medium containing 15% FBS (fetal bovine serum), 5 ng/ml FGF(fibroblast growth factor), 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. HUVECs were seeded onto 6-well plates at 1×106 cells/well concentration in 2 ml DMEM medium under a humidified 5% CO2 atmosphere to 80% confluency. 2-6 generations of Cells were used in the experiments.

Quantitative real-time RT-PCR
RNA was extracted using TRIzol reagent and cDNA was obtained using RevertAidTM First Strand cDNA Synthesis Kit according to the manufacturer's instructions. Specific primers were designed using Primer Express 3.0 software (Table 1). Real-time PCR was performed with the 7300 Real Time PCR System (Applied Biosystems) and SYBR Green (TaKaRa). The cycling program was set as follows: thermal activation for 30 s at 95 °C and 40 cycles of PCR (melting for 5 s at 95°C, followed by annealing/extension for 31 s at 60°C). Relative expression normalized to the internal control GAPDH was assessed by comparative analysis of the quantitative real-time PCR results using the 2 −ΔΔCT method [21].

ELISA
The concentration of TNF-α, IL-1β, IL-6 and IL-8 in the culture supernatants was measured by ELISA kit (R&D) according to manufacturer's instructions.

Cell transfections
HUVECs were transfected in Opti-MEM with Lipofectamine RNAiMAX according to the manufacturer's protocol. For transfection, all siRNAs (PPAR-α siRNA 005, siRNA 006, siRNA 007 or the negative control) were re-suspended to a final concentration of 20 nM. After 24 h incubation, the transient transfected cells were treated with 1 μg/ml LPS and 40 μM Pd-Ia for 24h. Relative expression of TNF-α and IL-1β was assessed by Quantitative Real-time PCR.

Statistical analysis
Experimental data are presented as mean ± SEM. Differences were analyzed by one-way ANOVA followed by Fisher's least significant difference test. A value of p < 0.05 was regarded as significant.

Pd-Ia inhibited pro-inflammatory genes expression in LPSstimulated HUVECs
To evaluate the effects of Pd-Ia on LPS-induced inflammatory responses, we determined the pro-inflammatory genes expression in LPS-stimulated HUVECs. HUVECs were treated with LPS and different concentrations of Pd-Ia for 24 h. The LPS stimulation significantly increased the mRNA expression of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, chemokine IL-8,MCP-2 and adhesion molecular VCAM-1 (Figure 1). Pd-Ia at concentration of 10, 20 and 40 μM could dose-dependently decreased the mRNA levels of TNF-α, IL-1β and IL-6 ( Figure 1), The mRNA levels of IL-8, MCP-2 and VCAM-1 were significantly decreased by Pd-Ia, but not in a dosedependent manner (Figure 1). Consistent with mRNA expression, the protein levels of TNF-α, IL-1β, IL-6, IL-8 that were upregulated upon LPS stimulation in culture supernatants were also decreased by Pd-Ia ( Figure 2). These results suggested that Pd-Ia exert anti-inflammatory effects through inhibiting pro-inflammatory genes expression in LPSstimulated HUVECs.

Pd-Ia upregulates PPAR-α expression and enhances its phosphorylation in LPS-stimulated HUVECs
Nuclear receptors superfamily PPARs, including PPAR-α, PPARβ/δ and PPAR-γ, are involved in the inflammatory responses in a variety of cell types [22,23]. To investigate which nuclear receptor was associated with the anti-inflammatory effects of Pd-Ia, HUVECs were treated with LPS and different concentrations of Pd-Ia for 24h. LPS stimulation significantly downregulated the mRNA level of PPAR-α and Pd-Ia treatment dose-dependently increased the PPAR-α level (Figure 3). The expression of PPAR-β was no changed by LPS stimulation and Pd-Ia treatment (Figure 3). LPS stimulation significantly downregulated the mRNA level of PPAR-γ but no effect was found by Pd-Ia treatment (Figure 3). These results suggested that Pd-Ia specifically upregulates PPAR-α expression. Next, we examined the effect of Pd-Ia on PPAR-α at protein level in LPS-induced HUVECs. Consistent with mRNA expression, Pd-Ia at the concentration of 20 and 40 μM dose-dependently upregulated the expression of PPAR-α and the phosphorylation of PPAR-α (p-PPAR-α), that was downregulated by LPS stimulation (Figure 4), These results suggested that Pd-Ia could upregulate PPAR-α expression and enhance PPAR-α activity in LPSinduced HUVECs.

Anti-inflammatory effects of Pd-Ia on LPS-induced inflammation is PPAR-α -dependent
To further investigate whether the anti-inflammatory effect of Pd-Ia is PPAR-α-dependent, the PPAR-α siRNA was used to explore the role of PPAR-α in LPS-induced HUVECs. Our results showed that the PPAR-α siRNA006 was more effective than siRNA005 or siRNA007 to suppress both the mRNA and protein expression of PPAR-α in HUVECs ( Figure 5). The knock-down efficiencies of PPAR-α were 50.7% and 51.3% as determined by quantitative real-time PCR and western blot analysis, respectively ( Figure 5). Therefore, we selected PPAR-α siRNA006 in the following experiments. PPAR-α knockdown could significantly increase the mRNA levels of TNF-α and IL-1β that were reduced by 40 μM Pd-Ia (Figure 6), suggesting that the anti-inflammatory effect of Pd-Ia on LPS-induce inflammatory response is PPAR-α dependent.

Pd-Ia inhibits LPS-induced phosphorylation of NF-κB through PPAR-α
NF-κB has been reported to play an important role in the regulation of inflammatory cytokine production [24]. Accordingly, to evaluate whether NF-κB is involved in the inhibition of inflammatory responses by Pd-Ia, we investigated the effect of Pd-Ia on the phosphorylation of NF-κB p65 (p-NF-κB p65), which is required for the activation of NF-κB. LPS induced the p-NF-κB P65 level began to increase at 15 min, peaked at 60 min and gradually decreased at 120 min (Figure 7). In contrast, Pd-Ia (40μM) greatly suppressed the LPSinduced increase in the p-NF-κB p65 from 5min to 120min ( Figure  7). The inhibitory effect of Pd-Ia on the p-NF-κB p65 was apparently suppressed when the cells were treated with a PPAR-α antagonist MK886 from 5min to 120min (Figure 7). These results suggest that the inhibitory effect of Pd-Ia on the LPS-induced increase in p-NF-κB p65 is through PPAR-α signalling pathway.

Discussion
The major finding of this study is that the anti-inflammatory effect of Pd-Ia is due to its ability to activate PPAR-α in LPS-stimulated HUVECs. This can be supported by the following pieces of evidence. First, Pd-Ia selectively upregulated the transcript level of PPAR-α but not that of PPAR-β and PPAR-γ; Second, Pd-Ia increased the protein expression of PPAR-α and enhanced PPAR-α phosphorylation that was reduced by LPS stimulation; Third, PPAR-α siRNA attenuated the inhibitory effect of Pd-Ia on LPS induced expression of TNF-α and IL-1β, which suggests that the action of Pd-Ia was PPAR-α-dependent; Fourth, PPAR-α antagonist MK886 suppressed the effect of Pd-Ia on the LPS-induced phosphorylation of NF-κB p65.
Some studies have suggested that activation of PPAR-α by its agonists can inhibit LPS-induced pro-inflammatory mediators increase in a variety of cell types, In LPS-stimulated microglia cells, PPAR-α agonists inhibited the secretion of the proinflammatory cytokines IL-1β, TNF-α, IL-6, and IL-12p40 and the chemokine MCP-1 [25]. In the adipocytes, PPAR-α activators suppress the pathogenetical secretion of TNF-α [26]. In activated mesangial cells, LPS treatment induced the significant appearance and substantial activation of PPARα, thus attenuates the proinflammatory response [27]. In endothelial cells, PPARα acts as a master regulator of inflammation by suppressing TNF-α and IL-6 expression and involved in inflammatory signalling include NF-κB-dependent pathway [28]. In EAhy926 endothelial cells, PPARα activators inhibit cytokine-induced nuclear translocation of NF-κB and expression of VCAM-1 [29]. In the present study, we showed that in LPS-stimulated HUVECs, Pd-Ia suppressed proinflammatory cytokines TNF-α, IL-1β, IL-6, chemokines IL-8, MCP-2 and adhesion molecules VCAM-1 expression, The inhibitory effect of Pd-Ia was mediated by activation of PPAR-α. Whether activation of PPAR-α may be a possible mechanism for anti-inflammatory effect of Pd-Ia in HUVECs, Next we detected the expression of TNF-α and IL-1β following Pd-Ia treatment in the presence and absence of PPARα siRNA, The results showed that interfering PPAR-α attenuate the antiinflammatory effect of Pd-Ia. This may confirmed that inhibiting proinflammatory genes expression by Pd-Ia is associated with PPAR-α pathway.
It is well established that LPS triggers inflammatory responses through the activation NF-κB [30]. NF-κB is a key promoter of inflammation, Once stimulated by LPS, NF-κB translocate from the cytoplasm to the nucleus where it activates the transcription of pro-inflammatory genes [31,32]. In the present study, our results displayed that Pd-Ia suppressed the expressions of pro-inflammatory genes that were upregulated by LPS in HUVECs. On the other hand, PPAR-α activation has been shown to antagonize the NF-κB signalling pathway, which in turn lead to downregulation of pro-inflammatory cytokines, involved in the vascular inflammation in atherosclerosis [33]. Activation of PPAR-α limits the expression of pro-inflammatory cytokines in atherosclerosis pathological processes [34]. Activation of PPAR-α inhibits inflammatory responses through the TLR4/NF-κB signalling pathway in vascular smooth muscle cells in concert to protect against atherosclerosis [35]. In agreement with these reports, our results showed that Pd-Ia inhibited the LPS induced phosphorylation of NF-κBp65, which was abolished by PPAR-α antagonist MK886 at tested timepoints. Taken together, all these findings supported that Pd-Ia exert anti-inflammatory effects by activating PPAR-α and inhibiting NF-κB pathway. Here, a schematic diagram was drawn to describe the anti-inflammatory role of Pd-Ia in LPS-induced HUVECs and the underlying mechanisms ( Figure 8).
In conclusion, our results demonstrated that Pd-Ia exerted antiinflammatory effects in LPS-induced HUVECs which involves in the activation of PPAR-α and the suppression of NF-κB activity. This may offer a therapeutic strategy for the management of endothelial inflammatory diseases. Further appropriate studies are warranted to evaluate the effect in vivo.