IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (−544 to −389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.


Introduction
Non-small cell lung cancer (NSCLC) is common type of malignancy, and the leading death cause of NSCLC patients is associated with the cancer metastasis [1][2][3].Recently, accumulating evidence has demonstrated that NSCLC is an inflammation-related cancer [4][5][6], and the overexpression of inflammatory cytokines or mediators in NSCLC microenvironment can induce the cancer cell proliferation, migration and invasion [7][8][9][10], but the precise mechanism, e.g., IL-17-mediated NSCLC metastasis has not been characterized.
IL-17A, also known as IL-17, is a pro-inflammatory cytokine [10].Many scholars have reported that the expression increase of IL-17 in the cancer tissues can improve cell proliferation resulting in cancer growth [11,12] or elevating MMP2 and MMP9 production leading to NSCLC metastasis [13].It is recognized that cancer cell metastasis is a complex process that involves the extracellular matrix (ECM) degradation and basement membrane penetration [14].Several studies have demonstrated that MMP1, MMP2, MMP9, MMP13 and MMP19 in some cancer tissues such as NSCLC or gastric cancer are all raised, and these MMP members can degrade basement membrane and open the channel for cancer's invasion and metastasis [13,[15][16][17].In the early stage of our experiments, we found that MMP19 expression was greatly higher than other MMP members both in the detected NSCLC tissues and in the NSCLC cells stimulated with IL-17.However, little is known about how IL-17 can induce MMP19 expression causing NSCLC metastasis.
Signal transducer and activator of transcription 3 (STAT3) is a member of STAT family, and can activate its target gene transcription in many cancer cells upon various stimulus inducing the change of cell behaviors [18][19][20].For example, the STAT3 activated by IL-17 or IL-6 can facilitate cancer cell angiogenesis, migration, invasion and metastasis via increasing the expression of vascular endothelial growth factor (VEGF), O-GlcNacylation, MMP or the switch of epithelial-mesenchymal transition (EMT) to mesenchymalepithelial transition (MET) [19][20][21][22][23].Our previous test of this work has found that the NSCLC cells under IL-17 stimulation can markedly activate STAT3 and up-regulate MMP19 expression, but their relationship and effects of STAT3 activation on MMP19 induction in the NSCLC cells exposed to IL-17 are not clear.
Current documents have reported that the protein posttranslational modification (PTM), such as phosphorylation or acetylation is essential for regulating protein function and cell behaviors [23][24][25][26].Previous researches have revealed STAT3 can subject to phosphorylation (e.g., p-STAT3 at Ser727 or Tyr705) and acetylation, and its phosphorylation or acetylation is necessary for STAT3 activation promoting cancer tumorigenesis or metastasis and STAT3-driven gene transcription in the microglia and other cancers [23,27,28], but the role of STAT3 acetylation and the interaction of STAT3 acetylation and phosphorylation in MMP19 expression as well as NSCLC cell metastasis in response to IL-17 remain undefined.p300 is a transcriptional co-activator with histone acetyltransferase (HAT) activity [29], and p300 can acetylate the lysine of histones or transcription factors resulting in cell behavior alteration [29][30][31][32].Cai et al. [31] have confirmed that p300 can epigenetically regulate hepatocellular carcinoma (HCC) progression through acetylating metabolic enzymes, and Hou et al. [32] have pointed out that p300 can enhance NSCLC cell proliferation, migration and invasion via inducing EMT.Given that CREPT/RPRD1B promotes tumorigenesis by STAT3-driven gene transcription in p300dependent manner [28], p300 augments the cell metastasis of HCC and NSCLC [31,32], and p300 overexpression is also proved in our detected NSCLC samples, whether p300 can acetylate STAT3 and affect its-Y705 phosphorylation, and the effect of the interaction between these two posttranslational modifications on MMP19 expression, cell migration and invasion in IL-17-stimulated NSCLC cells require to be ascertained.
In the present study, we first examined the expression of IL-17 receptor A (IL-17RA, a major receptor), p300, Y705-phosphorylated STAT3 (i.e., p-STAT3), acetylated STAT3 (Ack-STAT3) and MMP19 both in NSCLC tissues and in the NSCLC cells stimulated by IL-17.Then, we explored in vitro that the role and mechanism of p300, STAT3, MMP19 expression as well as p300-mediated STAT3 acetylation or its phosphorylation and their interaction in regulating MMP19 gene transcription and expression, cell migration and invasion in the NSCLC upon IL-17.Moreover, we utilized nude mouse metastasis model to evaluate that the influence of silencing p300, STAT3 or MMP19 gene plus IL-17 treatment on NSCLC metastasis and related protein expression.

Human specimens and cell lines
The cancer tissues and adjacent normal tissues of 10 NSCLC patients were from Jiangsu Cancer Hospital.The patients suffering from other diseases, e.g., infection, autoimmune, inflammatory diseases and other cancers were excluded.This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (2019-SRFA-052) and performed in compliance with the Declaration of Helsinki.Informed consent for this procedure was obtained from each participant.The NSCLC tissue arrays (n = 52, paired) were provided by Outdo Biotech Co., Ltd.(Shanghai, China).The human NSCLC cell lines such as NCI-H1299 (RRID: CVCL_0060), NCI-H1975 (RRID: CVCL_1511) or PC-9 (RRID: CVCL_B260), and normal bronchial epithelium cell line BEAS-2B (RRID: CVCL_0168) were purchased from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC).The above-mentioned cell lines were authenticated via short-tandem repeat (STR) analysis by General Biosystems.Besides, all cell lines used in the experiments were routinely tested for mycoplasma by using MycoAlert TM mycoplasma detection kit and showed mycoplasma-free.
Cell culture and IL-17 stimulation Cells were maintained with 10% fetal bovine serum (FBS) added Dulbecco's Modified Eagle's Medium (DMEM).For IL-17 stimulation, the NSCLC cell lines were suffered from 12 h serum starvation, and then were given 0, 25, 50 and 100 ng/ml IL-17 for different time.

RT-PCR and real-time PCR
Total RNA from NSCLC tissues or cells was isolated using TRIzol.The cDNA was produced and RT-PCR assay was carried out.Besides, real-time PCR was done and the results were normalized to β-actin and analyzed using the 2 −ΔΔCt method.RT-PCR and real-time PCR primers are shown in Suppl.Tables S1 and S2.
Co-immunoprecipitation (Co-IP) NSCLC tissues or cells extracts were incubated with protein G-Sepharose beads and pre-immune IgG.After centrifugation, the supernatant was incubated with 2 μg of relevant Ab overnight.Thereafter, the protein G-Sepharose was added and incubated for 2 h.The precipitates were washed and eluted with 2 × SDS-PAGE sample buffer, and then analyzed by immunoblotting (IB).

IB experiment
Total protein from the tissues and cells was extracted by RIPA cell lysis buffer, and the proteins (30 μg/lane) were separated by SDS-PAGE and transferred onto PVDF membrane.
Afterwards, the membrane was incubated with primary Ab overnight followed by HRP-conjugated corresponding Ab for 1 h.The chemiluminescent substrate was applied to the blots, and the signals were captured by ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).Autoradiograms were quantified by densitometry with the Quantity One software (Bio-Rad, Hercules, CA, USA).

Cytoplasm and nucleus extraction
The cytoplasmic and nuclear protein in 8 × 10 5 H1299 cells was extracted using the corresponding extraction kit according to the manufacturer's instruction.The extracted proteins were examined by IB test.
Wound healing assay NSCLC cells (1 × 10 5 /well) were incubated for 48 h, and a 100 μL pipette tip was used to scratch the monolayer and create a constant gap.The detached or dead cells were washed away with PBS, and then the medium with 50 ng/ml IL-17 was added.The movement of cells filling the gap was recorded by photographing at 0 and 24 h.The images were analyzed by ImageJ software.

Cell migration or invasion assay
For migration, cells (5 × 10 4 /well) were seeded in top chambers of the Transwell plates in the FBS-free media with membrane inserts without Matrigel coated, and the complete media were added to the wells (lower compartment), followed by incubation for 24 h.The cells that migrated to the lower membrane were fixed and stained with 0.1% crystal violet for 30 min and counted.For invasion, the upper compartment of the insert was coated with Matrigel and the cells on the lower side of the insert membrane were fixed and stained.
Mass spectrometry (MS) detection H1299 cells (8 × 10 5 ) were transfected with HA-STAT3 plasmids for 48 h plus IL-17 (50 ng/mL) for 3 h.The anti-HA immunoprecipitants were subjected to SDS-PAGE and silver staining.The protein bands were collected.MS detection was performed at Center of Hygienic Analysis and Detection of Nanjing Medical University, Nanjing, China.

Plasmid construction and cell transfection
The plasmids of pGL3-MMP19 full length (FL, −1163 to +313 nt) promoter, and the four truncated promoters (−662 to +313 nt, −312 to +313 nt, −104 to +313 nt, +62 to +313 nt) were constructed by inserting these fragments of MMP19 promoter into pGL3-basic vector respectively.The primer sequences of pGL3-MMP19-FL and the truncated promoters were listed in Table S3.For plasmid transfection, the cells were incubated with the mixture of 2 µg plasmids and 4 µl lipofectamine 2000 for 48 h, and then the mixture was washed away.
Luciferase reporter assay pGL3-MMP19-FL and four truncated MMP19 promoter plasmids were separately transfected into H1299 cells with pRL-SV40 for 48 h.Then, the cells were treated with or without 50 ng/mL IL-17 for 3 h, and MMP19 promoter activity was determined by dual-luciferase reporter kit.
ChIP, re-ChIP and chromatin immunodepletion (ChID) assay ChIP assay was carried out by using anti-preimmune IgG, STAT3 or p300 according to the protocol from the manufacturer.For re-ChIP, the primary ChIP production was immunoprecipitated again with another ChIP-grade Abs, and the immunoprecipitated DNA was amplified by RT-PCR and real-time PCR.For ChID, the second-round immunoprecipitation was done with anti-Ack Ab, and MMP19 promoter fragment immunodepleted with anti-Ack in the supernatant was measured by real-time PCR.The ChIP-PCR primers for MMP19 promoter fragment were exhibited in Suppl.Table S2.

Metastatic model experiment
Female nude BALB/c mice (4-5 weeks) were maintained under a SPF environment, and the animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-2108034).Before the inoculation of mice, the H1299 cells transfected stably with LV-shCTR, LV-shp300, LV-shSTAT3 and LV-shMMP19 plus IL-17 (50 ng/mL) stimulation were selected with puromycin (2 μg/mL).Thereafter, the H1299 cells (1 × 10 6 ) were respectively injected into the nude mouse via tail vein (100 μL/mouse, 5 mice/group).At 7 weeks, all mice were sacrificed, and the pulmonary metastatic nodules were counted and the tissue sections were observed by HE staining.Meantime, the expression of p300, p-STAT3, Ack-STAT3 and MMP19 in the lung metastatic tissues was examined by IP/IB.

Statistical analysis
Statistical analysis was performed by GraphPad Prism software (San Diego, CA, USA).Two-tailed t-test was using to compare data between two groups, and multiple group comparison was analyzed by one-way ANOVA with Dunnett's corrections.Data were shown as means ± SD from three independent experiments, and p < 0.05 was considered statistically significant.The correlations of IHC staining scores between the interest proteins were determined by computing Pearson's correlation coefficient.Chi-square test was employed to evaluate the association between the protein expression and clinicopathological parameters of NSCLC patients.
Cell migration and invasion or p300, MMP19, p-STAT3 and Ack-STAT3 expression are elevated or reduced in IL-17stimulated or IL-17RA-silenced NSCLC cells Numerous researches indicate that IL-17 up-regulation in NSCIC tissues contributes to NSCLC metastasis [10,13,21], but the mechanism of IL-17-induced NSCLC metastasis has not been fully known.To solve this problem, we first detected IL-17RA in three NSCLC cell lines and normal bronchial epithelium cell line BEAS-2B, and meanwhile, we also examined the expression levels of p300, p-STAT3, STAT3, and MMP19 in these cell lines.The results showed that mRNA and protein of IL-17RA were highly expressed in H1299 and PC9 cells (Figs. 1A and 1B), and the protein of p300, p-STAT3, STAT3, and MMP19 could be seen and the expression levels of these proteins were similar in the four cell lines (Suppl.Fig. S3A).Next, we exploited H1299 and PC9 cells with IL-17 to observe cell migration and invasion and proved that the cell behavior was markedly enhanced at 50 and 100 ng/mL IL-17 (Figs. 1C and 1D, Suppl.Figs.S3B and S3C).Because the changes of cell migration and invasion in the two cells treated with 50 or 100 ng/mL IL-17 had no statistical significance (p > 0.05), then we further detected the cell migration in H1299 and PC9 cells stimulated with 50 ng/mL IL-17 and confirmed that the cell migration was augmented at 24 h, particularly markedly in H1299 cells (Suppl.Figs.S3E and S3F).Moreover, we transfected siIL-17RA into H1299 cells to silence the IL-17RA gene (Suppl.Fig. S3D) followed by 50 ng/mL IL-17 stimulation for 24 h and found that H1299 cell migration and invasion did not enhance (Figs.1E and 1F), implying IL-17-induced these changes truly depend on IL-17-IL-17RA combination.
Because the −662 to −312 nt of the MMP19 promoter has STAT3 binding elements predicted by JASPAR, we then designed the primers that contained the two elements (−544 to −389 nt and −564 to +313 nt) to do ChIP assay.The results exhibited that IL-17-activated STAT3 could bind to the −544 to −389 nt region of the MMP19 promoter (Fig. 3D), and STAT3 gene overexpression or knockdown could up-regulate or down-regulate its binding to this site of MMP19 promoter (Figs. 3E and 3F).These findings reveal that STAT3 binding element locates within −544 to −389 nt of the MMP19 promoter.
Moreover, ChIP and re-ChIP manifested that p300 gene overexpression or knockdown also increased or decreased its binding to −544 to −389 nt of the MMP19 promoter (Figs. 3G and 3H).Meanwhile, p300 and STAT3 could form a complex binding to the same site of MMP19 promoter in a STAT3-dependent way (Fig. 3I), implying that p300 also promotes IL-17-induced MMP19 gene transcription.

IL-17 stimulation, p300 overexpression or knockdown and activity inhibition can regulate p300-STAT3 combination, STAT3 acetylation and phosphorylation
It is accepted that p300 can modify the lysine of histones and transcription factors [29][30][31], but whether p300 can acetylate STAT3 promoting MMP19 gene transcription in IL-17treated H1299 cells remains unclear.Hence, we did IP/IB tests and found a robust interaction between p300 and STAT3 in H1299 cells under IL-17 exposure.Meantime, STAT3 acetylation and its Y705-phosphorylation (i.e., p-STAT3) were also enhanced (Fig. 4A), but IL-17RA-silenced cells followed by IL-17 stimulation obtained the opposite effect (Fig. 4B).Furthermore, to investigate that p300 can acetylate STAT3 and affect its p-STAT3, we transfected pcDNA3.1/p300into H1299 cells and confirmed that the levels of p300 binding to STAT3, Ack-STAT3 and p-STAT3 were all increased (Fig. 4C).However, the same results did not appear in H1299 cells with shp300 or 10 μmol/L C646 (p300 inhibitor) plus 50 ng/mL IL-17 (Figs. 4D and 4E).Besides, we proved that the distribution of Ack-STAT3 and p-STAT3 in H1299 cells exposed to IL-17 was mainly located in the cell nucleus (Fig. 4F).These findings support that IL-17-up-regulated p300 can acetylate STAT3 and impact its phosphorylation via p300 binding to STAT3.
STAT3 acetylation site mediated by p300 is K631 in H1299 cells in response to IL-17 Since we have found that IL-17-induced STAT3 acetylation is p300-dependent (Fig. 5B), we thus continued to search which lysine (K) of STAT3 could be acetylated.The mass spectrometry (MS) detection uncovered that STAT3-K97, -K354, -K601 and -K631 were all acetylated in H1299 cells upon 50 ng/mL IL-17 (Fig. 5A).Next, to further confirm the effective site of p300-acetylated STAT3, we mutated the four lysine of STAT3 by altering K to non-acetylatable arginine (R) (Figs.5C and 5D), and then co-transfected these mutants into H1299 cells with HA-p300.The experiments showed that STAT3 acetylation level was markedly reduced in the cells con-transfected STAT3-K631R with HA-p300 (Fig. 5E), implicating that the effective site of p300-mediated STAT3 acetylation is K631.

STAT3 acetylation can up-regulate its binding to MMP19 promoter or MMP19 promoter activity, p-STAT3, MMP19 expression, cell migration and invasion
Because p300 expression can increase STAT3 binding to MMP19 promoter, we assume that p300-acetylated STAT3 can augment this binding capacity leading to cell behavior changes.To give weight to this speculation, we performed ChIP, re-ChIP and ChID assays, and found that acetylated-STAT3 binding to −544 to −389 nt of MMP19 promoter in IL-17-stimulated H1299 cells was greatly enhanced (Fig. 6A).Additionally, the levels of MMP19 promoter activity, mRNA, protein and p-STAT3 expression (Figs. 6B-6D) as well as cell migration and invasion (Figs.6E and 6F) were also increased or decreased in the cells co-transfected with STAT3 (WT), p300 or STAT3 (K631R) and p300 plasmids.These results disclose that p300-mediated STAT3-K631 acetylation can elevate its binding capability to specific sites of MMP19 promoter, MMP19 promoter activity, p-STAT3 and MMP19 expression as well as cell migration and invasion.

STAT3-Y705 phosphorylation inhibition or mutation can affect IL-17-induced STAT3 acetylation, MMP19 expression, cell migration and invasion
Our foregoing tests have demonstrated that p300-dependent STAT3 acetylation can boost its Y705-phosphorylation (p-STAT3), whether p-STAT3 can influence its acetylation (Ack-STAT3) or cell migration and invasion is worth exploring.Our data displayed that the Ack-STAT3 and p-  -7D) as well as cell migration and invasion (Figs.7E and 7F) were all down-regulated in H1299 cells pre-treated with 10 μmol/L Stattic (p-STAT3-Y705 inhibitor) [23] plus IL-17 stimulation.Moreover, we respectively transfected the plasmids of phosphorylationactivated-STAT3(Y705D) or -inactived-STAT3(Y705F) followed by IL-17, and found that despite STAT3(Y705F) did not facilitate STAT3 acetylation (Fig. 7G), other abovedescribed parameters could accordingly up-regulate or down-regulate (Figs.7H-7L), indicating that the interplay between STAT3 acetylation and its phosphorylation can synergistically promote MMP19 induction, cell migration and invasion in IL-17-exposed to H1299 cells.Lung metastatic nodules and relative protein expression are alleviated in the metastatic model of BALB/c nude mice Given that we have discovered the pro-metastasis role of p300, STAT3 and MMP19 in vitro, we wonder these protein functions in vivo.Thus, we infected H1299 cells with LV-packaging shCTR, shp300, shSTAT3 or shMMP19 plasmids and followed by 50 ng/mL IL-17 stimulation.After the corresponding gene knockdown was verified (Figs.8A and 8B), we injected intravenously these cells treated with the different LV-shRNA into BALB/c nude mice, respectively.corresponding groups were also declined (Figs. 8E and 8F).These results support that p300, STAT3 and MMP19 expression can indeed accelerate NSCLC cell metastasis.
Generally, the cell reaction stimulated by cytokine, e.g., IL-17 needs binding to its receptor on the cells [13,35].Hence, we first examined IL-17RA expression in three NSCLC cell lines and normal bronchial epithelium cell line (BEAS-2B) and proved that IL-17RA levels in H1299 and PC9 cells were more than BEAS-2B cells.Next, we observed the cell migration and invasion in H1299 and PC9 cells stimulated by IL-17 or transfected siIL-17RA plus IL-17, and demonstrated that IL-17 stimulation enhanced the cell migration and invasion, while IL-17RA gene knockdown inhibited these changes induced by IL-17, particularly in H1299 cells.Thereupon, we further used H1299 cells to measure the levels of p300, STAT3, p-STAT3, Ack-STAT3, and MMP19 expression in response to IL-17 or IL-17RA knockdown plus IL-17, and data showed that the changes of above-mentioned parameters were the similar to those of H1299 cell migration and invasion described previously.These results implicate that the cell reactions caused by IL-17 are dependent on IL-17 binding to IL-17RA in the cells.
Emerging studies have discovered that STAT3, p300 and MMP19 are overexpressed in breast cancer [36], esophageal cancer [37], colorectal cancer (CRC) [38] or NSCLC [15] and so on [16,31], promoting cancer cell growth or metastasis.Therefore, we explored the role of p300, STAT3 and MMP19 in IL-17-induced H1299 cell migration and invasion as well as their relationship, and affirmed that the overexpression of these genes could augment the cell migration and invasion, but the knockdown of these genes had contrary effects.Moreover, p300 or STAT3 overexpression increased MMP19 level, while p300 or STAT3 gene knockdown received opposite results.However, MMP19 expression up-regulation or down-regulation did not alter p300 or STAT3 level, supporting that MMP19 is a downstream effector of p300 and STAT3 in H1299 cells upon IL-17 stimulation.
It has been known that STAT3 and p300 can contribute to various cancer progression [18][19][20][21][22][39][40][41][42], and STAT3 activation (e.g., p-STAT3) can co-localize with MMP1, governing MMP1 induction in NSCLC [15].Meanwhile, activated-STAT3 also can regulate MMP2 gene transcription via binding to MMP2 promoter in esophageal squamous cell carcinoma (ESCC), leading to ESCC metastasis [40].Besides, p300 can enhance cell migration and invasion by inducing EMT in NSCLC [32] or elevating MMP production by forming EYA3-SIX5-p300 complex accelerating CRC growth and metastasis [41].Because of a limited understanding of how IL-17, STAT3 and p300 affect MMP19 gene activation, we checked the roles of IL-17, IL-17RA, STAT3 or p300 in MMP19 promoter activity.Our data manifested that IL-17 stimulation, STAT3 or p300 overexpression could elevate MMP19 promoter activity, while IL-17RA, STAT3 or p300 gene knockdown received the contrary effects in H1299 cells exposed to IL-17.Notably, this research revealed that activated-STAT3 could bind to the −544 to −389 nt of MMP19 promoter, and p300 could also bind to the same site of MMP19 promoter in a STAT3-dependent manner, indicating that STAT3 and p300 as a complex can co-localize the specific region of MMP19 promotor promoting its gene transcription.
Recently, documents have reported that p300 can acetylate histones and transcriptional factors regulating cancer progression [31,38,41,42].Growing evidence has exhibited that acetylated-STAT3 can boost its transcriptional activity via facilitating their access to the DNA template [43], and finally promote cancer metastasis [44].Since our former results showed p300 and STAT3 could bind to the same element of MMP19 promoter, whether p300 can interact with STAT3 and acetylate it resulting in the changes of aforementioned indicators requires to be identified.Toward this end, we performed assays and verified that IL-17 stimulation, p300 overexpression, or IL-17RA and p300 gene knockdown or p300 activity inhibition could up-regulate or down-regulate the interaction of p300 and STAT3 as well as the level of Ack-STAT3 and p-STAT3.Furthermore, mass spectrometry detection displayed that STAT3-K97, -K354, -K601 and -K631 were all acetylated in IL-17-treated H1299 cells, and mutated assay indicated that only STAT3-K631 was an effective site of p300-mediated STAT3 acetylation.In addition, ChIP, re-ChIP and ChID tests proved that p300acetylated STAT3-K631 not only could strengthen STAT3 binding to MMP19 promoter, but also increase p-STAT3 and MMP19 expression as well as cell migration and invasion in H1299 cells, while STAT3-K631 mutation (STAT3-K631R) did not show these changes.Protein can undergo site-specific phosphorylation, acetylation or methylation [23][24][25][26][27][45][46][47].Previous studies have confirmed that the canonical IL-6/JAK/STAT3 pathway relies on STAT3-Y705 phosphorylation catalyzed by JAKs, which can induce p-STAT3 translocating into cell nucleus and activate its target gene [23,43,48].Consistently, STAT3 acetylation belongs to another activating pattern, and Ack-STAT3 in cell nucleus also enhances target gene transcription [27,43,45].Moreover, the interaction between p-STAT3 and Ack-STAT3 can impact cancer metastasis [44,49].In this study, although we found that p300, p-STAT3 and Ack-STAT3 were increased in NSCLC tissues and in the cells stimulated with IL-17, and p300 overexpression or knockdown could elevate or reduce the levels of Ack-STAT3, p-STAT3 and MMP19 as well as the cell mobility, whether STAT3 phosphorylation can affect its acetylation and other parameters upon IL-17 has not been clear.Therefore, we treated H1299 cells with Stattic, a p-STAT3-Y705 inhibitor [23,50] or transfected with the mutants of p-STAT3(Y705F) or p-STAT3(Y705D), and displayed that p-STAT3 activity inhibition or p-STAT3 (Y705F) transfection could suppress p-STAT3, Ack-STAT3 and MMP19 expression as well as the cell migration and invasion, but p-STAT3(Y705D) transfection possessed the opposite effects, despite p-STAT3(Y705D) did not change Ack-STAT3 level.These data implicate that p300-mediated STAT3-K631 acetylation can increase its Y705phosphorylation, and STAT3 Y705-phosphorylation also elevates STAT3-K631 acetylation, improving MMP19 production and NSCLC cell migration and invasion via their cooperative interaction.Besides, our animal metastatic model demonstrated that the lung metastatic nodules and MMP19, p-STAT3 or Ack-STAT3 expression were significantly inhibited in the nude mice inoculated with H1299 cells transfected with LV-shp300 or LV-shSTAT3 or LV-shMMP19 plus IL-17 stimulation.Our findings suggest that expression of IL-17-induced p300, Ack-STAT3, p-STAT3 and MMP19 can promote NSCLC cell metastasis.
In summary, our studies found that p300, p-STAT3, Ack-STAT3 and MMP19 were all up-regulated both in NSCLC tissues and in NSCLC cells exposed to IL-17.Functionally, the overexpression or knockdown of p300, STAT3 or MMP19 gene could increase or decrease NSCLC cell migration and invasion.Mechanistically, IL-17-induced p300 and STAT3 as two upstream molecules can bind to −544 to −389 nt of MMP19 gene promoter.In the process, p300 acetylated STAT3-K631 enhanced STAT3 transcriptional activity, Ack-STAT3, p-STAT3 and MMP19 expression as well as cell migration and invasion.Importantly, STAT3-K631 acetylation by p300 and STAT3-Y705 phosphorylation could interact, synergistically promoting MMP19 gene transcription (Fig. 4G).Additionally, the lung metastatic nodules, and MMP19, Ack-STAT3, p-STAT3 expression in metastatic tissues were greatly lessened after the nude mice were inoculated with H1299 cells by silencing p300, STAT3 or MMP19 gene plus IL-17 stimulation.Collectively, these data reveal that NSCLC cell metastasis is related to IL-17/IL-17RA/p300/STAT3/MMP19 axis, which offers a novel insight for NSCLC metastatic mechanism and potential targets for NSCLC treatment.

FIGURE 3 .
FIGURE 3. Identification of STAT3 binding to MMP19 promoter, and the effect of STAT3 or p300 overexpression on the binding of STAT3 to MMP19 promoter.(A) Full-length (FL) and truncated fragments of the MMP19 promoter were shown.(B) H1299 cells were transfected with MMP19 FL and truncated promoter respectively, and the cells were stimulated with IL-17 (50 ng/mL) for 3 h, MMP19 promoter activity was evaluated by luciferase reporter assay (**p < 0.01 vs. pGL3-MMP19-FL+IL-17).(C) MMP19 promoter plasmids above-mentioned were cotransfected with pCMV/STAT3, and MMP19 promoter activity was detected (**p < 0.01 vs. pGL3-MMP19-FL+pCMV/STAT3). (D) H1299 cells were stimulated by IL-17 (50 ng/mL) for 3 h, and ChIP assay was done with STAT3 Ab, and PCR was employed to measure the corresponding region of the MMP19 promoter.(E-H) H1299 cells were treated with 50 ng/mL IL-17 or transfected by STAT3 or p300 overexpression plasmids for 48 h or interferon plasmids for 48 h plus the same dose of IL-17 for 3 h.ChIP was carried out.The −544 to −389 nt fragment of the MMP19 promoter was detected by RT-PCR (E, G) and real-time PCR (F, H). **p < 0.01 vs. DMEM; : p < 0.05 vs. pcDNA3.1;:: p < 0.01 vs. pCMV; ## p < 0.01 vs. shCTR+IL-17.(I) H1299 cells were stimulated with 50 ng/mL IL-17 for 3 h.The anti-STAT3 Ab was used for ChIP, and anti-p300 Ab was used for re-ChIP, or vice versa.The MMP19 promoter fragment described previously was examined by real-time PCR (**p < 0.01 vs. IgG).Data from triplicate experiments are expressed as means ± SD and analyzed by one-way ANOVA (B, C, F, H, I).

FIGURE 8 .
FIGURE 8. Effects of lung metastasis and relative protein expression in BALB/c nude mice inoculated with H1299 cells by silencing p300, STAT3 or MMP19 gene plus 50 ng/mL IL-17 stimulation.(A) H1299 cells were infected with LV-shCTR, LV-shp300, LV-shSTAT3 or LV-shMMP19, and the infected efficiency was assessed by GFP (scale bar: 200 μm).(B) H1299 cells infected with above-mentioned LV-shRNA were selected with 2 μg/mL puromycin and then stimulated with 50 ng/mL IL-17 for 3 h.The corresponding protein expression was examined using IB to verify the gene knockdown.(C) BALB/c nude mice (n = 5/group) were intravenously injected H1299 cells previously described, and at 7 weeks after inoculation, the mice were sacrificed and lung tissues were collected to count the number of lung metastatic nodules (**p < 0.01 vs. LV-shCTR+IL-17).(D) Morphological sections of lung metastatic nodules were observed by HE staining (scale bar: top, 200 μm or bottom, 100 μm).(E) The p300, STAT3 and MMP19 expression of the lung metastatic tissues in different groups were detected by IB (**p < 0.01 vs. LV-shCTR+IL-17).(F) STAT3 acetylation and phosphorylation levels in the corresponding groups were measured by IP/IB (**p < 0.01 vs. LV-shCTR+IL-17).Representative pictures are shown, and data from each group of mice are expressed as means ± SD and analyzed by one-way ANOVA (C, E) or t-test (F).