MALAT1 Induces Fibroblast Activation to Promote Cancer Progression via Blunting Autophagic Flux and Instigating IL-6 Secretion in Gastric Cancer Cells

Autophagy defection contributes to inammation dysregulation, which plays an important role in gastric cancer (GC) progression. Various studies have demonstrated that long noncoding RNA could function as novel regulators of autophagy. However, the epigenetic regulatory mechanisms by which blockage of autophagy caused by long noncoding RNAs develops cancer progression remain unclear. This study aimed to investigate the role of the long noncoding RNA MALAT1 in the autophagy related inammation dysregulation of GC progression and elucidate the underlying molecular mechanisms.

In ammatory mediators within tumour microenvironment (TME) play important roles in promoting gastric cancer (GC) progression. The various cytokines within the GC TME are secreted from in ammatory cells, broblasts, and GC cells [1]. Moreover, GC cells could receive extracellular signals, which could further modulate TME via paracrine secretion of cytokine. The cross talk between GC cells and stroma cells facilitate cancer progression. Cancer-associated broblasts (CAFs), a major component of the tumour stroma, are a critical source of various molecules secreted in TME, which stimulate cancer cells progression. Similarly, the uctuation of in ammatory mediators (growth factors, interleukin) by cancer cells in TME also altered resident broblast phenotypes and lead to normal broblast (NF) activation, considered as the main CAF source [2,3]. Increasing evidence demonstrated interleukin-6 (IL- 6) was abundant in GC TME, facilitating GC progression [4]. Most studies have reported that IL-6 could be released from CAFs and promote GC cells proliferation or metastasis in a paracrine way. However, IL-6 secretion from GC and effect on modulating TME has not been studied in detail. Here, we have found that autophagy inhibition in GC could upregulate IL-6 expression and secretion.
Autophagy is an important biological process that appears to be a double-edged sword with respect to cytokine signalling and modulating tumour progression in certain instances [5]. Activated autophagy could protect cells from in ammatory damage by inhibiting autophagy and aggravating in ammatory responses in many tissues [5,6]. It is widely accepted that autophagy defects contribute to in ammation and autophagy inhibition under the condition of chronic in ammation devoted to oncogenesis [7,8].
Several reports have suggested that long noncoding RNA (lncRNA) could function as novel autophagy regulators. Silencing lncRNA-FA2H-2 facilitates impairment of oxidized low-density lipoprotein-induced autophagy ux to activate in ammation for increased IL-6 and other cytokine production [9]. Defective autophagy increases in ammatory mediator (such as TNF-α and HGF) production to promote hepatocellular carcinoma [7]. Impaired autophagy could promote chemoresistance in gastric cancer via lncRNA ARHGAP5-AS1 accumulation [10]. Although recent studies have shed light on some autophagy impairment mechanisms in GC, the molecular components that mediate the process are yet to be fully identi ed.
LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to activate autophagy in pancreatic ductal adenocarcinoma [11], retinoblastoma [12], and multiple myeloma [13] to promote tumour progression. However, in this study we found that MALAT1 upregulation in GC could inhibit autophagic ux, which led to Sequestosome1(SQSTM1) protein accumulation and IL-6 overexpression. SQSTM1 is a scaffold and stress-inducible protein with multiple domains (such as ZZ, LIR, and PBI), which not only acts as an indicator of autophagy ux but also mediates in ammation response [14]. Hence, SQSTM1 protein accumulation might be responsible for of IL-6 overexpression in GC cells. Majority of studies have revealed the effect of CAFs on GC cell growth or metastasis within the interaction between CAFs and cancer cells. However, the in uence on CAFs exerted by GC cells has not been studied in detail. Autophagy inhibition in cancer cells led to expansion and release of cytokines. The dysregulated cytokines could activate the transition from NFs to CAFs via paracrine signalling [15]. Here, we found that impairment of autophagy caused by increased MALAT1 could activate NF to CAF conversion through expansion and secretion of IL-6. These data suggest a critical role for MALAT1 in interaction between CAFs and GCs cells. Furthermore, the underlying mechanisms were investigated to identify potential therapeutic strategies targeting GC.

Materials And Methods
Cell lines. Human GC cell lines MKN-45, MGC-803, and GES-1 were purchased from the Shanghai Institute for Biological Sciences of Chinese Academy of Sciences. GC cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL) at 37℃ in 5% CO 2 in air at saturation humidity.
Isolation and Culture of broblasts. CAFs and adjacent NFs were isolated from freshly resected tissues from GC patients at the Department of Surgery, Ruijin hospital a liated with Shanghai JiaoTong University, School of Medicine. The tissues were well cultured in Dulbecco's modi ed Eagle's medium (DMEM) with 10% FBS, 100 U/mL penicillin and 100 ug/mL streptomycin. A homogeneous group of broblasts were developed after two weeks of culture, which were cultured >10 times so that the minimum number of clones could be selected. Identi cation test for CAFs and NFs were performed as described previously ( Supplementary Fig.4A B).
RNA interference and Plasmids. Small interfering RNAs (siRNAs) that speci cally target human MALAT1 and SQSTM1 were purchased from Ribobio Technology (Guangzhou, China) and GenePharma Western blot. Cells were lysed in RIPA buffer containing complete protease and phosphatase inhibitor cocktail (Sigma, USA). The protein concentration of the cell lysates was quanti ed by a BCA Protein Assay Kit (Pierce, Rockford). The same amount of protein samples was resolved onto 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk at 37 °C for 2 h, the membranes were incubated with the primary antibodies diluted in TBST buffer overnight at 4 °C , followed by incubation with the HRP-conjugated secondary antibody for 2 h at room temperature. GAPDH antibody was used to verify equal protein loading. The protein band images were captured and analysed by a Tanon detection system with ECL reagent (Thermo) and the antigen-antibody reaction was visualized by enhanced chemiluminescence (ECL, Thermo, USA). The antibodies used in this study were obtained from Cell Signaling Technology.
Transfection of mRFP-GFP-LC3 lentivirus vector. The mRFP-GFP-LC3 lentivirus vector was purchased from Genechem (Shanghai, China), which was transfected to GC cells according to the manual. Puromycin (1 μg/ml) was used to select stably expressing mRFP-GFP-LC3 cells. GC cells treated with different plasmids were xed and analysed using uorescence microscopy.
Transmission electron microscopy (TEM). GC cells were xed in 2% glutaraldehyde containing 0.1 mol/l phosphate-buffered saline at 4 °C for 2 h, incubated in 1% osmium tetroxide containing 0.1 mol/l phosphate-buffered saline for 1.5 h at 4 °C, dehydrated in graded ethanol, saturated in graded ethanol, embedded, cut into ultrathin sections, stained with lead citrate, and nally viewed using Philip CM-120 TEM (Philips, Netherlands).
RNA stability assay. Transcription inhibitor Actinomycin D (Sigma-Aldrich, USA) was added to culture medium of GC cells transfected with different plasmids for 0, 2, 4, and 6 h. Individual total RNA was harvested for qRT-PCR analysis. The relative mRNA decay rate was measured and t into an exponential curve.
RNA immunoprecipitation-quantitative PCR (RIP-PCR). RIP assays were performed by using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer's instructions. Brie y, cells were lysed in lysis buffer and the cleared lysates were immunoprecipitated with the indicated anti-ELAVL1 and anti-IgG antibodies (Cell Signaling Technology). Immunoprecipitated and input RNA were isolated and reverse transcribed for qRT-PCR ampli cations with PTEN 3′-UTR-speci c primers. mRNA relative expression level was normalized to input mRNA expression. The primers used for ampli cation are listed in Supplementary Table1. Enzyme-linked Immunosorbent Assay (ELISA). The human angiogenesis array (Raybiotech, USA) was used to analyse the soluble mediators according to the manufacturer's protocol. Human IL-6 ELISA kit (Raybiotech) was used to determine the concentration of human IL-6 in medium of different treatment according to the manufacturer's instructions.
Immuno uorescence (IF)/Immunohistochemistry (IHC). For IF assay, GC cells were xed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100, and blocked with 5% BSA for 2 h before incubation with primary antibodies including anti-ELAV1, anti-DAPI, anti-NF-KB, anti-FAP (Cell Signaling Technology), and anti-α-SMA (Abcam, USA) overnight at 4 °C. After incubation with uorescent secondary antibody for 2 h, images were acquired by uorescence microscope. Immunohistochemical analysis was conducted as described previously.
Collagen contraction assays. A total of 1×10 5 NFs were suspended in 100 μl DMEM, which was mixed with 100 μl of collagen mix containing 68.75 μl DMEM and 31.25 μl Type 1 Rat Tail Collagen (Solarbio, China), and added to one well of a 96-well plate at 37 °C for 30 min. After incubation with media derived from different treatments, the gels were photographed and the contractions were evaluated.
Cell-proliferation /EdU assay. Cell viability was measured using Cell Counting Kit-8 (Dojindo, Japan). The cell proliferation and colony-formation assays were performed as described previously. EdU assay was performed using Cell-Light EdU Apollo 567 In Vitro Imaging Kit (Ribobio, Guangzhou, China) according to the manufacturer's instructions.
Xenograft assay. All the experiments were performed in accordance with the o cial recommendations of the Chinese animal community. Four-week-old male BALB/C nude mice were purchased from the Institute of Zoology, Chinese Academy of Sciences of Shanghai. NFs and MGC-803/MALAT1 or MGC-803/NC cells mixed at the ratio of 1:4 in 100 lL PBS were injected subcutaneously. During the experiment, the tumour volume was measured weekly using the formula V = (length × width 2 )/2.
Statistical methods. Student's t-test or one-way ANOVA were used for statistical analysis when appropriate. All statistical analyses were performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). A twotailed value of P<0.05 was considered statistically signi cant.

MALAT1 blocks autophagic ux in GC cells
Anomalous autophagy activity led to variation of in ammation process [16]. The effect of aberrant MALAT1 expression on autophagic ux was investigated. We found that MALAT1 overexpression LC3-II cloud accumulation results from autophagy activation or reduced turnover from autophagosome to autolysosomes. Moreover, accumulation of SQSTM1 was an indicator of autophagy impairment. Therefore, autophagy inhibitors, 3-methyladenine (3-MA) and ba lomycin A1 (BafA1), were used to treat cells to block autophagy initiation and maturation, respectively. MALAT1 effect on LC3-II and SQSTM1 accumulation was not compromised by 3-MA treatment ( Figure 1B, P<0.05). In contrast, LC3-II and SQSTM1 accumulation was not affected by BafA1 treatment in MALAT1 overexpression group compared to negative group ( Figure 1C, P<0.05). Subsequently, mRFP-GFP-LC3 lentivirus vector was introduced to determine MALAT1 effect on autophagy ux. When autolysosomes formed, green uorescence faded, leaving only RFP signal as RFP signal is more stable than green uorescence in acidic conditions. MALAT1 overexpression in MKN-45 and MGC-803 led to yellow puncta enrichment rather than red ones, indicating autolysosome maturation blockage ( Figure 1D, E, P<0.05). Additionally, we used TEM to evaluate autophagosomes and found out that the number of autophagic vesicles increased in MKN-45/MALAT1 and MGC-803/MALAT1 cells compared to that in control cells ( Figure 1F, G). Taken together, these results suggested that increased MATLA1 in GC cells could impair autophagy ux. MALAT1 activates AKT/mTOR pathway to inhibit autophagy in GC cells GESA dataset analysis was performed to suggest that autophagy was negatively association with MALAT1 expression (Figure2A, NES=-1.459, FDR q-value=0.06). Activation of mTOR is crucial to inhibit autophagy ux, which led to substantial autophagosome-lysosome fusion inhibition and lysosome dysfunction so that autophagy degradation was impaired [17,18]. Hence, both phosphorylated-mTOR (p-mTOR) and its key substrate, phosphorylated-p70 S6 kinase (p-p70S6K), were detected while assessing MALAT1 effect on mTOR pathway activation. As shown in Figure 3B, a signi cant p-mTOR and p-p70S6K level increase was observed in MKN-45 and MGC-803 cells transfected with MALAT1 overexpression vectors. Silencing MALAT1 resulted in p-mTOR and p-p70S6K level reduction, which indicated that MALAT1 could activate mTOR pathway (Figure2B, P<0.05). Autophagy ux impaired by MALAT1 in GC led to SQSTM1 accumulation and rapamycin was used to inhibit mTOR activation to better understand whether MALAT1 impaired autophagy degradation to elevate SQSTM1 accumulation via mTOR pathway.  Fig.2A). Based on the current evidence on the opposite effects of MALAT1 and ELAVL1 on PTEN mRNA expression and the reported correlation between MALAT1 and ELAVL1, we assumed that increased MALAT1 could competitively interact with ELAVL1 to expose PTEN 3′-UTR such that PTEN mRNA destabilization was augmented. To determine the above assumption better, rescue assay was carried out. As shown in Figure  3F, These results showed that ELAVL1 could bind more MALAT1 mRNA fractions than PTEN 3′-UTR enrichments under MALAT1 overexpression condition. Additionally, we found that increased MALAT1 led to more ELAVL1 protein being distributed within the nucleus where MALAT1 was located ( Figure 3I), indicating that ELAVL1 nucleocytoplasmic shuttling was abrogated and resulted in PTEN mRNA destabilization. The combined data implied that MALAT1 could competitively interact with ELAVL1 to destabilize PTEN mRNA.

Inhibition of autophagy promotes IL-6 secretion via accumulation of SQSTM1
Increasing evidence showed that autophagy ux inhibition aggravates the in ammatory response [20].  Figure 4E, F, P<0.05). In addition, increased MALAT1 had no effect on SQSTM1 mRNA expression, which had been shown in rst part of results. As mentioned above, increased MALAT1 impaired autophagic ux, resulting in elevated SQSTM1 accumulation within GC cells, with SQSTM1 being involved in both autophagy and in ammation response. Therefore, we assumed that MALAT1 might activate NF-κB pathway to increase IL-6 expression via SQSTM1. SQSTM1 siRNA-treated MKN-45/MALAT1 and MGC-803/MALAT1 cells abrogated the enhanced phosphorylated-NF-κB and IL-6 expressions. Similarly, increased SQSTM1 via transfection with SQSTM1 plasmid could reverse NF-κB/IL-6 inactivation pathway caused by MALAT1 siRNAs (Figure 4G, P<0.05). Taken together, increased MALAT1 could elevate SQSTM1 accumulation to activate NF-κB so that IL-6 expression could be increased.

Impairment of autophagy in GC cells facilitates transition from NFs to CAFs
Existing evidence have revealed that in ammatory cytokines in TME can induce conversion of NFs to CAFs. Thus, autophagic ux impairment-induced in ammatory cytokine effect on NF activation was investigated. We found that the CM collected from MKN-45/MALAT1 and MGC-803/MALAT1 cells markedly induced NFs to acquire myo broblast phenotype characterized by a-SMA and FAP expression ( Figure 5A-C). Furthermore, the broblast contraction abilities were markedly enhanced after treatment with CM derived from GC cells with increased MALAT1 (Figure 5D To demonstrate the function of activated broblast converted from NFs (Activated-NFs), NFs treated with CM derived from GC cells with increased MALAT1 were prepared to perform proliferation assay and EdU dye assay. Then, MKN-45 and MGC-803 cells were incubated by CM collected from broblasts pre-cocultured with MKN-45/MALAT1 and MGC-803/MALAT1 cells, respectively. As shown in Figure 6A-C, GC cells exhibited proliferation and colony formation enhancement after treatment ( Figure 6A-C, P<0.05). To further determine whether IL-6 from activated-NFs play crucial role in promoting GC cells proliferation, IL-6 blocking antibody was used to treat Activated-NF/MKN-45 and Activated-NF/MGC-803 group. Then we found that impairment of IL-6 with blocking antibody could signi cantly attenuate GC cells proliferation ( Figure 6D, P<0.05). Additionally, we further investigated whether activated-NFs could promote tumour growth in vivo. Co-injection of activated-NFs or NFs cells with MGC-803 was performed in nude mice. MGC-803 treated activated-NFs cells generated tumours with larger volume and weight than those generated by MGC-803 treated with NFs ( Figure 6E, F, P<0.05). Furthermore, immunohistochemistry staining results showed that the FAP and a-SMA (CAF activation markers), SQSTM1 (autophagy marker) and IL-6 expressions were highly increased in MGC-803/activated-NF group ( Figure 6G, P<0.01), which is consistent with in vitro experiment results.

IL-6 derived from CAFs promote MALAT1 expression in GC cells
The Cancer Genome Atlas (TCGA) data demonstrated that MALAT1 was highly expressed in GC ( Supplementary Fig.3A), and survival curve analysis with GEO dataset showed MALAT1 expression was negatively correlated with post-progression survival time of GC patients (Supplementary Fig.3B).
Increasing evidence showed that MALAT1 were aberrantly overexpressed and could act as oncogene in GC [21]. The crosstalk between CAFs and GC cells could aggravate dysregulation of gene expression [22,23]. However the mechanism of upregulation of MALAT1 within TME was rarely reported. Therefore, coculture CAFs or NFs with GC cells were performed to determine whether CAFs could upregulate MALAT1 expression in GC cells via paracrine signaling. As shown in gure1A, relative expression of MALAT1 was signi cantly higher in MKN-45 or MGC-803 co-cultured with CAFs group than that co-cultured with NFs group (Figure7A P<0.05), which means cytokine from CAFs might induce MALAT1 expression in GC. GESA dataset analysis was performed to suggest that IL-6/STAT3 pathway signaling was positive association with MALAT1 expression (Figure 7B Taken together, aberrant MALAT1 expression was partly attributed to IL-6 derived from CAFs via activation of STAT3 pathway within GC TME. Additionally, we also found that overexpression of IL-6 was detected in MKN-45 and MGC-803 cells treated with rIL-6 ( Supplementary Fig.4E).

Discussion
In the present study, we showed, for the rst time, that increased MALAT1 in GC cells could impair autophagic ux to aggravate IL-6 secretion to activate NF to CAF conversion via paracrine signalling, which resulted in GC cell progression. Increased MALAT1 could destabilize PTEN mRNA to activate AKT/mTOR pathway for blocking autophagic ux, leading to IL-6 overexpression induced by SQSTM1/NF-κB pathway, and the secreted IL-6 from GC cells stimulate NF to CAF conversion ( Figure 8).
The interaction between GC and stromal cells could cause a positive feedback to foster an in ammatory microenvironment and promote GC progression.
It has been widely reported that increased MALAT1 can promote cancer cell proliferation or migration. Previously we had found MALAT1 could function as oncogene to promote proliferation of GC cells [24]. However, MALAT1 effect on the interaction between stromal and cancer cells have been rarely studied. Autophagy is a biological process involved with interaction between different types of cells through production of in ammatory mediators [6,20], which can regulate complex multicellular interactions within TME. Several studies have reported that MALAT1 could promote autophagy in various cancers, including retinoblastoma [13], lung cancer [25], and pancreatic cancer [11]. However, autophagy inhibition caused by increased MALAT1 has been rarely reported and investigated. For autophagy regulation, it is widely accepted that the mammalian target of rapamycin complex 1 (mTORC1) from the autophagy-inhibiting PI3k-Akt pathway [26] and increased MALAT1 could activate PI3K-AKT pathway in numerous cancers including GC [27]. In this study, we demonstrated that increased MALAT1 could inhibit autophagy ux through activating AKT/mTOR pathway. Not only LC3-I to LC3-II protein conversion was increased along with MALAT1 augmentation in GC cells, SQSTM1 protein accumulation was also detected, suggesting LC3 protein conversion resulted from autophagy impairment rather than autophagy induction. Additionally, rescue assays were performed to further con rm that increased MALAT1 could inhibited autophagy ux with 3-MA and BafA1 treatment. Besides AKT/mTOR pathway being activated by increased MALAT1, expression of PTEN, the negative regulator of AKT/mTOR signalling, was also changed. Our study found that increased MALAT1 could destabilize PTEN mRNA to shorten its half-life in GC. AREs were rich in the PTEN 3′-UTR, to which RBP could bind to modulate mRNA stability. ELAVL1 is a ubiquitously expressed RBP that regulates many post-transcriptional steps including mRNA stability and translation. ELAVL1 has been reported to stabilize COX-2, β-catenin and BECN1 mRNA via binding to target AREs of 3′-UTR [28,29]. ELAVL1 not only could bind to 3′-UTR, but also interact with lncRNA to form a functional complex. ELAVL1/MALAT1 complex was found to repress CD133 expression and suppress epithelial-mesenchymal transition in breast cancer [19]. However, whether ELAVL1 could bind to PTEN 3′-UTR regulating mRNA stability had not been reported, and whether MALAT1 could modulate PTEN mRNA expression via competitive interfering with interaction between ELAVL1 and 3′-UTR was not investigated.
In the present study, we found that MALAT1 could interact with ELAVL1 directly and restrain ELAVL1 in the nucleus away from cytoplasm, where it could stabilize PTEN mRNA, as shown by RIP and IF assays. Based on collected evidence, we con rmed that increased MALAT1 could impair autophagy ux in GC via stimulating PTEN/AKT/mTOR signalling pathway. As the consequence of autophagy impairment caused by MALAT1, SQSTM1 accumulation was increased. SQSTM1 has been reported to be a signi cant activate factor in in ammatory responses [30,31]through many signalling pathways including stimulating the NF-κB activation [32,33]. Therefore, we observed whether SQSTM1/NF-κB activation was responsible for IL-6 upregulation induced by increased MALAT1 in GC. From results of rescue assays, we clearly found that SQSTM1 knockdown could reverse NF-κB activation and IL-6 upregulation caused by MALAT1, and restored SQSTM1 could reverse the NF-κB/IL-6 inhibition induced by silencing MALAT1 in GC cells.
CAFs secret in ammatory mediators to modulate components in TME and changes in TME can also regulate CAF function. We have shown previously that miR-149 can inhibit CAF activation via targeting IL-6 expression, which indicated that IL-6 has an important role in CAF activation process [34]. In this study, we found that increased MALAT1 in GC cells results in IL-6 expression and secretion, and IL-6 augmentation activates NF to CAF conversion. The IL-6 effect on activating NFs was found in GC. IL-6 could also mediate interaction between cancer cells and CAFs not only by supporting tumour cell growth, but also by promoting broblast activation in oesophageal cancer [35]. Although IL-6 could stimulate NF to CAF conversion, the underlying molecular mechanisms was rarely known. Most studies attributed that IL-6 mediate microRNA-dependent pathway to CAF activation [36][37][38], which could not fully describe the underlying mechanisms. The mechanism of cytokines, like IL-6, on stimulating CAF activation should be further investigated. Chronic in ammation leads to NF activation and their conversion into CAFs, producing pro-tumorigenic cytokines, interacting with the cancer cells, and altering their gene expression pro le, which result in cancer progression. In this study, activated CAFs induced by IL-6 could express α-SMA, acquire highly contractile phenotype, and functionally, activated CAFs could facilitate GC cell proliferation, which resulted in co-evolution of CAFs with cancer cells. Additionally, MALAT1 has been reported to be aberrantly overexpressed in GC samples; however, the mechanism of upregulation of MALAT1 within TME was rarely reported. The interaction between CAFs and GC cells could aggravate dysregulation of gene expression. We found that CAFs could upregulate MALAT1 expression in GC cells via paracrine signalling. Moreover, IL-6 derived from CAFs might be responsible for high expression of MALAT1 in GC via promoting STAT3 binding to MALAT1 promoter. In this way, the positive feedback loop contributed to a positive feedback to foster an in ammatory microenvironment and promote GC progression.

Conclusion
Our results indicate that MALAT1 could inhibit autophagic ux and instigate IL-6 via regulating PTEN/AKT/mTOR and SQSTM1/NF-κB pathways, which convert broblasts to CAFs to promote GC progression Figure8 . However, the mechanism for CAF activation induced by IL-6 needs to be further investigated. Our study illustrated a new molecular mechanism underlying the interaction between cancer cells and broblasts, which may contribute to provide novel prevention and therapeutic strategies for GC.