Background
Endoplasmic reticulum (ER) is a eukaryotic organelle majorly responsible for proper biosynthesis, folding and transportation of proteins. Correctly folded and assembled proteins will be transported to the cell membrane or released into the extracellular space through the endomembrane system [
1]. Protein folding in ER is exquisitely sensitive to internal and external adverse stresses, which lead to the interruption of normal protein folding process, accumulation of unfolded or misfolded proteins and entry into a special condition termed endoplasmic reticulum stress (ER stress) [
2‐
5]. The induction of ER stress starts from the liberation of three ER stress sensors (ATF6, PERK or IRE1α) away from GRP78 (BIP) and could separately trigger downstream signaling events [
6] by reprogramming cellular protein translation and gene expression to rescue back to normal ER homeostasis, which is termed unfolded protein response (UPR) [
7,
8]. However, prolonged UPR signaling cannot restore homeostasis and be toxic by leading to overwhelming ER stress. Under such conditions, IRE1α and ATF6α branches would be generally attenuated and PERK signaling branch dominates the UPR featured by a series of sequential events including the phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α), enhanced translation of activating transcription factor 4 (ATF4), transcriptionally upregulation of DNA damage inducible transcript 3 [DDIT3, also known as C/EBP-homologous protein (CHOP)], activation of death receptor 5 (DR5) and finally execution of apoptosis via caspase-8 involved apoptotic cascade [
6,
9]. Tumor cells inevitably face diverse harsh stresses involving hypoxia, nutritional deficiencies and drug toxicity, which would commonly lead to ER stress and the following UPR. Scientists are curious about how tumor cells overcome such endless ER stresses and survive in such harsh microenvironment. Since unmitigated UPR signaling shown by persistent eIF2α phosphorylation is harmful to tumor cells, the survival mechanism shall be tightly related to the modulation of eIF2α phosphorylation.
Currently, the regulation of eIF2α phosphorylation is mainly through two ways: one way is through eIF2α kinases including PERK, while the other way is by regulating the dephosphorylation process of eIF2α. PERK is the only kinase among the three known UPR sensors and responsible for the phosphorylation of eIF2α at Ser51, which is the crucial step to initiate the PERK signaling branch of UPR under ER stress condition [
10]. Factors that interfere with or enhance PERK kinase activity can influence eIF2α phosphorylation. For example, ER chaperone GRP78 (BIP) in tumors can associate with PERK to prevent PERK-catalyzed phosphorylation of eIF2α and avoid ER stress-induced apoptosis, thereby promoting malignant phenotype, metastasis and chemotherapy resistance of tumor cells [
11]. Endogenous miRNAs can also indirectly regulate the phosphorylation of eIF2α by targeting PERK or GRP78, affecting ER homeostasis in tumor cells. For example, miR-30d, miR-181a and miR-199a-5p targeting GRP78 are downregulated in multiple cancers including colon, prostate and bladder, leading to the upregulation of GRP78 in these cancers to bind more PERK and inhibit its kinase activity for preventing eIF2α hyperphosphorylation-triggered apoptosis [
12]. On the other hand, GADD34 transcriptionally regulated by DDIT3 under ER stress conditions could cooperate with protein phosphatase 1 (PP1) to promote eIF2α dephosphorylation [
11]. This is an important feedback regulatory mechanism against the ER burden of mRNA translation in response to the ER stress-induced constraint of protein synthesis for avoiding the prolonged phosphorylation of eIF2α to activate downstream apoptotic pathways for survival. However, under persistent ER stress condition, the continuous promotion of eIF2α dephosphorylation by GADD34 will lead to an increase in the level of protein translation load, which will result in the failure of UPR and finally lead to apoptosis instead [
11,
13]. Despite the known protein and miRNA regulators, our knowledge about the modulation of eIF2α phosphorylation is still limited.
Long non-coding RNAs (LncRNAs) are a group of noncoding RNAs with length exceeding 200 nucleotides and no protein-coding potential [
14]. LncRNAs play diverse roles in chromatin modification, post-transcriptional regulation, genomic imprinting, X chromosome inactivation and miRNA sponge modulation [
15,
16] involving pathophysiological processes including carcinogenesis, angiogenesis, muscle development or immune regulation with diverse mechanisms [
16,
17]. Recently, accumulating evidences have revealed LncRNAs are also key players during UPR signaling. FOXD3-AS1 could competitively bind to let-7e-5p to regulate RCN1. Silencing FOXD3-AS1 or upregulating let-7e-5p increased the expression profiles of GRP78, CHOP, and ATF4, consequently promoting ER stress-induced apoptosis [
18]. MEG3 increased the expression of ER stress-related proteins, including GRP78, IRE1, PERK, ATF6, and CHOP, consequently inhibiting growth and inducing the apoptosis of cancer cells. In addition, MEG3 competitively combines with miR-7-5p or miR-103a-3p to promote ER stress-mediated apoptosis [
19]. However, these reports showed that LncRNAs either indirectly modulated ER stress through targeting miRNAs or hardly presented evidence for direct regulation despite the checking of biomarkers for ER stress-induced UPR. Seeing the lack of evidence of direct regulation, we would like to ask whether LncRNAs themselves directly participate and play critical roles in UPR signaling?
Cutaneous squamous cell carcinoma (cSCC), the second most common cancer with an annual incidence exceeding one million worldwide, originates from epidermal keratinocytes [
20,
21]. Malignant cSCC is highly aggressive and can actively metastasize to the lymph nodes followed by spreading throughout the body. Patients with cSCC have a high recurrence rate and poor prognosis with a 5 year survival rate of only 22–56% and a 1 year survival rate of only about 50% for patients with recurrence and metastasis [
22]. An urgent need to explore the detailed mechanism in cSCC pathogenesis and identify a potential therapeutic target for establishing novel treatment modality of cSCC is necessary. In this study, LncRNA in non-homologous end joining pathway 1 (LINP1) was identified to be significantly upregulated in cSCC tumors and cell lines. Functional studies revealed that LINP1 functions as an oncogene to promote cell proliferation, colony formation, migration and invasiveness but inhibits cell apoptosis in cSCC cells and tumors. Transcriptomic sequencing showed LINP1 may functionally be related with UPR and regulates apoptosis. Loss of LINP1 activates PERK-eIF2α branch-mediated UPR and induces apoptosis in cSCC by upregulating UPR mediator DDIT3 and death receptor DR5. Mechanistic study identified LINP1 directly interacts with eIF2α to repress its phosphorylation for inhibiting PERK-eIF2α branch-mediated UPR signaling and the subsequent apoptosis. Our findings demonstrate that upregulated LINP1 acts as a key regulator to repress UPR-induced apoptosis signaling by inhibiting eIF2α phosphorylation in cSCC, which finally contributes to the development of cSCC.
Materials and methods
Patient samples
This study was approved by the Institutional Review Board of Shanghai Outdo Biotech Co. Ltd. (Shanghai, China), all patients provided written informed consent for the use of surgical samples. The tissue array (HSkiC100PT01) that included 57 cSCC specimens and 7 normal cutaneous specimens was purchased from Shanghai Outdo Biotech Co. Ltd. Fresh samples obtained during surgery were immediately frozen in liquid nitrogen for subsequent total RNA and protein extractions and paraffin embedding. Tumors were classified according to the SCC Broders Pathological Classification [
43]: stage I (well differentiated) with 75–100% differentiated cells, stage II (moderately differentiated) with 50–75% differentiated cells and stage III and IV (poorly differentiated) with 0–50% differentiated cells.
Animal studies
This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanfang Hospital affiliated with the Southern Medical University (Approval code L2018024). All experiments were performed in accordance with the guidelines of the Asian Federation of Laboratory Animal Science Associations (AFLAS) and the National Regulations for the Administration of Affairs Concerning Experimental Animals (8 January 2011). Details of Mouse transportation, housing, and breeding were conducted according to the recommendations of “The use of non-human animals in research.” Methods of euthanasia accord with international conventions or refer to current guidelines of the AVMA Panel on Euthanasia. During sample collections, mice were euthanized by cervical dislocation to prevent suffering.
Cell lines
cSCC lines HSC-1 (Male, HonSun Biological Co. Ltd.), A431 (Female, CellCook Biotech Co. Ltd.) and the human benign epidermal keratinocyte cell line HaCaT (Male, CellCook Biotech Co. Ltd.) were grown in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (ExCell Bio, FSP500) and maintained at 37 °C with 5% CO
2 in a humidified atmosphere. The authentication information of all the cell lines used in this study were listed in Additional file
1: Fig. S7–S9. All the cell lines have been tested and shown negative for mycoplasma contamination.
Isolation of primary human keratinocytes
Fresh foreskin tissue was placed in a container containing 1 × PBS (pH 7.4) on ice. The tissues were cleaned twice with 1 × PBS containing penicillin (500 units/mL) and streptomycin (50 μg/mL) and disinfected with 75% ethanol. Blood vessels and fatty tissue from the foreskin were removed using scissors and tweezers and the tissues were incubated with 3 mL 1 × dispase (1.2–2.4 U/mL) at 4 °C for 12–18 h. The epidermis was detached and placed into 2–3 mL of 0.05% trypsin. It was then incubated at 37 °C for 15 min in a 50 mL falcon tube. Digestion was terminated with 2 mL DMEM containing 10% FBS. After passing 200 mesh cell filter, the cells were transferred to centrifuge tube 400 g for 10 min. The supernatant was discarded and the cells were washed and precipitate with PBS for 2 times. Then, the cells were resuspended with 2 mL primary keratinocytes special medium and cultured for the following assays.
RNA isolation and qPCR
The total RNA was extracted from cells or tissues by Trizol reagent (TransGen Biotech Co., Ltd.) according to the manufacturer’s instructions. And subsequently reversely transcribed into cDNA using TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, AU341). mRNA expression analysis was performed using PerfectStart Green qPCR SuperMix (TransGen Biotech, AQ601) on a LightCycler 96 Detection System (Roche) using GAPDH for normalization. Primers used in this study are listed in Additional file
6: Table S5. 2
−ΔΔCt illustrated the fold changes in the target gene expression between the experimental group and the control group. All the qPCR experiments were repeated 3 times.
Transcriptomic sequencing analysis
Transcriptomic sequencing was performed at RiboBio Co., Ltd. using the Illumina HiSeq 2500 instrument. RNA-Seq data was aligned to the reference genome (human assembly GRCh37/hg19) using Tophat2 (
http://ccb.jhu.edu/software/tophat/index.shtml). HTSeq (
http://www-huber.embl.de/HTSeq) was then applied on the aligned data set to determine differentially expressed genes with a “significant” status. The Gene Ontology and KEGG analyses of the differentially expressed genes were performed using DAVID (
https://david.ncifcrf.gov/). For Clustering analysis of public GEO (
http://www.ncbi.nlm.nih.gov/geo, accession GSE139505), z-score transformation of the normalized expression of top 10 upregulated and downregulated genes were calculated as previous described [
39]. The output z values were used to generate a heat map.
In situ hybridization (ISH) and Fluorescence in situ hybridization (FISH)
Antisense single-stranded DNA probe (Additional file
6: Table S5) was synthesized and end-labelled with digoxigenin (DIG) (Roche). ISH or FISH was performed in formalin-fixed paraffin-embedded melanoma sections or slides covered with cultured melanoma cells. The pre-hybridization, hybridization, anti-DIG-HRP IgG fraction monoclonal (Jackson, 200-032-156) incubation (1:200) and stained with DAB (Servicebio, G1211) was performed as described in previous studies. Stained ISH or FISH sections were imaged with a ZEISS Axio Vert.A1 microscope and at least 10 representative images were collected for statistical analysis. The ISH or FISH staining was performed “blind” with respect to the different treatments [
44]. Co-localization of LINP1 with eIF2α in cSCC cells was detected using FISH for LINP1 and immunofluorescence staining for eIF2α and observed by confocal microscope.
Cell transfection
cSCC cells in exponential growth phase were used for cell transfection. Before transfection, the cells were cultured in 60 or 100 mm dishes with complete medium for 24 h until they were 90% confluent. Transient transfection of cells with siRNA oligos or DNA plasmids was performed with TransIntro EL Transfection Reagent (TransGen Biotech, FT201) as suggested by the manufacturer. Then, the cells were cultured with DMEM medium following the instructions. 36 h after transfection, cell lysates were subjected to western blot and total RNA were extracted and purified from cSCC cells using the Trizol reagent according to the manufacturer’s instructions.
HPLC–MS analysis
A 20 μg sample of immunoprecipitated protein mix was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue R250 and then processed with Trypsin Profile IGD Kit (Sigma, PP0100). The resulting digest was treated with ZipTip C18 (Merck Millipore, ZTC18S096) then subjected to analysis by Thermo Fisher Scientific orbitrap fusion LC-MS/MS in positive ion, linear, delayed-extraction mode. Calibration was carried out using a standard peptide mixture. The mass spectra were subjected to sequence database search with Proteome Discoverer v2.1 software (Thermo Scientific).
Generation of LINP1 knockout cell strains
For LINP1 knockout, the single guide RNAs (sgRNA) were designed using the online CRISPR design tool (CRISPOR,
http://crispor.tefor.net/) [
45]. A ranked list of sgRNAs was generated with specificity and efficiency scores. Two sgRNAs were selected which flank the genomic locus of LINP1. All sgRNAs were accessed using the online, off-target searching tool (Cas-OFFinder;
http://www.rgenome.net/cas-offinder). The pair of oligos was annealed and ligated to Bbs I-digested pSpCas9BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988) respectively [
46]. Such two pX459 plasmids containing each target sgRNA sequences were cotransfected into cells with Lipofectamine 2000 (Thermo Fisher Scientific). After isolation of clonal cell lines by dilution, 100 cells were seeded into each well of a 96-well plate. After the selection of single colonies, colonies with genomic knockout of LINP1 were determined by Sanger sequencing with isolated genomic DNA and LINP1 expression levels in each clone were validated by qPCR. The sgRNAs and primers for CRISPR design and genomic validation are shown in Additional file
6: Table S5.
ChIP-qPCR analysis
The chromatin immunoprecipitation (ChIP) procedure was performed using the EZ ChIP
™ Chromatin Immunoprecipitation kit manual (Merck Millipore, Cat. no. 17-371) following the manufacturer’s instructions. 5 μg antibodies against ATF4 (CST) or isotype IgG (Merck Millipore) used as a negative control were added and the complex co-precipitates are captured by Protein G magnetic beads. Genomic DNA pellets were purified using phenol chloroform extraction and ethanol precipitation, and then resuspended in 20 μl water, at which point it is ready for PCR. Relative enrichment was calculated as the amount of amplified DNA normalized to input and relative to values obtained after normal IgG immunoprecipitation, which were set as 1. Primers used are listed in Additional file
6: Table S5.
Immunoblotting and IHC assays
Total cell protein extracts were prepared and assayed by western blot as previously described [
44]. The following primary antibodies and dilutions were used: eIF2α (Santa Cruz Biotechnology, sc-133132, 1:2000), p-eIF2α (Cell Signaling Technology, #3398, 1:2000) GRP78 (Santa Cruz Biotechnology, sc-13539, 1:2000), XBP1 (Santa Cruz Biotechnology, sc-8015, 1:2000), ATF4 (Cell Signaling Technology, #11815, 1:2000), DDIT3 (Santa Cruz Biotechnology, sc-7351, 1:2000), DR5 (Santa Cruz Biotechnology, sc-166624, 1:2000), Caspase-8 (Santa Cruz Biotechnology, sc-81656, 1:2000), Caspase-3 (Santa Cruz Biotechnology, sc-56053, 1:2000), Caspase-7 (Santa Cruz Biotechnology, sc-56063, 1:2000) and GAPDH (Santa Cruz Biotechnology, sc-25778,1:5000). The following secondary antibodies were also used: anti-mouse IgG-horseradish peroxidase (HRP), anti-rabbit IgG-HRP, and anti-goat IgG-HRP (Santa Cruz Biotechnology). Bound antibodies were visualized with the Luminata Forte Western HRP substrate (Millipore).
Xenograft tumors were formalin-fixed and paraffin-embedded and sectioned for IHC staining. The following antibodies were used: GRP78 (Santa Cruz Biotechnology, sc-13539, 1:100), XBP1 (Santa Cruz Biotechnology, sc-8015, 1:100), DDIT3 (Santa Cruz Biotechnology, sc-7351, 1:100) and DR5 (Santa Cruz Biotechnology, sc-166624, 1:100). Stained sections were imaged using BX53 microscope (Olympus) to get representative images for statistical analysis.
Cell proliferation and colony forming assays
An equal number of cells (5000 per well) transfected with siRNAs were plated in 96-well plates using 5 wells for technical replicates. After 0, 24, 48, and 72 h, the cells were incubated with 10 μL CCK-8 solution in cell counting kit (TransGen Biotech, FP101) at 37 °C for 1.5 h. The incubated plate was then placed into a microplate reader in order to determine the optical density (OD) value at the wavelength of 450 nm. For the colony forming assay, transfected cells were incubated in 6-well plates with 1500 cells per well, which were maintained in DMEM. Medium was replaced 2 times. The cells were cultured for 10 days before they were washed twice with PBS, fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet. Visible colonies were photographed and counted.
Apoptosis assay
Cells were seeded on a 60 mm dish and transfected with siRNAs and cultured for 36 h. Apoptotic cells were quantitated using the TransDetect Annexin V/PI cell apoptosis detection kit (TransGen Biotech) according to the manufacturer’s instructions. Briefly, the cells were harvested and washed with phosphate buffer saline (PBS). Then, cells were resuspended in binding buffer and incubated with Annexin V/propidium iodide (PI) for 15 min at room temperature in dark before analysis using a flow cytometer.
Transwell assay
In vitro migration assay was performed using transwell chambers. 1 × 105 cells transfected with siNC or siLINP1 were seeded into the 8 μm upper chambers of 12-well plates (Merk Millipore) in serum-free DMEM. During culture at 37 °C for 48 h, the cells in the upper chambers were attracted by the DMEM medium supplemented with 10% fetal bovine serum in the lower chamber. The chambers were washed with PBS twice and fixed with 3.7% formaldehyde. Cells were permeabilized using 100% methanol at room temperature and stained with 0.1% crystal violet. After scraping the cells remained in the wells off with cotton swabs, images were captured from each membrane and the number of migratory cells was counted under a microscope.
Matrigel invasiveness assay
For the assessment of invasive ability, Cells transfected with siNC or siLINP1 were concentrated to 2 × 105 cells in cell suspension and then added to the upper chamber (Merck Millipore) coated with Matrigel for the invasion assay. Other treatments were performed as in the migration assay.
RNA-pulldown assay
Biotin-labeled RNAs were transcribed in vitro using RNA max-T7 biotin-labeled transcription kit (Ribo Biotechnology Co., Ltd.). The above RNAs were denatured at 90 °C for 2 min and then renatured with RNA structure buffer at RT for 20 min. A431 cell pellets (5 × 106).were resuspended in 1 ml RIP buffer (150 mM KCl, 25 mM Tris pH 7.4, 0.5 mM DTT, 0.5% NP40, 1 mM phenylmethyl sulfonyl fluoride and 1 × PIC) and sonicated with 10 cycles (30 s interval, 30 s sonication). After centrifugation at 13,000 rpm for 10 min, total cell lysate was mixed with 3 μg of renatured RNA respectively and incubated with rotation for 1 h at RT. Each pull-down reaction were mixed with thirty microlitres of washed streptavidin agarose beads (Life Technologies) at RT for 1 h. After washing thoroughly three times, the RNA–protein binding mixture was boiled in SDS buffer and the eluted proteins were detected by western blot or mass spectrometry.
RNA immunoprecipitation (RIP) assay
Cells were cross-linked with 1% formaldehyde and collected for lysis by radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM phenylmethyl sulfonyl fluoride, 1 × Proteinase inhibitor cocktail and 1% RNase Out). The lysate was incubated with eIF2α antibody or normal IgG control for overnight. The RNA/protein complex was recovered with protein G Dynabeads and washed with RIPA buffer several times. After reverse cross-link with proteinase K at 45 °C for 45 min, RNA was recovered with Trizol and analysed by RT–qPCR.
Xenograft mouse model
4–5 week-old female NCG mice (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt), purchased from the Guangdong Gempharmatech Biotechnology Co., Ltd., were used for establishing xenograft model. Equal number of NCG mice were assigned to siNC and siLINP1 groups respectively. 0.2 mL of above cell suspension that contained 4 × 106 cells were subcutaneously implanted into the left and right flanks of nude mice. The tumor diameters were measured and recorded every 2 days to generate tumor growth curves. The tumor volumes were determined by measuring their length (l) and width (w) and calculating the volume (V) as the formula: V = lw2/2. After tumor growth assessment, the tumors were excised and snap-frozen for RNA and protein extraction or paraffin-embedded for IHC staining.
In vitro phosphorylation assay
As previously described [
27], the phosphorylation reactions were performed in 50 µl kinase buffer (50 mM Tris–HCl PH 8.0, 1% SDS, 1 mM EDTA, 5 mM DTT, 10 mM PMSF, 1 mM NaF, 1 mM Na
3VO
4, and protease inhibitor cocktail) at 30 °C for 30 min by including 0.1 mM ATP and different combinations of 20 µg recombinant PERK protein, 50 ng recombinant or immunopurified eIF2α protein, and 1 µg LINP1 in vitro transcript. The final products were analysed by 10% SDS–PAGE.
Nuclear-cytosolic fractionation
2.5 × 107 cells were harvested by trypsinization, spun down at 500 g for 5 min and washed twice. Cell pellet was resuspended in 5 volumes of CER buffer supplemented with RNase Inhibitor and vortexed for 5 s to completely resuspend the cell pellet with 15 min incubate on ice. The cytoplasmic components (supernatant) and nuclear components (particles) were seperated after centrifugation at 4 °C 1500 g for 5 min. Subsequently, RNA extraction and qPCR was performed as described above.
Tunicamycin (TM) treatments
Tunicamycin (TM) (5 mg, BBI) treatment was performed in cSCC cells for 12 h after LINP1 knockdown or overexpression.
Statistical analysis
SPSS 21.0 (IBM SPSS Inc. Chicago, IL, USA) statistical software was used to analyze the data. Statistical tests were performed for independent-samples with an unpaired t-test or one-way ANOVA tests. All statistical tests incorporated two-tailed tests and homogeneity of variance tests, and were considered to reflect significant differences if *P < 0.05, **P < 0.01, or ***P < 0.001. Details of statistical analyses including sample numbers (n) are included in the respective figure legends.
Discussion
Tumor cells commonly grow in harsh microenvironment and face the challenges of cellular stresses including oxidative stress, metabolic stress, genotoxic stress and ER stress, etc. Properly handling such adverse situations would be beneficial to tumor cells to maintain homeostasis and enhance their survival and further progression [
34‐
36]. However, chronic exposure to stresses or unmitigated responses would compromise the beneficial effects, promote diseases and cancer and even cell death. Tumor cells have to evolve the adaptive capacities to deal with adverse environmental conditions to ensure positive-selection of the survival of stress-adapted cells and negative-selection of damaged cells [
37]. Exploring the underlying signaling mechanisms is fascinating to disclose the survival secrets and contribute to develop novel anti-tumor drugs.
Long noncoding RNAs (LncRNAs) are tissue-specifically expressed and play key regulatory roles in important physiological and pathological processes such as carcinogenesis, angiogenesis, muscle development or immune regulation [
15,
38]. LncRNAs can act as a scaffold or guide to regulate protein–protein and the following signaling events, as decoys to lower in vivo active concentration of miRNAs, and as chromatin modifiers to modulate gene expression by enhancers [
15,
38]. Intensive studies in cancer field have revealed many LncRNAs are widely expressed in tumors and act as oncogene or tumor suppressor to influence tumor progression. Regarding the number of LncRNAs in tens of thousands, their functional mechanisms are far from being fully understood.
LINP1 locates on the short arm of human chromosome 10 with 2 exons. There is almost no homolog of LINP1 in other species except for a predicted non-coding transcript with a homologous region of only 186 bp in length and 84% similarity in cynomolgus monkeys, suggesting that LINP1 and its homolog transcripts may be unique to primates. The lack of homolog in rodents limits the means to validate LINP1 function and mechanism using knockout animal model. LINP1 was first found to be highly expressed in triple-negative breast cancer (TNBC) and participates in non-homologous end joining (NHEJ) pathway by acting as a scaffold to bind the ATP-dependent DNA helicase complex Ku80-Ku70 heterodimer and catalytic subunit DNA-PKcs to the damage site to promote DNA junction repair. Knockdown of LINP1 enhances the sensitivity of TNBC to chemotherapy. In cervical cancer, LINP1 translocates from the cytoplasm to the nucleus to bind Ku80 and DNA-PKcs to promote DNA damage repair during radiotherapy. Loss of LINP1 significantly enhances ionizing radiation-induced apoptosis. In addition, LINP1 is also up-regulated in a variety of cancers, significantly affecting cancer-related processes such as cell proliferation, migration, invasion, and apoptosis and participating in disease pathogenesis. For example, LINP1 is highly expressed in patients with acute myeloid leukemia (AML), but down-regulated in patients with complete remission after treatment; LINP1 enhances HNF4a-AMPK/WNT5A signaling pathway activation and promotes AML progression, and knockdown of LINP1 significantly inhibits AML cells of glucose absorption and survival. LINP1 is highly expressed in esophageal squamous cell carcinoma tissues and cell lines. In vivo and in vitro experiments show that knockdown of LINP1 causes tumor cells to arrest in the G2/M phase, inhibits cell proliferation and in vivo xenograft tumor growth and significantly promotes cell apoptosis. The current in-depth mechanism study of LINP1 is limited to the repair of DNA damage in the NHEJ pathway induced by treatments of ionizing radiation and chemical drugs.
However, the fluorescence in situ hybridization data from two of the above reports clearly indicated LINP1 was mainly located in the cytoplasm of cells and only a small portion in the nucleus. Even in the case of partial LINP1 translocating from the cytoplasm to the nucleus under radiotherapy treatment, the main subcellular localization of LINP1 is still dominated by the cytoplasm. A number of studies have shown that LINP1 can participate in multiple important cellular processes (such as proliferation, migration, invasion, apoptosis, etc.) and play strong regulatory roles without the presence of DNA damage-inducing agents. Such phenomenon suggests that DNA damage repair may be only one of the functions performed by LINP1 in tumor cells and its critical functions in cells are far from full elucidation.
In this study, LINP1 is identified to be significantly higher-expressed in cSCC tumors compared with normal skin tissues and positively correlated with tumor staging. In addition to the confirmation of its oncogenic role in cSCC by routine oncogene verifications, our transcriptomic analysis after LINP1 depletion indicated its function may link with ER stress-induced UPR (Fig.
3D, E). KEGG clustering of proteins identified by RNA pulldown followed by HPLC-MS also indicated UPR signaling-related categories (Fig.
5A, B). Especially, the subcellular localization of LINP1 is mainly in cytoplasm with only a small portion of LINP1 present in nucleus (Fig.
3A, B). These evidences prompted us LINP1 may be tightly involved in ER stress response. Critically, LINP1 is physically associated with the initiator of UPR signaling, eIF2α, which is validated by RNA pulldown and RIP assays (Fig.
5C, D) and spatially supported by confocal microscopy observation (Fig.
5F). Interestingly, further in vitro and in vivo investigations discovered LINP1 not only interacts with eIF2α but also constrains PERK-mediated eIF2α phosphorylation to avoid overaction of UPR and the following apoptosis (Fig.
5H, J). Although previous reports also observed the cytoplasm-dominated subcellular localization pattern of LINP1, they focused on the role of nucleus-localized LINP1 and elucidated that LINP1 associates with Ku80 and DNA-PKcs and functions in NHEJ DNA repair pathway [
24,
25]. Our findings replenish the functions of LINP1 in maintaining cell homeostasis and contribute to the full understanding of the diverse roles of LncRNAs.
The regulation of eIF2α phosphorylation depends on the gambling between pro-phosphorylation process by kinases and dephosphorylation by phosphatases [
10]. miRNAs and cofactors are indirectly involved in such gambling by targeting kinases or phosphatases [
11,
12]. Here, we present a new finding that LINP1 directly attenuates the kinase-mediated phosphorylation of eIF2α and enforces the survival capacity by arming the cell with an extra shield to avoid unmitigated UPR. Such a novel regulatory hierarchy disclosed by this study enriches our understanding about the fine-tuning network in maintaining cellular homeostasis to improve the adaptation ability and survival of cells against harsh microenvironments especially for tumor cells, which may suggest new targets for drug development. This study also reminds us the unlimited potential for LncRNAs and reevaluates their importance in signal transduction and stress responses. Accompanied by the in-depth explorations of LncRNAs, it looks the current theory is not capable to fully predict the exact roles of LncRNAs in different tissues and environment. Post-translational protein modifications influence the structure, electrophilicity and even interaction network of proteins and are responsible for the functional divergence of the same protein. Specially, recent advances have primarily disclosed the involvement of LncRNAs in modulating protein modifications. PURPL physically interacts with ULK1 and differentially regulates its phosphorylation by promoting the association with mTOR and departure from AMPK to suppress autophagic cell death for maintaining the survivability of melanoma cells [
39]. LincRNAFEZF1-AS1 represses p21 expression to promote gastric cancer proliferation through LSD1-Mediated H3K4me2 demethylation [
40]. Lnc-DC interacts with STAT3 to induce STAT3 phosphorylation and avoid SHP1-mediated STAT3 dephosphorylation [
41]. In triple-negative breast cancer (TNBC), highly-expressed LINK-A actives BRK kinase to phosphorylate HIF-1α and prevent hydroxylation for enhancing p300-HIF-1α association and the resulting HIF-1α transcriptional activity [
42]. Here, we provide our finding that LINP1 physically interacts with eIF2α and regulates its phosphorylation. Our finding is in line with the above reports and supports to open a novel unclear territory, where LncRNAs may be “big” players in diversifying the regulatory levels of protein modifications and the following pathophysiologic processes.
Collectively, our study showed LINP1 is higher-expressed and acts as an oncogene in cSCC to promote the proliferation, colony formation, migration and invasiveness of tumor cells. Transcriptomic sequencing and molecular validations confirmed that loss of LINP1 activates UPR signaling and the following apoptosis by inducing PERK/eIF2α signal branch UPR mediator DDIT3 and DR5 expression. Mechanistic study showed that LINP1 interacts with eIF2α to constrain the eIF2α phosphorylation and the induction of DDIT3 for inhibiting UPR signaling-mediated apoptosis, which finally contributes to cSCC progression (Fig.
8G). Our findings highlight the oncogenic and UPR signaling-suppressive roles of LINP1 in cSCC and emphasize a novel regulatory mechanism to constrain UPR signaling and the following apoptosis, which may provide novel intervention targets for designing new therapeutic modality of cSCC.
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