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www.nature.com/cddiscovery ARTICLE OPEN The long non-coding RNA PVT1 promotes tumorigenesis of cutaneous squamous cell carcinoma via interaction with 4EBP1 ✉ ✉ ✉ 1 1 1 1 1 1 1 Rong Li , Dan Huang , Mei Ju , Hong-ying Chen , Chao Luan , Jia-an Zhang and Kun Chen © The Author(s) 2023 The long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) plays an oncogenic role in multiple cancers due to its high expression. However, the expression and associated regulatory mechanisms of PVT1 in cutaneous squamous cell carcinoma (cSCC) remain unclear. Our results revealed that PVT1 was highly upregulated in cSCC tissues and cSCC cell lines. To determine the functional role of PVT1 in cSCC, we constructed a stable knockdown cell model of PVT1 in the A431 and COLO16 cell lines using a lentiviral approach. Xenograft tumor experiments of nude mice in vivo, and colony formation, CCK-8, and EdU assays in vitro demonstrated that knockdown of PVT1 could widely suppress cell proliferation in vivo and in vitro. In addition, PVT1 knockdown induced cell cycle arrest and promoted apoptosis, as detected by ﬂow cytometry analysis. Wound healing and transwell assays revealed that PVT1 knockdown signiﬁcantly inhibited the migration and invasion of CSCC cell lines. To gain insight into the tumorigenic mechanism and explore the potential target molecules of PVT1, we employed label-free quantitative proteomic analysis. The GO, KEGG enrichment, and protein–protein interaction (PPI) networks suggested that 4E-binding protein 1 (4EBP1) is the possible downstream target effector of PVT1, which was validated by western blot analysis. PVT1 silencing markedly decreased 4EBP1 protein expression levels and directly bound 4EBP1 in the cytoplasm of cSCC cells. 4EBP1 overexpression counteracted the effects of PVT1 knockdown on tumorigenesis in cSCC cells, including cell proliferation, apoptosis, migration, and invasion. Our ﬁndings provide strong evidence that PVT1 is an oncogene which plays a role in tumorigenesis of cSCC, that PVT1 may interact with 4EBP1 in the cytoplasm as an underlying mechanism in cSCC carcinogenesis, and that PVT1 combined with 4EBP1 may serve as a potential new therapeutic target for cSCC. Cell Death Discovery (2023) 9:101 ; https://doi.org/10.1038/s41420-023-01380-7 INTRODUCTION play a primary functional role in diverse biological processes . Cutaneous squamous cell carcinoma (cSCC) is the second most Recently, a number of lncRNA molecules have been identiﬁed to be common non-melanoma skin cancer (NMSC), deﬁned as a frequently involved in the pathophysiological processes of various malignant proliferation of the skin epithelium accounting for 20 to diseases or cancers through lncRNA–DNA, lncRNA–RNA, and 50% of all skin cancers . Although most cSCCs can be successfully lncRNA–protein interactions [7, 8]. lncRNA plasmacytoma variant eradicated by surgical excision, a proportion are more likely to recur translocation 1 (PVT1), encoded by the human PVT1 gene, is and metastasize, resulting in a lower survival rate with some special abnormally expressed in many tumors, and aberrantly altered clinicopathological characteristics. Although the lifetime incidence of expression of PVT1 has been closely associated with tumorigenesis cSCC is ~7–11%, which is lower than that of basal cell carcinoma in a variety of tumors, such as pancreatic, pulmonary, hepatocellular, (BCC) (28–33%), the incidence rates of cSCC have been growing cervical, and gastric cancer [9–13]. There is increasing evidence to proportionally in recent years, and cSCC patients have signiﬁcant suggest that PVT1 expression is highly associated with poor long-term morbidity, mortality, and economic burden [2–4]. Until prognosis in various tumors . PVT1 induces apoptosis and now, almost 4% of patients with cSCC developed local recurrence or radioresistance through DNA damage repair-related pathways in metastasis after complete resection of the primary tumor, which is poorly differentiated nasopharyngeal squamous cell carcinoma . signiﬁcantly higher than the rates for other NMSCs. Thus, cSCC Furthermore,PVT1servesasan oncogenethatpromotescell remains a major health challenge, with various well-established risk proliferation in cervical squamous cell carcinoma by inhibiting factors. A deeper understanding of the pathogenesis of cSCC is transforming growth factor-β . However, despite its known roles essential for more effective diagnosis and treatment of patients. in other squamous cell carcinomas, the function and molecular Further insights into the molecular mechanisms of tumorigenesis in mechanism of PVT1 in cSCC remain unknown. Our previous study cSCC have created new opportunities for preventive and therapeutic used microarrays to identify dysregulated cSCC-speciﬁc lncRNAs and interventions . found that lncRNA PVT1, a top overexpressed lncRNA detected by Long non-coding RNAs (lncRNAs), a class of largely non-protein- the p29508 probe in microarray analysis, was potentially implicated coding RNAs 200 to 100,000 nucleotides in length, are thought to in carcinogenesis and progression of cSCC . Department of Physiotherapy, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, 210042 Nanjing, China. email: email@example.com; firstname.lastname@example.org; email@example.com Received: 29 October 2022 Revised: 21 February 2023 Accepted: 23 February 2023 Ofﬁcial journal of CDDpress 1234567890();,: R. Li et al. Fig. 1 LncRNA PVT1 is upregulated in human cSCC tissues and cells. A The expression level of lncRNA PVT1 is signiﬁcantly elevated in clinical cSCC tissues and para-tumor tissues (n = 20). B LncRNA PVT1 expression is highly upregulated in the three human cSCC cell lines A431, COLO16, and SCL-1. C Representative images of lncRNA PVT1 expression were taken by RNA-FISH in the A431 and COLO16 cell lines (×400 magniﬁcation, scale bar = 25 µm). D Quantiﬁcation of PVT1 distribution percentage was measured by nucleocytoplasmic fractionation, demonstrating that the lncRNA PVT1 was mainly distributed in the cytoplasm of A431 and COLO16 cell lines. T tumor, P paratumour. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The 4E-binding protein 1 (4EBP1), belonging to a family of studies have reported that MYC, androgen receptor, ATF4, and eukaryotic initiation factor 4E (eIF4E)-binding proteins, acts as a HIF-1α can bind to and modulate the activity of the 4EBP1 tumor suppressor by modulation the mammalian target of promoter to mediate 4EBP1 expression [26–28]. However, the rapamycin (mTOR) pathway in some cancers, and may have precise function and molecular mechanism of 4EBP1 remain additional oncogenic roles under other circumstances [17–19]. largely unclear in cSCC. However, the exact role of 4EBP1 in regulating carcinogenesis in In this study, we aimed to investigate the role of PVT1 in the cancers is disputed [17–21]. As a mechanistic target of the mTOR oncogenic progression of CSCC and identiﬁed 4EBP1, which is the pathway, high phosphorylation of 4EBP1 leads to active 5’cap- underlying downstream target of PVT1, using advanced label-free dependent mRNA translation by binding to eIF4E . 4EBP1 quantitative proteomics strategies. Identiﬁcation of the underlying exerts a tumor-suppressive function in prostate cancer, as tumorigenic mechanisms of PVT1 via binding to 4EBP1 could help silencing 4EBP1 promotes cell proliferation and accelerates the elucidate its critical role in cSCC carcinogenesis and have phases of progression . In contrast, 4EBP1 is indispensable for important implications for therapeutically targeting cancer. the regulation of angiogenesis and tumor growth under some cancer conditions [19–22], highlighting distinct roles in different tumors, which tend to play a dual role as an oncogenic function or RESULTS tumor suppressor, respectively. The clinical signiﬁcance of 4EBP1 PVT1 expression is elevated in cutaneous squamous cell expression also depends on tumor type, which echoes the carcinoma ﬁndings of the aforementioned reports. Many clinical studies To detect the functional role of lncRNA PVT1 in human cSCC, we have shown that high 4EBP1 expression is closely related to tumor ﬁrst examined PVT1 expression in 20 clinical specimens of cSCC metastasis and proliferation in many cancers, including breast patients. As shown in Fig. 1A, PVT1 was found to be both more cancer, ovarian cancer, and liver cancer [23–25]. In addition, high highly expressed in clinical cSCC tissues than in distant normal expression of 4EBP1 in liver and breast tumors is closely correlated tissues. To support these ﬁndings, we assessed the lncRNA with poor prognosis and low survival rates [23, 25]. Thus, many expression level of PVT1 by RT-qPCR in three typical human cSCC Cell Death Discovery (2023) 9:101 R. Li et al. cell lines A431, COLO16, and SCL-1, and normal HaCaT cells. slower than that of the control with sh-NC group (Fig. 4A–C). Compared to HaCaT cells, the expression level of PVT1 was highly Regarding average tumor weight, sh-NC tumors were 0.42 ± 0.07 g, upregulated in the three human cSCC cell lines (Fig. 1B). The whereas sh-PVT1#3 tumors were 0.142 ± 0.06 g (Fig. 4D). PVT1 increase in the expression of PVT1 was more prominent in the two expression levels in xenograft tumor tissues were assessed by RT- cell lines A431 and COLO16, which were subsequently selected for qPCR, and were found to be decreased in the sh-PVT1#3 group (Fig. experiments to clarify the biological function of PVT1. To 4E). HE staining of the tumor sections conﬁrmed the characteristics of characterize PVT1 localization in vitro, we performed RNA-FISH cSCC (Fig. 4F). Furthermore, we used immunohistochemical staining and nucleocytoplasmic fractionation in the A431 and COLO16 cell to detect cell proliferation and apoptosis using Ki-67 and TUNEL lines (Fig. 1C, D). The results showed that the lncRNA PVT1 was staining in mouse tumor samples (Fig. 4F). predominantly located in the cytoplasm. These ﬁndings suggest Therefore, PVT1 knockdown also exhibited strong suppression that elevated PVT1 expression is associated with cSCC progression, of tumor proliferation and promotion of apoptosis in an animal indicating that lncRNA PVT1 may play a crucial role in the model of cSCC, which was consistent with the in vitro results. multistage carcinogenesis process of cSCC. LncRNA PVT1 interacts with 4EBP1 in cSCC cells LncRNA PVT1 knockdown suppresses cSCC cells proliferation Accumulating evidence suggests that lncRNA-binding proteins are and promotes apoptosis in vitro closely associated with various developmental and pathological To investigate the potential biological role of lncRNA PVT1 in vitro, processes in tumors. We hypothesized that the lncRNA PVT1 may we constructed stable PVT1-knockdown cell lines in both A431 promote cSCC tumorigenesis, depending on its binding proteins, to and COLO16 cells using shRNA targeting PVT1 (sh-PVT1#1, sh- regulate downstream gene expression. A431 cell line is the PVT1#2, and sh-PVT1#3) or a normal control shRNA (sh-NC). The commonly used cSCC cell line and caused a more extensive efﬁciency of the PVT1-knockdown shRNA sequences was validated regulation of biological function, so it was consequently selected by RT-qPCR in A431 and COLO16 cells (Fig. 2A). As shown, PVT1 forsubsequentproteomicsexperimentation. To identify the potential expression was obviously impaired by sh-PVT1#1 and sh-PVT1#3 interacting proteins of lncRNA PVT1, label-free quantitative proteo- in both cSCC cell lines compared with the sh-NC controls. In a mics based on mass spectrometry was used to detect signiﬁcant subsequent study, we used the sequences sh-PVT1#1 and sh- DEPs in A431 sh-NC and sh-PVT1#3 groups (Fig. 5A). LS-MS mass PVT1#3 to establish stable PVT1-knockdown models in A431 and spectrometry identiﬁed 117 DEPs, including 60 upregulated and 57 COLO16 cells. downregulated, after silencing PVT1 in A431 cSCC cells (Fig. 5A, B). To Cell proliferation of cSCC cells in vitro was detected using CCK- further identify the associated target proteins of PVT1, KEGG 8, colony formation, and EDU assays. The CCK-8 assay showed that enrichment (Fig. 5C), GO enrichment (Fig. 5D), and PPI network the knockdown of PVT1 dramatically suppressed the viability of analyses (Fig. 5E, F) were performed using a related online database. both cSCC cells at 72 h and 96 h (Fig. 2B). The results of colony According to the outcomes of the PPI network, 4EBP1 had the formation assay also revealed that PVT1 knockdown inhibited the highest degree and most interactions in the DEPs, and was the key growth of A431 and COLO16 cells in the sh-PVT1#1 and sh-PVT1#3 protein centered around four protein nodes. Thus, we identiﬁed groups (Fig. 2C). EDU assay results were also observed to be 4EBP1 as the most promising protein among several candidates for consistent with the above experiments on proliferation (Fig. 2D, E). PVT1-binding factors. To conﬁrm the veracity and reliability of DEPs Detection of cell cycle progression revealed that PVT1 knockdown from proteomics, we used western blotting to validate the expression led to an accumulation of cSCC cells in the G1-phase (Fig. 2F, G). of several proteins in both A431 and COLO16 cSCC cells (Fig. 5G). The These ﬁndings suggest that PVT1 knockdown is involved in the results revealed that RAP2B, ANXA3, and 4EBP1 expression were inhibition of cSCC tumor proliferation and arrest of cell cycle decreased in sh-PVT1#3 groups compared to sh-NC groups, which progression in cSCC cells. was consistent with the results of the proteomics analysis. To To evaluate the apoptosis of cSCC cells, Annexin V-PE/7-AAD elucidate the potential regulatory mechanism between PVT1 and double staining was performed using ﬂow cytometry. The data 4EBP1, we detected 4EBP1 mRNA expression by RT-qPCR in stable indicated that the knockdown of PVT1 signiﬁcantly promoted cell PVT1-knockdown cSCC cells (Fig. 6A). Interestingly, the results apoptosis in vitro (Fig. 2H, I). Taken together, PVT1 knockdown showed that PVT1 knockdown had no effect on 4EBP1 mRNA inhibited cSCC cell proliferation and promoted apoptosis in vitro. expression, while the protein expression of 4EBP1 was markedly downregulated in PVT1-knockdown cSCC cells. This suggests that LncRNA PVT1 knockdown suppresses cSCC cells metastasis PVT1 may modulate 4EBP1 at the protein level and does not function and invasion in vitro through a competitive endogenous RNA (ceRNA) mechanism. To The functional role of lncRNA PVT1 knockdown on cSCC validate whether PVT1 binds to 4EBP1 in cSCC cells, we performed metastasis and invasion was investigated using transwell and immunoﬂuorescence combined with an RNA-FISH assay to predict its scratch-wound healing assays. Compared with the sh-NC groups subcellular localization. Both the 4EBP1 and PVT1 regions showed in vitro, the wound closure of the PVT1-silencing groups was similar localization patterns with the predominant overlap in the decreased in A431 and COLO16 cells (Fig. 3A, B). To further cytoplasm, suggesting underlying functional interactions (Fig. 6B). validate whether PVT1 is critical for cell metastasis and invasion in Next, we performed an RNA pull-down assay in A431 and COLO16 cSCC, transwell assays with Matrigel-coated or uncoated wells cSCC cells using the Flag-MS2bp-MS2bs system to support the were performed. PVT1 knockdown efﬁciently reduced the transit ﬁnding that 4EBP1 is the binding protein of PVT1. As shown in (Fig. of migrated and invaded cells in the sh-PVT1#1 and sh-PVT1#3 6C), 4EBP1 was constitutively associated with PVT1 in both A431 and cSCC cells (Fig. 3C, D). Thus, we concluded that the knockdown of COLO16 cSCC cells. Taken together, these results indicate that PVT1 PVT1 markedly inhibited the cell metastasis and invasion behavior directly binds to 4EBP1 in the cytoplasm of cSCC cells and can of cSCC in vitro. regulate 4EBP1 expression, which can provide novel molecular mechanistic insights into cSCC. LncRNA PVT1 knockdown inhibits cSCC proliferation and enhances apoptosis in vivo 4EBP1 overexpression reversed the effects of lncRNA PVT1 To further evaluate the tumorigenic effect of lncRNA PVT1 in vivo, we knockdown in cSCC cells constructed a xenograft tumor model of A431 cSCC cells in nude To identify the role of 4EBP1 in PVT1-mediated oncogenic mice. The 10 female BALB/c nude mice (4–6-weeks-old) were progression, we established a 4EBP1-overexpression model in randomly divided into two groups, and injected with sh-NC or sh- PVT1-knockdown A431 and COLO16 cells by transfecting cells with PVT1#3 A431 cells. The tumor growth of the sh-PVT1#3 group was the 4EBP1-overexpression vector (oe4EBP1) or an empty vector. To Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 2 LncRNA PVT1 knockdown regulates the viability and apoptosis of A431 and COLO16 cells in vitro. A The efﬁciency of PVT1 knockdown in A431 and COLO16 cells was validated by RT-qPCR. Cell proliferation and quantiﬁcation in vitro were detected by CCK-8 assay (B), colony formation (C), and EDU assay (200×magniﬁcation, scale bar = 50 µm) (D, E). Flow cytometry was performed to quantify cell cycle (F, G) and cell apoptosis (H, I) in A431 and COLO16 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns no statistical signiﬁcance. Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 3 LncRNA PVT1 knockdown inhibits metastasis and invasion of A431 and COLO16 cells in vitro. A Representative images (×200 magniﬁcation, scale bar = 50 µm) and quantiﬁcation (B) of scratch-wound assays in A431 and COLO16 cells. Images (×200 magniﬁcation, scale bar = 50 µm) and quantiﬁcation of the number of migrated (C) and invaded cells (D) by transwell assays in vitro are exhibited. *P < 0.05, **P < 0.01, ***P < 0.001. Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 4 The LncRNA PVT1 knockdown inhibits xenograft tumor growth and enhances apoptosis in nude mice. A, B Tumor engraftment 7 3 sizes in nude mice injected with sh-NC or sh-PVT1#3 A431 cells on the right subcutaneous axilla (1 × 10 cells/ml). C Tumor volume (mm ) was assessed every 3 days. D Quantitation of weight of tumors dissected from xenograft tumor models. E LncRNA PVT1 expression in xenograft tumor tissues measured by RT-qPCR. F CSCC histology by HE staining and Ki-67 and TUNEL measured by immunohistochemistry (×200 magniﬁcation, scale bar = 50 µm). *P < 0.05, **P < 0.01. verify whether the transfection of 4EBP1 affected PVT1 knockdown, previously described. 4EBP1 overexpression reversed the PVT1- lncRNA PVT1 expression was measured by RT-qPCR following 4EBP1 knockdown-arrested cell cycle in both A431 and COLO16 cells (Fig. transfection in stable knockdown cSCC cells (Fig. 7A). The results 7F). Moreover, 4EBP1 overexpression considerably reduced apoptosis showed that co-transfection with 4EBP1 did not affect the in the oe4EBP1/sh-PVT1#3 groups of cSCC cells (Fig. 7G). Transwell PVT1 silencing. The efﬁciency of the 4EBP1-overexpression protein assays with matrigel-coated or uncoated wells were performed to was validated by WB in PVT1-knockdown A431 and COLO16 cells detect cell invasion and metastasis in cSCC cells. As shown in (Fig. (Fig. 7B). As shown, 4EBP1 protein expression was obviously elevated 7H–K), overexpression of 4EBP1 partially rescued PVT1-knockdown- in the oe4EBP1/sh-PVT1#3 groups in both cSCC cell lines compared inhibited cell metastasis and invasion in vitro. To further validate the with the NC/Vector controls. To explore whether PVT1 regulates cSCC downstream targets of 4EBP1, we measured several indicators tumorigenesis through 4EBP1 in cSCC cells, we performed rescue accounting for cell phenotypic functions, including proliferation experiments with respect to cell proliferation, apoptosis, metastasis, (cyclin D1, p21), apoptosis (caspase3, Bax, Bcl2), metastasis (Snail, N- and invasion in vitro. In a subsequent study, we detected cell cadherin, E-cadherin) and invasion (MMP-2, MMP-9) indexes. All proliferation using CCK-8 and colony formation assays. The results of indicators were detected by RT-qPCR in stable co-transfection A431 the two experiments showed that 4EBP1 overexpression increased and COLO16 cells. The results showed that cyclin D1, Bcl2 were the growth of A431 and COLO16 cells in the oe4EBP1/sh-PVT1#3 downregulated and p21, caspase3, Bax were upregulated in sh- groups compared with sh-PVT1#3/Vector groups (Fig. 7C–E). Flow PVT1#3/Vector groups (Fig. 8A), suggesting PVT1 knockdown can cytometry was performed to assess cell cycle and apoptosis, as inhibit proliferation and promote apoptosis. 4EBP1 overexpression Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 5 Label-free quantitative proteomics and bioinformatics analysis in A431 cell. A, B A total of 117 signiﬁcant DEPs was detected by label-free quantitative proteomics in A431 cSCC cells. C, D KEGG and GO analysis of DEPs associated with cSCC. KEGG enrichment presents the top 20 enriched pathway of DEPs in cSCC. GO enrichment shows the aspects of DEPs categorized by biological process (BP), molecular function (MF), and cellular component (CC). E, F Images of up- and downregulated PPI networks are shown. 4EBP1 protein is indicated by red arrows. G RAP2B, ANXA3, and 4EBP1 protein expression measured by WB. Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 6 LncRNA PVT1 interacts with 4EBP1 in A431 and COLO16 cells. A 4EBP1 mRNA expression is measured by RT-qPCR in PVT1- knockdown A431 and COLO16 cells. B Immunoﬂuorescent images combined with RNA-FISH staining in A431 and COLO16 cells (×200 magniﬁcation, scale bar = 50 µm). C 4EBP1 was identiﬁed as the PVT1-interacting protein by RNA pull-down assays. Proteins pulled down by the PVT1 probe, or its negative probe were separated by SDS-PAGE and subjected to silver staining. A speciﬁc band, marked with an arrow, was identiﬁed as 4EBP1 protein in the PVT1 group. WB detection of 4EBP1 after PVT1 RNA pull-down assay. ns no statistical signiﬁcance. reversed the expression of these targets induced by PVT1 knock- trend following 4EBP1 overexpression (Fig. 8B). These ﬁndings were down in both A431 and COLO16 cells (Fig. 8A). Moreover, Snail, N- all in accordance with observed phenotype experiments. Thus, these cadherin,MMP-2,and MMP-9weredownregulated and E-cadherin outcomes further support our hypotheses that 4EBP1 can regulate were upregulated in sh-PVT1#3/Vector groups (Fig. 8B), suggesting several downstream functional proteins to typically reverse the cell PVT1 knockdown can inhibit metastasis and invasion. Also, these functions induced by PVT1 knockdown, indicating PVT1 modulate metastasis and invasion markers expression displayed a reversed 4EBP1 to promote cSCC carcinogenesis. These data further Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 7 4EBP1 overexpression reverses the effects of lncRNA PVT1 knockdown in A431 and COLO16 cells. A Expression of the lncRNA PVT1 as measured by RT-qPCR in stable PVT1-knockdown A431 and COLO16 cells co-transfected with 4EBP1-overexpression constructs. B Overexpression of 4EBP1 validated by WB in PVT1-knockdown A431 and COLO16 cells. C–K Knockdown of PVT1 inhibits cell proliferation, metastasis, and invasion and promotes apoptosis in A431 and COLO16 cells, but knockdown of PVT1 combined with 4EBP1 overexpression reverses this effect. Images of migrated and invaded cells by transwell assays in vitro are exhibited (×200 magniﬁcation, scale bar = 50 µm). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Cell Death Discovery (2023) 9:101 R. Li et al. Fig. 8 The mechanism of lncRNA PVT1/4EBP1 promotion of cSCC progression and tumorigenesis. A Expression of the proliferation (cyclin D1, p21) and apoptosis (caspase3, Bax, Bcl2) indexes measured by RT-qPCR in stable PVT1-knockdown A431 and COLO16 cells co-transfected with 4EBP1-overexpression constructs. B Expression of the metastasis (Snail, N-cadherin, E-cadherin) and invasion (MMP-2, MMP-9) indexes measured by RT-qPCR in stable PVT1-knockdown A431 and COLO16 cells co-transfected with 4EBP1-overexpression constructs. C Schematic model illustrates the mechanism by which PVT1 interacts with 4EBP1 to promote tumorigenesis of cSCC. *P < 0.05, **P < 0.01, ***P < 0.001. Cell Death Discovery (2023) 9:101 R. Li et al. demonstrated that lncRNA PVT1 plays an important role in selectively enhancing or preventing the translation of speciﬁc tumorigenesis of cSCC by mediating 4EBP1 through direct transcripts . However, its precise role in cSCC has not yet interaction. been clariﬁed. Notably, PVT1 knockdown dramatically decreased 4EBP1 protein expression, while its mRNA level remained unchanged. These results suggest that PVT1 regulates 4EBP1 DISCUSSION at the protein level, and does not function via a ceRNA Despite it being the second most common NMSC, the pathogen- mechanism. Moreover, dysregulation of 4EBP1 may have an esis of cSCC remains unclear. In several recent studies, the oncogenic role in cSCC progression. To further explore the expression and function of dysregulated lncRNAs in cSCC functional role and mechanism of 4EBP1 action, a study was tumorigenesis have attracted increased attention [29, 30]. In this conducted to conﬁrm the interaction between lncRNA PVT1 and study, we demonstrated that the lncRNA PVT1 is frequently 4EBP1 protein by immunoﬂuorescence combined with RNA-FISH upregulated and ampliﬁed in clinical cSCC specimens compared staining in cSCC cells. A signiﬁcant overlap with the cytoplasmic with distant normal tissue samples. Consistent with previous localization pattern of lncRNA PVT1 and 4EBP1 was found in results , expression of the lncRNA PVT1 was also signiﬁcantly cSCC cells. In addition, the RNA pull-down assay revealed that elevated in both cSCC cell lines. Knockdown of PVT1 markedly lncRNA PVT1 is directly associated with the 4EBP1 protein, inhibited cSCC cell proliferation, metastasis, and invasion, while further supporting our previous results and hypothesis. We enhancing apoptosis both in vivo and in vitro. In addition, we demonstrated that overexpression of 4EBP1 can partially rescue elucidated lncRNA PVT1 as a critical modulator of cSCC lncRNA PVT1-knockdown-inhibited cell proliferation, metastasis, tumorigenesis. Moreover, we revealed a novel molecular mechan- and invasion in vitro, while reverse cell apoptosis was promoted ism by which lncRNA PVT1 can connect with the 4EBP1 protein by silencing PVT1. The Rescue experiments revealed that 4EBP1 and regulate its expression, thus resulting in enhanced tumor may exert a pro-tumor role in cSCC cellular functions, while progression of cSCC. Our ﬁndings provide new insights into the overexpression of 4EBP1 enhances the malignancy of cSCCs. therapeutic implications of potential molecular targeted treat- Recent studies have reported that 4EBP1 can positively regulate ments in patients with cSCC. the translation of a subset of mRNAs and enhance speciﬁc Increasing evidence has suggested that ampliﬁcation and protein synthesis under metabolic stress conditions to support dysregulation of lncRNAs play essential roles in the development uncontrolled tumor growth [22, 47–49]. 4EBP1 may also increase of malignant transformation in cSCC [31–34]. Several studies have the translation of pro-angiogenic factors under hypoxia through also demonstrated that PVT1 functions as an oncogene and affects a cap-independent mechanism, and 4EBP1 overexpression can the malignancy of various tumors via multiple pathways . The promote HIF-1α and VEGF, which are known to mediate cap- results of our previous study showed that PVT1 is the most independent translation . In addition, another study revealed overexpressed lncRNA detected by microarray analysis and may be that high expression of 4EBP1 is closely related to poor potentially implicated in carcinogenesis and progression of cSCC. prognosis in neuroblastoma via MYCN upregulation, and may lncRNA PVT1 expression is highly upregulated in cSCC tissues and confer advantages to tumor cell survival and proliferation under cells, and elevated expression of lncRNA PVT1 is relevant to the nutrient deprivation and metabolic stress [22, 49]. In addition to biological functions of cSCC. Moreover, we performed further neuroblastoma, 4EBP1 overexpression has been reported to be a functional experiments, and the data indicated that depletion of factor forpoorprognosisinall combined TCGA tumortypes . lncRNA PVT1 suppressed cSCC cell proliferation, metastasis, and Given the pro-cancer signiﬁcance of 4EBP1 in cSCC, it is possible invasion, and promoted apoptosis both in vivo and in vitro. Together, that 4EBP1 may aid cSCC cells to cope with metabolic stress, these results support the hypothesis that PVT1 plays an oncogenic such as hypoxia and nutrient deprivation, by regulating mRNA role in the tumorigenesis of cSCC and might be a potential biomarker translation, which is also consistent with the reported studies for the diagnosis and treatment of cSCC. mentioned above. These lines of evidence reveal that lncRNA LncRNAs have been reported to be involved in various PVT1 promotes cSCC tumorigenesis by interacting with and biological processes in tumors through multiple coordinative regulating the 4EBP1 protein. The direct interaction between mechanisms, such as direct binding with DNA, RNA, and speciﬁc lncRNA PVT1 and 4EBP1 can lead to malignant transformation of proteins . The conventional view holds that lncRNAs cSCC cells, thereby inducing tumor progression. primarily act as competitive endogenous RNAs (ceRNAs) to The detailed mechanism underlying the crosstalk between the facilitate cSCC tumor progression by regulating miRNA–lncRNA lncRNA PVT1 and 4EBP1 is not yet well deﬁned and warrants interactions [37–39]. In contrast, accumulating evidence has further investigation. Taken together, our data showed that indicated that lncRNAs, which are predominantly located in the lncRNA PVT1 regulates cSCC carcinogenesis by binding to cytoplasm, can exert their function by binding with targeted 4EBP1, which lays a ﬁrm foundation for future cSCC screens that proteins and modulating the expression of corresponding deeply explore the molecular regulatory details of pathogenesis. proteins [40–44]. Although lncRNA PVT1 contributes to malig- nant regulation in cSCC cells, the exact mechanism by which lncRNA PVT1 promotes carcinogenesis in cSCC remains unclear. CONCLUSION Thus, we report that lncRNA PVT1 mediates cSCC tumorigenesis In summary, we demonstrated that lncRNA PVT1 acts as an by regulating 4EBP1. Considering that the majority of lncRNA oncogenic regulator in cSCC tumorigenesis by binding to 4EBP1. PVT1 was localized in the cytoplasm of cSCC cells, we conducted Most importantly, our study elucidated a novel regulatory label-free quantitative proteomics to identify potential proteins mechanism between lncRNA PVT1 and 4EBP1 to mediate cSCC interacting with lncRNA PVT1. Based on bioinformatics analysis, progression, which is important for understanding cSCC. Our we revealed that 4EBP1 is a potential protein in several study sheds new light on the molecular mechanism of the PVT1/ candidates for PVT1-binding factors, which shows the most 4EBP1 interaction in the tumorigenesis of cSCC, and provides a connection in PPI networks. 4EBP1 exerts a complex dual role in promising targeted therapeutic strategy for cSCC in the future. tumor progression and molecular function due to the different metabolic conditions of the tumor microenvironment . Conversely, 4EBP1 acts as a tumor suppressor by inhibiting MATERIALS AND METHODS eIF4E and blocking mRNA translation in various cancers. In Cell culture The CSCC cell lines A431 and COLO16 and the human immortal contrast, 4EBP1 exerts its tumor promoter effects by accelerating keratinocyte line (HaCaT) were provided by the central laboratory of the tumor adaptation to metabolic and genotoxic stress by Cell Death Discovery (2023) 9:101 R. Li et al. Institute of Dermatology, Chinese Academy of Medical Sciences & Peking performed; cells were seeded in 6-well plates (1500 cells/well). The cell Union Medical College. The CSCC cell line, SCL-1, was purchased from the culture medium was changed every 3 days. After the formation of Cell Bank of the Shanghai Institute, Shanghai, China. Cells were maintained observed colonies (>50 cells), cells were ﬁxed with 4% formaldehyde, at 37 °C/5% CO in a hatch chamber. CSCC and HaCaT cells were grown in stained with 0.1% crystal violet for 20 min, photographed, and counted. DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS) (VivaCell, EdU staining assays were performed using the BeyoClick EdU Kit Shanghai, China) supplemented with 1% penicillin/streptomycin (Gibco, (Beyotime, China), according to the manufacturer’s protocol. EdU USA). These cell lines were authenticated by proﬁling. incorporation (%): EdU-positive cells (%) = EdU-positive cells/(EdU-posi- tive+ EdU-negative cells) × 100. Clinical patient samples Twenty cSCC, para-tumor, and distant normal tissues were obtained from Flow cytometry patients following surgery at the Institute of Dermatology, Chinese The cell cycle and apoptosis were detected by ﬂow cytometry in vitro. The Academy of Medical Sciences, and Peking Union Medical College (Nanjing, cell cycle assay was performed using a Cell Cycle Analysis Kit (Beyotime, China). All tissues were diagnosed independently by two experienced China), according to the manufacturer’s instructions. The PE Annexin V pathologists and centrally reviewed to verify the diagnosis of cSCC. The Apoptosis Detection Kit I (BD Pharmingen, USA) was used to detect the clinical patient tissue samples were stored at −80 °C. The study was apoptotic ability of cSCC cells in vitro. Brieﬂy, A431 and COLO16 cells were reviewed and approved by the Ethics Committee of our institution (No. incubated with 5 μL annexin V-PE and 5 μL 7-AAD for 15 min at room 2016-KY-013). The clinical data of the patients are listed in Supplementary temperature (RT) in the dark and then assayed by ﬂow cytometry. Table 1. Apoptosis rate (%) = late apoptosis rate (%) + early apoptosis rate (%). The two experiments were conducted using FACSVerse™ (Becton, Dickinson, and Company) with BD FACSuite™ software. RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) Cell migration and invasion assay Total RNA was extracted from cultured cell lines and tissues using Trizol Scratch-wound and Transwell assays were performed to detect cSCC cell reagent (Invitrogen, USA). cDNA was reverse-transcribed using the migration and invasion in vitro. A431 and COLO16 cells were inoculated PrimeScript™ RT Kit (Takara, Japan). RT-qPCR was performed using the into six-well plates until reaching 80–90% conﬂuency. A vertical scratch SYBR-green Premix Ex Taq™ Kit (Takara, Japan), and all samples were wound was then made with a 10 mL aseptic suction head. After rinsing analyzed using the Roche Lightcycler 480 Real-Time PCR System. GAPDH twice with PBS to remove the cell fragments, cells were cultured in was used as the internal control. To calculate the relative lncRNA and (−ΔΔCt) serum-free medium and photographed at 0 h and 48 h for recording. In mRNAs expression levels, the 2 method was applied to data from at the Transwell experiment, A431 and COLO16 cells were inoculated into least three independent experiments. The primers used in this study are the upper chamber (2 × 10 cell/well) of Matrigel (BD Biosciences, USA)- listed in Supplementary Table 2. uncoated or coated Transwell inserts (Corning Costar, USA). The upper chamber was ﬁlled with 200 μl serum-free DMEM, and the lower RNA ﬂuorescence in situ hybridization (RNA-FISH) chamber was ﬁlled with 600 μl DMEM supplemented with 20%FBS. After FISH assay for the lncRNA PVT1 was performed in A431 and COLO16 cells inoculation, cells from the transwell migration assay and cells from the using lncRNA FISH kits (Servicebio, Wuhan, China), according to the transwell Matrigel invasion assay were ﬁxed and stained separately at manufacturer’s instructions. After the A431 and COLO16 cells were ﬁxed by 24 h and 48 h. in situ hybridization ﬁxture for 15 min, the ﬁxative solution was removed, and the cell climbing slices in six-well plates were washed twice with PBS. Animal experiment in vivo CY3-labeled lncRNA PVT1 probes were designed and synthesized by Under standard laboratory conditions, ten 4–6-week-old female BALB/c ServiceBio. DAPI was used for nuclear counterstaining, and images were nude mice (Gempharmatech, Nanjing, China) were randomly divided into photographed using a confocal scanning microscope. U6 and 18 S two groups (n = 5/group). To establish xenograft tumor models, nude mice ribosomal RNA (18 S rRNA) were used as internal controls for the nucleus were injected with 100 μl sh-NC A431 cells (1 × 10 cells/ml) or sh-PVT1#3 and cytoplasm, respectively. A431 cells (1 × 10 cells/ml) in the right subcutaneous axilla. Tumor volume was measured every 3 days and calculated using the following formula: Subcellular fractionation 3 2 Tumor volume (mm ) = length × width × 0.5. After 22 days of treatment, Nucleocytoplasmic fractionation of the A431 and COLO16 cell lines was ten nude mice were sacriﬁced, and the tumors were removed. The performed using the NE-PER Nuclear and Cytoplasmic Extraction kit dissected tumor tissues from the mice were photographed, weighed, ﬁxed, (Thermo Fisher Scientiﬁc), according to the manufacturer’s protocol. U6 and embedded in parafﬁn for immunohistochemistry (IHC) staining. and GAPDH were used as the internal controls for nuclear and cytoplasmic expression, respectively. The primers used were described previously for Immunohistochemistry and hematoxylin-eosin histology (HE) RT-qPCR. Tumor tissues isolated from nude mice were used for HE histological staining and immunohistochemical staining of Ki-67 (1:1000; Abcam, Lentivirus transfection USA) and TUNEL (1:1000; Abcam, USA). HE staining of mouse tissues was Lentivirus vectors of lncRNA PVT1 were designed based on NR_003367.3 of performed for histological observation, according to the standard Homo sapiens PVT1 oncogene, and the overexpression vector of 4EBP1 histological protocols. After deparafﬁnization, hydration, and blocking, was designed based on the NM_004095 of the human 4EBP1 gene. A431 mouse tumor tissue slides were incubated with primary antibody against and COLO16 cells were seeded in six-well plates at 1 × 10 cells/well for Ki-67 and TUNEL at 4 °C overnight, and then incubated with the 24 h prior to transfection and infected with lentivirus at the multiplicity of appropriate horseradish peroxidase (HRP)-conjugated secondary anti- infection (MOI) 50 and MOI 20 for 24 h. After 72 h, the transfected cSCC body (DAKO, USA) the next day at RT for 1 h. A DAB Horseradish cells were selected continuously with puromycin (1 μg/ml; Invitrogen) for Peroxidase Color Development kit (Beyotime, China) was used for 7 days. Stably-transfected cSCC cells were detected and validated by RT- visualization. qPCR and subsequently stored at −80 °C. The shRNA sequences of the PVT1 and 4EBP1-overexpression vectors were constructed by GeneChem Immunoﬂuorescence (Shanghai, China) and are listed in Supplementary Table 3. For immunoﬂuorescence, cell climbing slices of A431 and COLO16 cells were prepared and ﬁxed, as previously described for RNA-FISH. FAM- Cell proliferation assay labeled 4EBP1 (1:100; ProteinTech, Wuhan, China) was used for immuno- Cell proliferation of cSCC cells in vitro was detected using CCK-8, colony ﬂuorescent FISH analysis to detect subcellular localization. formation, and EDU assays. A431 and COLO16 cells in the logarithmic growth phase were seeded in 96-well plates (3000 cells/well) and cultured Western blotting for 24 h. After incubation in a medium containing 10% CCK-8 reagent Total protein was extracted from cultured cell lines using RIPA lysis buffer (MedChemExpress,USA) at 37 °C for 50 min, the absorbance at 450 nm was (Beyotime, China) and a BCA protein kit (Beyotime, China) was used to detected by spectrophotometry as the OD value, which was recorded at 0, assess protein concentrations. Protein samples of cSCC cells were 24, 48, 72, and 96 h, respectively. Then, the colony formation test was Cell Death Discovery (2023) 9:101 R. Li et al. separated on 4–20% SDS-PAGE gels and transferred onto a PVDF 4. Manyam BV, Garsa AA, Chin RI, Reddy CA, Gastman B, Thorstad W, et al. A multi- membrane (Millipore, USA). After blocking with 5% milk-TBST, membranes institutional comparison of outcomes of immunosuppressed and immuno- were incubated with primary antibody at 4 °C overnight and then probed competent patients treated with surgery and radiation therapy for cutaneous with HRP-conjugated anti-mouse/rabbit secondary antibodies (1:2000; squamous cell carcinoma of the head and neck. Cancer. 2017;123:2054–60. Cat# ab205719, Cat# ab6721 Abcam, USA) at RT for 1 h. Protein bands were 5. García-Foncillas J, Tejera-Vaquerizo A, Sanmartín O, Rojo F, Mestre J, Martín S, detected using the SuperSignal West Pico PLUS Kit (Thermo Scientiﬁc, et al. Update on management recommendations for advanced cutaneous USA). The primary antibodies included 4EBP1 (1:2000; Cat#ab32024), squamous cell carcinoma. Cancers. 2022;14:629. RAP2B (1:1000; Cat#ab101369), ANXA3 (1:1000; Cat#ab127924), and 6. Bridges MC, Daulagala AC, Kourtidis A. LNCcation: lncRNA localization and GAPDH (1:10,000; Cat#ab8245), all of which were purchased from Abcam function. J Cell Biol. 2021;220:e202009045. (Cambridge, USA). 7. Kopp F, Mendell JT. Functional classiﬁcation and experimental dissection of long noncoding RNAs. Cell. 2018;172:393–407. 8. Sun Q, Hao Q, Prasanth KV. Nuclear long noncoding RNAs: key regulators of gene Label-free quantitative proteomics and bioinformatics expression. Trends Genet: TIG. 2018;34:142–57. analysis 9. Zhou C, Yi C, Yi Y, Qin W, Yan Y, Dong X, et al. LncRNA PVT1 promotes gemcitabine To determine the differential expression of the protein by stable PVT1 resistance of pancreatic cancer via activating Wnt/β-catenin and autophagy knockdown, mass-spectrometry-based label-free quantitative proteo- pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes. mics was used to detect differentially expressed proteins (DEPs). 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Long non-coding RNA PVT1 (PPI) network (https://string-db.org/), and Kyoto Encyclopedia of Genes predicts poor prognosis and induces radioresistance by regulating DNA repair and Genomes (KEGG) database (http://www.genome.jp/kegg/). The and cell apoptosis in nasopharyngeal carcinoma. Cell Death Dis. 2018;9:235. related data from the label-free quantitative proteomics are listed in 16. Hu Y, Li R, Chen H, Chen L, Zhou X, Liu L, et al. Comprehensive analysis of lncRNA- Supplementary Table 4. mRNAs co-expression network identiﬁes potential lncRNA biomarkers in cuta- neous squamous cell carcinoma. BMC Genomics. 2022;23:274. 17. Hua H, Kong Q, Zhang H, Wang J, Luo T, Jiang Y. Targeting mTOR for cancer RNA pull-down assay therapy. J Hematol Oncol. 2019;12:71. An RNA pull-down assay was performed using a Pierce™ Magnetic RNA- 18. Hsieh AC, Nguyen HG, Wen L, Edlind MP, Carroll PR, Kim W, et al. 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Deep computational analysis of human cancer and non-cancer Attribution 4.0 International License, which permits use, sharing, tissues details dysregulation of eIF4F components and their interactions in adaptation, distribution and reproduction in any medium or format, as long as you give human cancers. bioRxiv:2020:2020.10.12.336263 [Preprint]. Available from: appropriate credit to the original author(s) and the source, provide a link to the Creative https://www.biorxiv.org/content/10.1101/2020.10.12.336263v1. Commons license, and indicate if changes were made. The images or other third party 49. Schaaf MB, Garg AD, Agostinis P. Deﬁning the role of the tumor vasculature in material in this article are included in the article’s Creative Commons license, unless antitumor immunity and immunotherapy. Cell Death Dis. 2018;9:115. indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// AUTHOR CONTRIBUTIONS creativecommons.org/licenses/by/4.0/. RL and KC conceived and designed the study. MJ, RL, and JZ collected the samples and acquired the data. CL and RL interpreted or analyzed data. MJ and RL prepared the manuscript, which was revised for important intellectual content by DH and HC. © The Author(s) 2023 Cell Death Discovery (2023) 9:101
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