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MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the SKP2/p27 axis

MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the... www.nature.com/cddiscovery ARTICLE OPEN MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the SKP2/p27 axis ✉ ✉ 1,3 1,3 1 2 2 1 1,2 1,2 Lingjun Ge , Gaichao Zhao , Chao Lan , Houji Song , Dan Qi , Pan Huang , Xiaoxue Ke and Hongjuan Cui © The Author(s) 2023 Gastric cancer (GC) is a major cause of human deaths worldwide, and is notorious for its high incidence and mortality rates. Mesoderm Posterior Basic Helix-loop-helix (bHLH) transcription factor 2 (MESP2) acts as a transcription factor with a conserved bHLH domain. However, whether MESP2 contributes to tumorigenesis and its potential molecular mechanisms, remain unexplored. Noticeably, MESP2 expression levels are decreased in GC tissues and cell lines compared to those in normal tissue. Further, in vitro and in vivo experiments have confirmed that MESP2 overexpression suppresses GC cell growth, migration, and invasion, whereas MESP2 knockdown results in the exact opposite. Here, we present the first report that MESP2 binds to transcription factor 7-like 2 (TCF7L2/TCF4) to inhibit the activation of the TCF4/beta-catenin transcriptional complex, decrease the occupancy of the complex on the S-phase kinase Associated Protein 2 (SKP2) promoter, and promote p27 accumulation. MESP2 knockdown facilitated tumorigenesis, which was partially suppressed by SKP2 knockdown. Taken together, we conclude that MESP2 binds competitively to TCF4 to suppress GC progression by regulating the SKP2/p27 axis, thus offering a potential therapeutic strategy for future treatment. Cell Death Discovery (2023) 9:79 ; https://doi.org/10.1038/s41420-023-01367-4 INTRODUCTION protein structure composed of a basic region [9] that interacts Gastric cancer (GC), a deadly malignancy, is the third main cause with DNA and a neighboring helix–loop–helix region that of global human cancer-related deaths, resulting in ~723000 mediates dimerization [10]. Most bHLH dimers recognize the E- deaths annually [1]. Nearly half of the cases occur in Eastern Asia, box, a hexameric sequence in the DNA with the consensus especially in China [1, 2]. Clinically, early disease development is sequence CANNTG [10]. These bHLH TFs modulate gene expres- rarely accompanied by GC symptoms [3], and patients have poor sion through dimer formation, combining activators or repressors rates of survival, mainly due to suboptimal treatment and late with ubiquitous proteins (E-proteins) [11]. Previous experiments diagnosis [1]. There is therefore, an urgent need for elucidating have showed that aberrant expression of the bHLH transcription the potential molecular mechanisms of GC progression and factor is related to tumorigenesis [12], suggesting that MESP2 may determining effective therapeutic strategies. also participate in human cancer progression. Mesoderm posterior 2 (MESP2), is a transcription factor with a The Wnt signalling pathway influences the regulation of basic helix–loop–helix (bHLH) motif that has been illustrated to intestinal development, differentiation, and adult tissue home- play pivotal roles during the development of mammalian somite ostasis, thus contributing to the progression of numerous human and heart. MESP2 links spatiotemporal information, produced by cancers [13]. Transcription factor 7-like 2 (TCF7L2/TCF4) is a key the determination wave front and the segmentation clock, to component of the Wnt signalling pathway, mostly acting as a multiple somite morphogenesis processes in mouse somitogen- transcription factor after entering the nucleus [14]. Furthermore, esis [4]. The controlled segmentation program of the Notch Wnt ligands trigger the typical Wnt/beta-catenin signalling path- signalling pathway reflects the important function of MESP2, and way, which in turn activates the LRP and Frizzled receptors, the determination of the segment boundary of the Wnt3a/beta- leading to beta-catenin stabilization and nucleus translocation. catenin signalling pathway was demonstrated by an MESP2 Subsequently, nuclear beta-catenin binds to TCF4, thereby expression assay of Wnt3a mutants [5]. In fact, boundary promoting the expression of relevant genes [15]. In the intestinal formation and somite compartmentalization seem to depend on epithelium, the expression levels of TCF4/beta-catenin down- the combined action of Wnt and Notch signalling. Interrupting stream target genes are regulated by mutational activation of Wnt either pathway restrains somite formation and the MESP2 protein signalling, then affecting malignant transformation of colorectal levels that indicate somite compartmentalization and boundary cancer [13]. The ultimate aim of the typical Wnt signalling in the formation [6]. The bHLH family is one of the most prominent beta-catenin pathway is the formation of the TCF4/beta-catenin among transcription factors [7] and is involved in cell differentia- complex, and this interaction is regarded as a valid target for tion and tissue development [8]. These TFs share a characteristic cancer treatment [16]. 1 2 State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Cancer Center, Medical Research Institute, Southwest University, Chongqing 400716, China. These authors contributed equally: Lingjun Ge, Gaichao Zhao. email: kexiaoxue@126.com; hcui@swu.edu.cn Received: 21 July 2022 Revised: 7 February 2023 Accepted: 13 February 2023 Official journal of CDDpress 1234567890();,: L. Ge et al. The F-box protein S-phase kinase-associated protein 2 (SKP2) is a tumor suppressor role of MESP2 in vivo, and discovered that subunit that recognizes substrates of the SCF–SKP2 E3 ligase, knockdown of MESP2 promoted tumor growth and volume targeting protein proteasome-mediated degradation, such as p27 (Fig. 3E, F). Furthermore, IHC staining implied that tumors formed [17]. The deletion of p27 and tumorigenesis often go hand-in- by MESP2 knockdown cells exhibited higher Ki67 and lower hand. The influence of p27 in tumorigenesis is supported by the p27 staining than those formed by control cells (Fig. 3G, −/− formation of spontaneous pituitary tumors in p27 -mice [18]. Supplementary Fig. 1C). Functionally, the CDK suppressor, p27, is a typical negative After assessing the functions of MESP2 in GC cell growth, we regulator of cell proliferation. It can inhibit the G1-S transition of confirmed its function in metastasis. Transwell assays demon- cyclinE/CDK2 complex and the S phase of cyclinA/CDK2 complex strated that migration and invasion behaviors were significantly [19]. Physiological regulation of G1-S transformation is crucial in inhibited by MESP2 overexpression (Fig. 2E), but enhanced by determining cell fate and is lost during oncogenic transformation. knockdown of MESP2 (Fig. 4B). Wound-healing assays also In addition, in a range of cancer types, the declined cytoplasmic confirmed the above results (Fig. 4A). Western blot experiments p27 protein expression is pertinent to high metastasis and poor using epithelial-mesenchymal transition (EMT)-related markers survival. The loss of p27 affects self-renewal of hematopoietic stem and matrix metalloproteinases (MMPs), displayed same trends. cells and the proliferation and differentiation of hematopoietic Upregulation of MESP2 decreased MMP2 and MMP9 levels (Fig. progenitor cells, making cells prone to tumor transformation [20]. 2F). Nevertheless, Knockdown of MESP2 reduced E-cadherin In this work, considering that MESP2 expression levels are protein levels, but upregulated N-cadherin, and MMPs protein reduced in GC tissues and cell lines, we focused on MESP2 levels (Fig. 4C, Supplementary Fig. 1B). Subsequently, we assessed regulation and function, which to our knowledge, are uninvesti- the influence of MESP2 on lung metastasis. The number of gated in cancer. Functionally, our study revealed that MESP2 binds pulmonary metastatic nodules in MESP2 downregulation group competitively with beta-catenin to TCF4, suppressing GC progres- greatly increased in comparison to the control group (Fig. 4D). sion by regulating the SKP2/p27 axis. Thus, MESP2 is a candidate Hematoxylin and eosin (HE) staining revealed that treatment with in the diagnosis and therapy of GC. shMESP2 effectively promoted tumor cell metastasis (Fig. 4E). Collectively, these results strongly imply that MESP2 can serve as a potential tumor suppressor in GC. RESULTS MESP2 as a diagnostic/prognostic marker for GC MESP2 induced the stabilization of p27 by inhibiting its To explore the functional importance of MESP2 in GC, we first ubiquitylation and subsequent degradation examined the intracellular localization of MESP2 in GC cells. The Meanwhile, we observed that MESP2 downregulation decreased findings showed that it was located in both the cytoplasm and the protein levels of p27 (Fig. 5A), which acted as a CDK inhibitor nucleus (Fig. 1A, B). Next, we analyzed MESP2 levels in 80 tissue and controlled cell proliferation and metastasis [21]. Multiple samples of clinical patients via IHC staining. MESP2 IHC staining studies, including our previous research, have showed that p27 can was notably lower in gastric tumor tissues in a grade-dependent combine and suppress the activity of the cyclin E/CDK2 complex to pattern compared to that in normal tissues (Fig. 1C–E). qRT-PCR block the G1 phase [22]. To further elucidate the regulatory and western blot experiments showed that mRNA and protein mechanisms of MESP2-stabilizing p27 expression, p27 mRNA levels expression of MESP2 were downregulated in HGC-27, MKN-45, in the experimental and control groups were assessed via qRT-PCR. SGC-7901 and BGC-823, contrasted with that of GES-1, a normal Contrary to p27 protein levels, MESP2 knockdown did not impact human gastric epithelial cell (Fig. 1F). Moreover, Analysis of MESP2 p27 mRNA levels significantly (Fig. 5B). Therefore, we speculated expression levels in GC patient tumors using the R2: Genomics that a post-transcriptional mechanism was involved in the changes Analysis and Visualization Platform (https://hgserver1.amc.nl/cgi- in p27 expression levels. We evaluated the protein stability of p27 bin/r2) consistently confirmed that MESP2 upregulation predicted by introducing a cyclohexamide (CHX), and found that MESP2 better patient survival (Fig. 1G–J). Taken together, our results overexpression significantly lengthened the half-life of p27 indicate that MESP2 may be an anti-oncogene. (Fig. 5C), revealing that the p27 expression is positively regulated by MESP2. Knockdown of MESP2 induced p27 degradation and Overexpression of MESP2 suppressed GC progression in vitro was blocked by Proteasome inhibitors MG-132 treatment (Fig. 5D), and in vivo, but downregulation resulted in the opposite supporting the ubiquitination-proteasome pathway to maintain To further investigate the impact of MESP2, GO analysis showed p27 protein levels. Additionally, the ubiquitylation assay showed that differentially expressed genes were mainly enriched in the that downregulation of MESP2 enhanced the poly-ubiquitylation of biological regulation and by the molecular function regulator p27 (Fig. 5E). Finally, transcriptome profiling showed that MESP2 (Supplementary Fig. 1A). depletion suppressed the p27 pathway (Supplementary Fig. 2A). Then we successfully overexpressed MESP2 in in HGC-27 and Based on these results, we concluded that MESP2 indeed improved MKN-45 cells lines (Fig. 2A). Besides, knocked down MESP2 the protein stability of p27 by suppressing its ubiquitylation and expression using the lentiviral shRNA, namely, shMESP2 nos. 1 subsequent degradation. and 2 (Fig. 3A, B). In MTT assays, MESP2 overexpression substantially suppressed cell growth (Fig. 2B). Conversely, knock- MESP2 inhibited activation of the TCF4/beta-catenin down of MESP2 promoted GC cell proliferation, compared with transcriptional complex by competing with beta-catenin for control cells (Fig. 3C). To further examine the molecular mechanism binding TCF4 of MESP2-mediated inhibition of cell growth, we utilized flow We further characterized the biological functions of MESP2 and cytometry to determine the effect of MESP2 on cell cycle deciphered the mechanisms of its roles in GC inhibition via progression. We showed that MESP2 overexpression arrested the transcriptome profiling, which revealed that MESP2 depletion cell cycle at the G0/G1 phase (Fig. 2C). Subsequently, BrdU staining activated TCF-dependent signalling in response to the Wnt signified that BrdU-positive rates in MESP2- upregulated cells were pathway (Fig. 6A). The known TCF4/beta-catenin transcriptional reduced (Fig. 2D). The changes noted in the knockdown group complex has great effects on the Wnt pathway. Through were different (Fig. 3D). Consistent with this finding, the levels of functional domain analyses, Studies have reported that ITF2 and critical G0/G1 regulon genes, such as CDK4, CCND1,and CCNE1, MESP2 exhibited the relatively well-conserved bHLH domain decreased with MESP2 upregulation, whereas knockdown of (Fig. 6B), and a previous study had showed that ITF2 suppressed MESP2 led to the reverse (Fig. 2F, Fig. 3B, Supplementary formation of the TCF4/beta-catenin complex by competing with Fig. 1B). We used a mouse xenograft model to verify the putative beta-catenin for TCF4 [13]. Hence, we assessed through an online Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 1 MESP2 expression is downregulated in GC tissues and related to high prognosis. A Subcellular fractionation assays display MESP2 levels. Cytoplasmic α-tubulin and nuclear Histone H3 were used as reference standards. B Intracellular localization analysis of Flag-MESP2 by immunofluorescence staining. Intracellular localization of Flag-MESP2 (red); Nuclei (blue) were stained with DAPI. Scale bar, 5 μm. C Representative images of MESP2 expression at different levels in gastric tumor (I, II, III) and normal tissues. Scale bar, 20 μm. D, E. IHC staining analyses of MESP2 expression levels in 80 samples of normal tissues and gastric tumor tissues. ***P < 0.001. F qRT-PCR and Western blot experiments showing MESP2 mRNA and protein levels in GC cell lines (HGC-27, MKN- 45, SGC-7901, BGC-823, GES-1). Data shown are means ± SD, n= 3, **P <0.01, ***P <0.001. G Box plot of MESP2 expression levels in GC tissues in the R2 database. H Kaplan-Meier analysis of progression-free survival using data from R2 databases with the log-rank test P-values indicated. I, J. Kaplan-Meier analysis of progression-free survival was performed for patients with different grades and subtypes of GC using data from R2 databases with the log-rank test P-values indicated. Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 2 MESP2 inhibits the proliferation, migration and invasion ability of GC cells both in vitro and in vivo. A qRT-PCR analysis of MESP2 mRNA levels in MESP2-overexpression cells. B MTT assays showing suppressed GC cell proliferation by MESP2 overexpression. C The HGC-27 and MKN-45 cell cycles were detected via flow cytometry after overexpression of MESP2. D Representative fluorescent images and quantification of BrdU staining of GC cells expressing GFP or MESP2. Scale bar, 20 μm. E The migratory capacity of GC cells transfected with GFP or MESP2, as determined via transwell assays. Scale bar, 20 μm. Data shown are means ± SD, n = 3, *P < 0.1, **P < 0.01. F Western blots showing the levels of G1 phase regulatory and pivotal transfer-associated proteins. prediction website whether MESP2 and TCF4 had a binding interaction between TCF4 and MESP2, we truncated the full length relationship (Fig. 6C). We further validated whether both proteins of TCF4 into a T1 fragment with beta-catenin binding. Similarly, integrated with each other under experimental conditions, and the full-length MESP2 was truncated into an Mb fragment with the immunofluorescence staining showed that MESP2 was co- bHLH domain (Fig. 6F). Next, for domain-mapping experiments, expressed with TCF4 in HGC-27 cells (Fig. 6D). Then, co-IP assays we transfected 293FT cells with Flag-TCF4 (amino acids (aa) 1–619) showed that TCF4 was immunoprecipitated by MESP2 (Fig. 6E). To or Flag-T1 (aa 1–55) or Flag-TΔ (aa 56–619) of TCF4 and gain further insight into the specific domains that mediated the immunoprecipitated these against HA to observe their interaction Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 3 Knockdown MESP2 promotes the proliferation ability of GC cells both in vitro and in vivo. A qRT-PCR was performed to assess MESP2 mRNA levels in MESP2-knockdown cells. B Western blot experiments showing G1 phase regulatory protein levels in MESP2-knockdown cells. C. MTT assays showing that MESP2 knockdown promoted GC cell proliferation. D Representative fluorescent images and quantification for BrdU staining of GC cells expressing shGFP, shMESP2#1, or shMESP2#2. Scale bar, 20 μm. E, F In vivo analyses of size and volume of xenograft tumors that were hypodermically injected with treated HGC-27 and MKN-45 cells. G Representative IHC micrographs of selected tumors. Scale bar, 20 μm. Data shown are means ± SD, n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001. Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 4 Knockdown MESP2 promotes the migration and invasion of GC cells in vitro and in vivo. A, B The migration and invasion capacities of GC cells transfected with shGFP alone, shMESP2#1 or shMESP2#2 were determined via Wound healing and transwell assays. Scale bar, 20 μm. C Western blot experiments showing the levels of several pivotal transfer-associated proteins. D Images showing that the shMESP2 mouse model had more pulmonary nodules (black arrows) than the others. E Histological analysis of pulmonary tumors as observed via H&E staining are shown. Scale bar, 2 mm. Data shown are means ± SD, n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001. with Flag-TCF4 or Flag-T1 or Flag-TΔ in vivo. Both TCF4 and T1 nuclear fraction increased remarkably after knocking down MESP2 bound to MESP2 or M1 (Fig. 6F). (Fig. 6I). Mutational activation of Wnt signalling aberrantly We detected no further significant changes in the total transactivates downstream TCF4/beta-catenin target genes, expression levels of TCF4 in MESP2-knockdown cells (Fig. 6H). It further contributing to malignant transformation in human is well known that the ability of the transcription factor to perform cancers. In addition, we examined that MESP2 overexpression its role always relies on transport between the cytoplasm and drastically decreased direct binding between beta-catenin and nucleus, which is controlled by specific sequences. Impressively, TCF4, on the basis that MESP2 did not bind to beta-catenin the bHLH domain of TCF4 contains specific sequences i.e. a (Fig. 6J). Luciferase reporter assay showed that TCF4 activity nuclear localization signal that overlaps two nuclear export signals decreased after overexpression of MESP2 (Fig. 6K). Altogether, [23]. Consequently, we found that the amount of TCF4 in the these observations support the proposal that MESP2 competes Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 5 MESP2 regulates p27 ubiquitination and degradation. A, B protein and mRNA levels of p27 in MESP2-knockdown cells were examined via western blotting and qRT-PCR. C. Western blots of cells overexpressing GFP or MESP2 after treatment with CHX (100 μg/ml) for a specific period. **P < 0.01. D GC cells with shGFP or shMESP2 were dealt with vehicle (−) or MG-132 (50 μg/ml) about 6 h prior to being immunoblotted for the protein expression levels of p27. E Effect of MESP2 knockdown on p27 ubiquitination in 293FT cells. with beta-catenin for binding to TCF4 to inhibit activation of the nucleotides and region are important in determining SKP2 TCF4/beta-catenin transcriptional complex. transcription, bioinformatics analyses using the promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/promoter/), MESP2 overexpression lessened the occupancy of TCF4/beta- JASPAR (http://jaspar.genereg.net/) were performed to identify catenin on SKP2 promoter, leading to subsequent the DNA bindings motifs of TCF4 (Fig. 7B). Further analyses of accumulation of p27 TCF4 binding sites revealed that the promoter of SKP2 contains four To narrow down potential MESP2 migratory targets that are binding motifs in a relatively concentrated and overlapped region relevant across GC, we analyzed the intersection between MESP2- (Fig. 7C). Then, we sought to identify whether MESP2 could affect the KD DEGs and migratory sample DEGs (GSE15459 and GSE34942). association of TCF4 with the SKP2 promoter. As expected, TCF4 could We found 30 overlapping genes. GO function enrichment analysis bind to the SKP2 promoter. Importantly, MESP2 overexpression for DEGs were performed using the DAVID (Fig. 7A). The results of significantly inhibited the association of TCF4 with the SKP2 GO function enrichment analysis for DEGs indicated that DEGs promoter (Fig. 7D). Next, luciferase reporter assay showed that were mainly enriched in post−translational protein modification transfection of TCF4 increased the luciferase activity of the SKP2 and extracellular matrix (ECM). Interestingly, for the cell compo- reporter in comparison with that in the control group (Fig. 7E). nent, the DEGs were enriched in SCF ubiquitin ligase complex Impressively, the luciferase activity of the SKP2 reporter was notably (Supplementary Fig. 2B). inhibited after co-transfection of MESP2 and TCF4 (Fig. 7E).Finally,we Previous studies have demonstrated that SKP2 is a major SCF found that knockdown of SKP2 and MESP2 suppressed the ubiquitin ligase that regulates p27 ubiquitination [17]. Thus, we proliferation and migration of GC cells (Figs. 7F–I), which further speculate that the accumulation of p27 by overexpressing ascertained that MESP2 regulated p27 ubiquitination through SKP2. MESP2 may be due to the inhibition of SKP2 transcription Consistently, these related protein expression levels were partially mediated by the TCF4/beta-catenin complex. To identify which inhibited (Fig. 7I). Similarly, there was a similar trend in the patient Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 6 MESP2 binds competitively to TCF4 to inhibit activation of the TCF4/beta-catenin transcriptional complex. A A plot of the TCF- dependent signalling in response to the Wnt pathway from gene set enrichment analysis (GSEA) of genes negatively correlated with MESP2 expression. B Schematic diagrams of the bHLH domain organization for human MESP2 and ITF2. C A prediction of the interaction between the MESP2 and TCF4 was made using the STRING website. D Immunofluorescence co-localization staining of Flag-MESP2 and TCF4 in HGC-27 and MKN-45 cells. Scale bar, 5 μm. E Interaction of endogenous MESP2 with endogenous TCF4. F Mapping of TCF4-binding domains on MESP2. HA-MESP2 (aa 1–397), HA-Mb (aa 79–137), HA-MΔ (aa 1–79,137–397), Flag-TCF4 (aa 1–619) Flag-T1 (aa 1–55) and Flag-TΔ (aa 56–619) were co- transfected in pairs into 293FT cells. G Proximity ligation assay indicating the interaction of MESP2 and TCF4 in HGC-27 and MKN-45 cells (red: PLA positive signal; blue: DAPI, scale bar = 5 μm). H TCF4 levels in MESP2-knockdown HGC-27 and MKN-45 cells. **P < 0.01. I Subcellular fractionation assays display TCF4 levels. Cytoplasmic α-tubulin and nuclear Histone H3 were used as reference standard. J Co-IP assays were used to examine TCF4 binding to beta-catenin after MESP2 overexpression. K Wnt-reporter luciferase activity in 293FT cells with or without MESP2 depletion and TCF4 overexpression. ***P < 0.001. Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 7 The depletion of MESP2 enhances the occupancy of TCF4/beta-catenin on the SKP2 promoter. A Venn diagram indicating 30 differential genes identified in all three cohorts. B JASPAR analysis of the DNA-binding motifs of TCF4. C Identification of TCF4 binding site in the SKP2 promoter. D Five primer sets were designed within the human SKP2 promoter. A Chromatin immunoprecipitation assay was carried out using Flag antibody. IgG was the negative control. E Luciferase activity was examined 48 h post-transfection. The pGL3-basic vector was used as the negative control. F MTT assays showing the proliferation ability of GC cells with knockdown of both MESP2 and SKP2. G Representative fluorescent images and quantification for BrdU staining of GC cells expressing shMESP2/shGFP or shMESP2/shSKP2. Scale bar, 20 μm. H Transwell assays showing the migration ability of GC cells with knockdown of both MESP2 and SKP2. Scale bar, 20 μm. I Western blots showing the levels of proliferation and transfer-associated proteins mentioned above. Data shown are means ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. Cell Death Discovery (2023) 9:79 L. Ge et al. database, although it did not show a significant difference compelling need for further research, as it remains to be (Supplementary Fig. 2C). determined whether the anti-GC activity of MESP2 depends on its abilities to inhibit the TCF4/beta-catenin complex formation in the cytoplasm or on dissociation in the nucleus. DISCUSSION In summary, we report that MESP2 functions as a completely Although investigators have achieved some success in treating new tumor suppressor in GC. Mechanistically, MESP2 competes patients with GC, there are currently strict criteria for determining with beta-catenin to bind to TCF4, and blocks TCF4/beta-catenin the prognosis of patients with GC [24]. Hence, targeting tumor complex transcriptional activity on the SKP2 promoter, which in suppressors in GC is a promising therapeutic strategy. Herein, we turn, enhances p27 stability and expression levels through detected for the first time, a decline in MESP2 expression in gastric SKP2 suppression (Fig. 8). Accordingly, this study reveals MESP2 tumor tissues that were simultaneously negatively correlated with as a novel player in TCF4/beta-catenin signalling. We believe that GC grade. Consistent with this result, MESP2 upregulation was this is the first comprehensive characterization of MESP2 role and intensively correlated with better prognosis, suggesting that mechanism in GC cells, implying that MESP2 is a prospective MESP2 may play a role in the inhibition of GC progression. It is therapeutic target for patients with GC. now becoming firmly established that MESP2 levels show signs of a periodic pattern that is necessary for the formation of segmental borders [25]. Other studies have demonstrated heritable muta- MATERIALS AND METHODS tions of MESP2 in human patients with heritable axial skeleton Cell lines and cultures All cell lines (HGC-27, MKN-45, SGC-7901, BGC-823, GES-1 and 293FT) were growth disorder [26]. Nevertheless, the function of MESP2 in derived from American Type Culture Collection (ATCC, Beijing, China) and human cancers have not been characterized till date. were cultured as described [31]. All cell lines tested negative for mycoplasma. Next, we found that a reduction in MESP2 expression levels increased proliferative and metastatic behaviors, and upregulation of MESP2 decreased relevant behaviors of GC cells. Indeed, MESP2 Protein extraction, western blotting, and overexpression strikingly inhibited GC cell proliferation via a G1 co-immunoprecipitation (co-IP) assays RIPA buffer, supplemented with protease and phosphatase inhibitors, was phase arrest, the cell cycle was regulated by cyclin-dependent utilized in cell lysis. Then sediments were removed by centrifugation at kinases, and the inhibitor p27 suppressed cell proliferation by 12,000 rpm and 4 °C for 10 min, and the supernatants were separated for hampering the cell cycle [27]. Accumulated evidence shows that western blot experiments. The Nuclear and Cytoplasmic Protein Extraction elevated p27 expression eventually causes growth inhibition in Kit (Beyotime, Shanghai, China) was utilized to analyze protein distribution. various human cancers [28]. We noticed a decrease in p27 levels, 30 µg of protein were used in equal amounts for western blot experiments. but p27 mRNA levels did not change after knockdown of MESP2. For co-IP assays, cell lysates with specific antibodies were incubated on a Reduced p27 expression is correlated with high rates of recurrence turn table at 4°C overnight. Then, Protein A/G Agarose (Santa Cruz, Dallas, and metastasis in a variety of malignancies. As documented, the Texas, USA) was added for antibody attachment and the immunopreci- p27 expression status is a key independent prognostic element in pitants were boiled for SDS-PAGE. Details about the antibodies used in this study are described in Supplementary Table 1. patients with GC [29]. Given that the impact of p27 can be disrupted in human cancers by excess proteolysis, C-terminal phosphorylation, or reduced translation, we found that deletion of immunohistochemical (IHC) staining MESP2 leading to p27 downregulation was due to SKP2-mediated Embedded tumor tissue sections in paraffin were probed with MESP2 ubiquitination through the proteasome pathway. Meanwhile, primary antibody (1:200, #bs-18796R, BIOSS, Beijing, China) or Ki67 primary antibody (1:200, #27309-1-AP, Proteintech, Wuhan, China), then tested as knockdown of SKP2 partially suppressed the growth and described [32, 33]. metastasis of GC cells enhanced via downregulation of MESP2. Furthermore, we provided evidence for the potential anti- cancer application of MESP2 as a marker for targeted therapy RNA extraction and qRT-PCR involving TCF4/beta-catenin signalling. After knockdown of Total RNA was extracted using TRIzoL as previously described [34]. NanoDrop ND-2000 instrument (Yeasen, Shanghai, China) was used for MESP2, we found that the activity of theTCF4/beta-catenin RNA concentration measurement. GoScript™ Reverse Transcription System transcription complex via the Wnt pathway and the expression Kit (Promega, Beijing, China) was used for complementary DNA synthesis. of its downstream target genes (MMPs) increased in GC cells. The qRT-PCR was used to examine cell mRNA levels. Details about the primers Wnt pathway dominates cell proliferation, cell differentiation, and used in qRT-PCR are described in Supplementary Table 2. EMT, which play increasingly crucial roles in human cancers [30]. Based on these results, we present a new hypothesis that TCF4/ shRNAs and Plasmids or lentiviruses for transfection or beta-catenin signalling is controlled inhibited by MESP2, but infection becomes significantly activated in the carcinoma stage due to shMESP2, shSKP2, and shGFP were purchased from Sangon and cloned MESP2 loss. Specifically, MESP2 suppressed formation of the TCF4/ into the pLKO.1 vector. Sequences of all the shRNAs and plasmids used are beta-catenin complex by competing with beta-catenin for binding provided in Supplementary Table 3. Lentiviruses were used for transfection to TCF4, which resulted in the inhibition of SKP2 transcriptional or infection as described [35]. activation and subsequent p27 accumulation. GSEA further confirmed that the action of MESP2 as a suppressor of TCF4/ Cell proliferation, migration, and invasion assays beta-catenin activity further regulated SKP2 /p27 signalling in GC. As previously mentioned, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazo- Interestingly, aside from MESP2, another bHLH transcription lium bromide (MTT) assays and bromodeoxyuridine (BrdU) staining were factor, ITF2, was also ascertained to inhibit activation of the TCF4/ used to assess cell proliferation [34]. Migration, invasion, and wound beta-catenin transcriptional complex. Different from MESP2, ITF2 healing assays were used for assessing the migratory and invasive directly interacted with beta-catenin, thereby reducing the binding behaviors of cells as described [35]. between TCF4 and beta-catenin [13]. These findings indicated that the different locations of the bHLH domains of MESP2 and ITF2 led Patient data analysis and patient tumor tissues to the differences in their biomolecular mechanisms. Of note, Patient data and gene expression data sets were obtained from R2: MESP2 was not co-precipitated with the TCF4/beta-catenin microarray analysis and visualization platform (http://hgserver1.amc.nl/ complex, which signified that TCF4, beta-catenin, and MESP2 did cgibin/r2/main.cgi). Kaplan–Meier analysis and survival curves were carried not form a trimeric complex. Considering the observation of MESP2 out by using GraphPad Prism (version 6.0, GraphPad Software, San Diego, in the cytoplasm and nucleus of GC cells, we have a particularly CA, USA). All cutoff values for separating high and low expression groups Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 8 Hypothesis diagram illustrates the mechanisms of MESP2 roles in GC. MESP2 and beta-catenin competitively bind to TCF4, which suppresses GC progression by regulating the SKP2/p27 axis. were determined by the online R2 database algorithm. Primary tumor were freely available online, and this study did not involve any experiment on specimens and prior approval were gained from the Ethics Committee of humans or animals performed by any of the authors. Daping Hospital (Chongqing, China). Tissue analysis was approved by the Ethics Committee of the Southwest University of China. All the patients Proximity ligation assay technique provided written informed consent to participate. A proximity ligation assay was performed using Duolink Proximity Ligation Assay reagents according to the supplier’s guidelines (Sigma- Animal studies Aldrich, Shanghai, China). Overexpred Flag-Mesp2 cells were cultured on All animal experiments were permitted by the Animal Care and Use the sliver. The cells were washed twice with 1 ml of PBS and fixed with 4% Committee of Southwest University and carried out in accordance with the paraformaldehyde. Then perforated with 0.3% Triton solution. After sealed Animal Care and Use Guidelines (Ministry of Science and Technology, with 10% goat serum (purchased by Beyotime, Shanghai, China), Paired Beijing, China). Five-week-old female nude mice were purchased and were combinations of mouse (anti-Flag,Proteintech, 66008-3-Ig, 1:50) and rabbit raised in SPF room for a week to adapt to the new environment. (anti-TCF4, Proteintech, 13838-1-AP, 1:50) antibodies were incubated with Subcutaneous xenotransplantation. Human GC lines (HGC-27) cells (1 × the samples for overnight at 4 °C temperature. Proximity ligation assay 10 cells) were stably transfected with shGFP or shMESP2, grown in 100 μl minus and PLA plus probes (containing the secondary antibodies medium (mixed with Matrigel at a 1:1 ratio), and injected hypodermically conjugated with oligonucleotides) were added and incubated 2 h at 4 °C. into the mice using a 1 ml Hamilton microliter syringe. Six female nude Afterwards, further oligonucleotides are added, allowed to hybridise to the mice were used in each group. After euthanizing the mice, the tumors PLA probes, and ligase joins the two hybridised oligonucleotides to a were removed, photographed, and weighed. closed circle. The DNA is then amplified, and detection of the amplicons In vivo metastasis assay. Five million HGC-27 cells that stably expressing was carried out using the Duolink® In Situ Red detection kit (including shGFP or shMESP2 were injected into the lateral tail vein of 5-week-old Hoechst 33342 dye nuclear staining were mounted with Vectashield female nude mice. Six female nude mice were used in each group. All mice mounting media), resulting in red fluorescence signals. were raised 25 days in SPF room and finally killed by cervical dislocation. All lung samples were collected. Chromatin immunoprecipitation GC cells were cultured in 10 cm culture dish followed by 1% formaldehyde Ubiquitination assay treatment for 10 min. Cells were pelletized and a supersonic device was The 293FT cells were co-transfected with relevant plasmid for 48 h. Then used for shearing DNA into fragments between 200 and 500 bps. After cells were dealt with MG-132 (50 μg/mL) for 6 h. Western blot and co-IP chromatin sonication and centrifugation, 2 µg of Flag antibody (#8146, assays were performed after cell lysis. CST, Shanghai, China) or normal IgG were added overnight. The precipitated DNA was analyzed via qRT-PCR and the primers used are listed in Supplementary Table 4. Expression microarray profiling Total RNAs were extracted from shGFP- and shMESP2-transfected HGC-27 cells using TRIzoL reagent. All samples were analyzed by Sangon Luciferase reporter assay (Shanghai, China) according to mRNA expression microarray protocols. The SKP2 promoter and TCF-reporter plasmid (TOP Flash and FOP Flash) were subcloned into pGL-basical vector (Promega), and 293FT cells were co-transfected with luciferase reporter and pGL-TK reporter for 48 h. The GSEA and GO analysis luciferase activity was measured using a Dual-Luciferase Reporter Assay kit GSEA analysis was performed by folding change data from differential (YEASEN, Shanghai, China) according to the manufacturer’s instructions at expression analysis into GSEA software (Broad Institute). GO analysis was 24 h post-transfection. performed with marker genes from each subcluster by tools from DAVID. The gene expression datasets (GSE15459 and GSE34942) analyzed in this study were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/ Statistical analysis geo/). GSE15459 and GSE34942 was based on platform GPL570 ([HG- All experiments were carried out with three technical and biological replicates. U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array). 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Skp2 over- Supplementary information The online version contains supplementary material expression is highly representative of intrinsic biological aggressiveness and available at https://doi.org/10.1038/s41420-023-01367-4. independently associated with poor prognosis in primary localized myxofi- brosarcomas. Clin Cancer Res: Off J Am Assoc Cancer Res. 2006;12:487–98. Correspondence and requests for materials should be addressed to Xiaoxue Ke or 20. Razavipour SF, Harikumar KB, Slingerland JM. p27 as a transcriptional regulator: Hongjuan Cui. new roles in development and cancer. Cancer Res. 2020;80:3451–8. 21. Jeannot P, Nowosad A, Perchey RT, Callot C, Bennana E, Katsube T, et al. p27 Reprints and permission information is available at http://www.nature.com/ promotes invadopodia turnover and invasion through the regulation of the reprints PAK1/Cortactin pathway. eLife. 2017;6:e22207. 22. Bencivenga D, Caldarelli I, Stampone E, Mancini FP, Balestrieri ML, Della Ragione Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims F, et al. p27 and human cancers: A reappraisal of a still enigmatic protein. Cancer in published maps and institutional affiliations. Lett. 2017;403:354–65. Cell Death Discovery (2023) 9:79 L. Ge et al. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the SKP2/p27 axis

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www.nature.com/cddiscovery ARTICLE OPEN MESP2 binds competitively to TCF4 to suppress gastric cancer progression by regulating the SKP2/p27 axis ✉ ✉ 1,3 1,3 1 2 2 1 1,2 1,2 Lingjun Ge , Gaichao Zhao , Chao Lan , Houji Song , Dan Qi , Pan Huang , Xiaoxue Ke and Hongjuan Cui © The Author(s) 2023 Gastric cancer (GC) is a major cause of human deaths worldwide, and is notorious for its high incidence and mortality rates. Mesoderm Posterior Basic Helix-loop-helix (bHLH) transcription factor 2 (MESP2) acts as a transcription factor with a conserved bHLH domain. However, whether MESP2 contributes to tumorigenesis and its potential molecular mechanisms, remain unexplored. Noticeably, MESP2 expression levels are decreased in GC tissues and cell lines compared to those in normal tissue. Further, in vitro and in vivo experiments have confirmed that MESP2 overexpression suppresses GC cell growth, migration, and invasion, whereas MESP2 knockdown results in the exact opposite. Here, we present the first report that MESP2 binds to transcription factor 7-like 2 (TCF7L2/TCF4) to inhibit the activation of the TCF4/beta-catenin transcriptional complex, decrease the occupancy of the complex on the S-phase kinase Associated Protein 2 (SKP2) promoter, and promote p27 accumulation. MESP2 knockdown facilitated tumorigenesis, which was partially suppressed by SKP2 knockdown. Taken together, we conclude that MESP2 binds competitively to TCF4 to suppress GC progression by regulating the SKP2/p27 axis, thus offering a potential therapeutic strategy for future treatment. Cell Death Discovery (2023) 9:79 ; https://doi.org/10.1038/s41420-023-01367-4 INTRODUCTION protein structure composed of a basic region [9] that interacts Gastric cancer (GC), a deadly malignancy, is the third main cause with DNA and a neighboring helix–loop–helix region that of global human cancer-related deaths, resulting in ~723000 mediates dimerization [10]. Most bHLH dimers recognize the E- deaths annually [1]. Nearly half of the cases occur in Eastern Asia, box, a hexameric sequence in the DNA with the consensus especially in China [1, 2]. Clinically, early disease development is sequence CANNTG [10]. These bHLH TFs modulate gene expres- rarely accompanied by GC symptoms [3], and patients have poor sion through dimer formation, combining activators or repressors rates of survival, mainly due to suboptimal treatment and late with ubiquitous proteins (E-proteins) [11]. Previous experiments diagnosis [1]. There is therefore, an urgent need for elucidating have showed that aberrant expression of the bHLH transcription the potential molecular mechanisms of GC progression and factor is related to tumorigenesis [12], suggesting that MESP2 may determining effective therapeutic strategies. also participate in human cancer progression. Mesoderm posterior 2 (MESP2), is a transcription factor with a The Wnt signalling pathway influences the regulation of basic helix–loop–helix (bHLH) motif that has been illustrated to intestinal development, differentiation, and adult tissue home- play pivotal roles during the development of mammalian somite ostasis, thus contributing to the progression of numerous human and heart. MESP2 links spatiotemporal information, produced by cancers [13]. Transcription factor 7-like 2 (TCF7L2/TCF4) is a key the determination wave front and the segmentation clock, to component of the Wnt signalling pathway, mostly acting as a multiple somite morphogenesis processes in mouse somitogen- transcription factor after entering the nucleus [14]. Furthermore, esis [4]. The controlled segmentation program of the Notch Wnt ligands trigger the typical Wnt/beta-catenin signalling path- signalling pathway reflects the important function of MESP2, and way, which in turn activates the LRP and Frizzled receptors, the determination of the segment boundary of the Wnt3a/beta- leading to beta-catenin stabilization and nucleus translocation. catenin signalling pathway was demonstrated by an MESP2 Subsequently, nuclear beta-catenin binds to TCF4, thereby expression assay of Wnt3a mutants [5]. In fact, boundary promoting the expression of relevant genes [15]. In the intestinal formation and somite compartmentalization seem to depend on epithelium, the expression levels of TCF4/beta-catenin down- the combined action of Wnt and Notch signalling. Interrupting stream target genes are regulated by mutational activation of Wnt either pathway restrains somite formation and the MESP2 protein signalling, then affecting malignant transformation of colorectal levels that indicate somite compartmentalization and boundary cancer [13]. The ultimate aim of the typical Wnt signalling in the formation [6]. The bHLH family is one of the most prominent beta-catenin pathway is the formation of the TCF4/beta-catenin among transcription factors [7] and is involved in cell differentia- complex, and this interaction is regarded as a valid target for tion and tissue development [8]. These TFs share a characteristic cancer treatment [16]. 1 2 State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Cancer Center, Medical Research Institute, Southwest University, Chongqing 400716, China. These authors contributed equally: Lingjun Ge, Gaichao Zhao. email: kexiaoxue@126.com; hcui@swu.edu.cn Received: 21 July 2022 Revised: 7 February 2023 Accepted: 13 February 2023 Official journal of CDDpress 1234567890();,: L. Ge et al. The F-box protein S-phase kinase-associated protein 2 (SKP2) is a tumor suppressor role of MESP2 in vivo, and discovered that subunit that recognizes substrates of the SCF–SKP2 E3 ligase, knockdown of MESP2 promoted tumor growth and volume targeting protein proteasome-mediated degradation, such as p27 (Fig. 3E, F). Furthermore, IHC staining implied that tumors formed [17]. The deletion of p27 and tumorigenesis often go hand-in- by MESP2 knockdown cells exhibited higher Ki67 and lower hand. The influence of p27 in tumorigenesis is supported by the p27 staining than those formed by control cells (Fig. 3G, −/− formation of spontaneous pituitary tumors in p27 -mice [18]. Supplementary Fig. 1C). Functionally, the CDK suppressor, p27, is a typical negative After assessing the functions of MESP2 in GC cell growth, we regulator of cell proliferation. It can inhibit the G1-S transition of confirmed its function in metastasis. Transwell assays demon- cyclinE/CDK2 complex and the S phase of cyclinA/CDK2 complex strated that migration and invasion behaviors were significantly [19]. Physiological regulation of G1-S transformation is crucial in inhibited by MESP2 overexpression (Fig. 2E), but enhanced by determining cell fate and is lost during oncogenic transformation. knockdown of MESP2 (Fig. 4B). Wound-healing assays also In addition, in a range of cancer types, the declined cytoplasmic confirmed the above results (Fig. 4A). Western blot experiments p27 protein expression is pertinent to high metastasis and poor using epithelial-mesenchymal transition (EMT)-related markers survival. The loss of p27 affects self-renewal of hematopoietic stem and matrix metalloproteinases (MMPs), displayed same trends. cells and the proliferation and differentiation of hematopoietic Upregulation of MESP2 decreased MMP2 and MMP9 levels (Fig. progenitor cells, making cells prone to tumor transformation [20]. 2F). Nevertheless, Knockdown of MESP2 reduced E-cadherin In this work, considering that MESP2 expression levels are protein levels, but upregulated N-cadherin, and MMPs protein reduced in GC tissues and cell lines, we focused on MESP2 levels (Fig. 4C, Supplementary Fig. 1B). Subsequently, we assessed regulation and function, which to our knowledge, are uninvesti- the influence of MESP2 on lung metastasis. The number of gated in cancer. Functionally, our study revealed that MESP2 binds pulmonary metastatic nodules in MESP2 downregulation group competitively with beta-catenin to TCF4, suppressing GC progres- greatly increased in comparison to the control group (Fig. 4D). sion by regulating the SKP2/p27 axis. Thus, MESP2 is a candidate Hematoxylin and eosin (HE) staining revealed that treatment with in the diagnosis and therapy of GC. shMESP2 effectively promoted tumor cell metastasis (Fig. 4E). Collectively, these results strongly imply that MESP2 can serve as a potential tumor suppressor in GC. RESULTS MESP2 as a diagnostic/prognostic marker for GC MESP2 induced the stabilization of p27 by inhibiting its To explore the functional importance of MESP2 in GC, we first ubiquitylation and subsequent degradation examined the intracellular localization of MESP2 in GC cells. The Meanwhile, we observed that MESP2 downregulation decreased findings showed that it was located in both the cytoplasm and the protein levels of p27 (Fig. 5A), which acted as a CDK inhibitor nucleus (Fig. 1A, B). Next, we analyzed MESP2 levels in 80 tissue and controlled cell proliferation and metastasis [21]. Multiple samples of clinical patients via IHC staining. MESP2 IHC staining studies, including our previous research, have showed that p27 can was notably lower in gastric tumor tissues in a grade-dependent combine and suppress the activity of the cyclin E/CDK2 complex to pattern compared to that in normal tissues (Fig. 1C–E). qRT-PCR block the G1 phase [22]. To further elucidate the regulatory and western blot experiments showed that mRNA and protein mechanisms of MESP2-stabilizing p27 expression, p27 mRNA levels expression of MESP2 were downregulated in HGC-27, MKN-45, in the experimental and control groups were assessed via qRT-PCR. SGC-7901 and BGC-823, contrasted with that of GES-1, a normal Contrary to p27 protein levels, MESP2 knockdown did not impact human gastric epithelial cell (Fig. 1F). Moreover, Analysis of MESP2 p27 mRNA levels significantly (Fig. 5B). Therefore, we speculated expression levels in GC patient tumors using the R2: Genomics that a post-transcriptional mechanism was involved in the changes Analysis and Visualization Platform (https://hgserver1.amc.nl/cgi- in p27 expression levels. We evaluated the protein stability of p27 bin/r2) consistently confirmed that MESP2 upregulation predicted by introducing a cyclohexamide (CHX), and found that MESP2 better patient survival (Fig. 1G–J). Taken together, our results overexpression significantly lengthened the half-life of p27 indicate that MESP2 may be an anti-oncogene. (Fig. 5C), revealing that the p27 expression is positively regulated by MESP2. Knockdown of MESP2 induced p27 degradation and Overexpression of MESP2 suppressed GC progression in vitro was blocked by Proteasome inhibitors MG-132 treatment (Fig. 5D), and in vivo, but downregulation resulted in the opposite supporting the ubiquitination-proteasome pathway to maintain To further investigate the impact of MESP2, GO analysis showed p27 protein levels. Additionally, the ubiquitylation assay showed that differentially expressed genes were mainly enriched in the that downregulation of MESP2 enhanced the poly-ubiquitylation of biological regulation and by the molecular function regulator p27 (Fig. 5E). Finally, transcriptome profiling showed that MESP2 (Supplementary Fig. 1A). depletion suppressed the p27 pathway (Supplementary Fig. 2A). Then we successfully overexpressed MESP2 in in HGC-27 and Based on these results, we concluded that MESP2 indeed improved MKN-45 cells lines (Fig. 2A). Besides, knocked down MESP2 the protein stability of p27 by suppressing its ubiquitylation and expression using the lentiviral shRNA, namely, shMESP2 nos. 1 subsequent degradation. and 2 (Fig. 3A, B). In MTT assays, MESP2 overexpression substantially suppressed cell growth (Fig. 2B). Conversely, knock- MESP2 inhibited activation of the TCF4/beta-catenin down of MESP2 promoted GC cell proliferation, compared with transcriptional complex by competing with beta-catenin for control cells (Fig. 3C). To further examine the molecular mechanism binding TCF4 of MESP2-mediated inhibition of cell growth, we utilized flow We further characterized the biological functions of MESP2 and cytometry to determine the effect of MESP2 on cell cycle deciphered the mechanisms of its roles in GC inhibition via progression. We showed that MESP2 overexpression arrested the transcriptome profiling, which revealed that MESP2 depletion cell cycle at the G0/G1 phase (Fig. 2C). Subsequently, BrdU staining activated TCF-dependent signalling in response to the Wnt signified that BrdU-positive rates in MESP2- upregulated cells were pathway (Fig. 6A). The known TCF4/beta-catenin transcriptional reduced (Fig. 2D). The changes noted in the knockdown group complex has great effects on the Wnt pathway. Through were different (Fig. 3D). Consistent with this finding, the levels of functional domain analyses, Studies have reported that ITF2 and critical G0/G1 regulon genes, such as CDK4, CCND1,and CCNE1, MESP2 exhibited the relatively well-conserved bHLH domain decreased with MESP2 upregulation, whereas knockdown of (Fig. 6B), and a previous study had showed that ITF2 suppressed MESP2 led to the reverse (Fig. 2F, Fig. 3B, Supplementary formation of the TCF4/beta-catenin complex by competing with Fig. 1B). We used a mouse xenograft model to verify the putative beta-catenin for TCF4 [13]. Hence, we assessed through an online Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 1 MESP2 expression is downregulated in GC tissues and related to high prognosis. A Subcellular fractionation assays display MESP2 levels. Cytoplasmic α-tubulin and nuclear Histone H3 were used as reference standards. B Intracellular localization analysis of Flag-MESP2 by immunofluorescence staining. Intracellular localization of Flag-MESP2 (red); Nuclei (blue) were stained with DAPI. Scale bar, 5 μm. C Representative images of MESP2 expression at different levels in gastric tumor (I, II, III) and normal tissues. Scale bar, 20 μm. D, E. IHC staining analyses of MESP2 expression levels in 80 samples of normal tissues and gastric tumor tissues. ***P < 0.001. F qRT-PCR and Western blot experiments showing MESP2 mRNA and protein levels in GC cell lines (HGC-27, MKN- 45, SGC-7901, BGC-823, GES-1). Data shown are means ± SD, n= 3, **P <0.01, ***P <0.001. G Box plot of MESP2 expression levels in GC tissues in the R2 database. H Kaplan-Meier analysis of progression-free survival using data from R2 databases with the log-rank test P-values indicated. I, J. Kaplan-Meier analysis of progression-free survival was performed for patients with different grades and subtypes of GC using data from R2 databases with the log-rank test P-values indicated. Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 2 MESP2 inhibits the proliferation, migration and invasion ability of GC cells both in vitro and in vivo. A qRT-PCR analysis of MESP2 mRNA levels in MESP2-overexpression cells. B MTT assays showing suppressed GC cell proliferation by MESP2 overexpression. C The HGC-27 and MKN-45 cell cycles were detected via flow cytometry after overexpression of MESP2. D Representative fluorescent images and quantification of BrdU staining of GC cells expressing GFP or MESP2. Scale bar, 20 μm. E The migratory capacity of GC cells transfected with GFP or MESP2, as determined via transwell assays. Scale bar, 20 μm. Data shown are means ± SD, n = 3, *P < 0.1, **P < 0.01. F Western blots showing the levels of G1 phase regulatory and pivotal transfer-associated proteins. prediction website whether MESP2 and TCF4 had a binding interaction between TCF4 and MESP2, we truncated the full length relationship (Fig. 6C). We further validated whether both proteins of TCF4 into a T1 fragment with beta-catenin binding. Similarly, integrated with each other under experimental conditions, and the full-length MESP2 was truncated into an Mb fragment with the immunofluorescence staining showed that MESP2 was co- bHLH domain (Fig. 6F). Next, for domain-mapping experiments, expressed with TCF4 in HGC-27 cells (Fig. 6D). Then, co-IP assays we transfected 293FT cells with Flag-TCF4 (amino acids (aa) 1–619) showed that TCF4 was immunoprecipitated by MESP2 (Fig. 6E). To or Flag-T1 (aa 1–55) or Flag-TΔ (aa 56–619) of TCF4 and gain further insight into the specific domains that mediated the immunoprecipitated these against HA to observe their interaction Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 3 Knockdown MESP2 promotes the proliferation ability of GC cells both in vitro and in vivo. A qRT-PCR was performed to assess MESP2 mRNA levels in MESP2-knockdown cells. B Western blot experiments showing G1 phase regulatory protein levels in MESP2-knockdown cells. C. MTT assays showing that MESP2 knockdown promoted GC cell proliferation. D Representative fluorescent images and quantification for BrdU staining of GC cells expressing shGFP, shMESP2#1, or shMESP2#2. Scale bar, 20 μm. E, F In vivo analyses of size and volume of xenograft tumors that were hypodermically injected with treated HGC-27 and MKN-45 cells. G Representative IHC micrographs of selected tumors. Scale bar, 20 μm. Data shown are means ± SD, n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001. Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 4 Knockdown MESP2 promotes the migration and invasion of GC cells in vitro and in vivo. A, B The migration and invasion capacities of GC cells transfected with shGFP alone, shMESP2#1 or shMESP2#2 were determined via Wound healing and transwell assays. Scale bar, 20 μm. C Western blot experiments showing the levels of several pivotal transfer-associated proteins. D Images showing that the shMESP2 mouse model had more pulmonary nodules (black arrows) than the others. E Histological analysis of pulmonary tumors as observed via H&E staining are shown. Scale bar, 2 mm. Data shown are means ± SD, n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001. with Flag-TCF4 or Flag-T1 or Flag-TΔ in vivo. Both TCF4 and T1 nuclear fraction increased remarkably after knocking down MESP2 bound to MESP2 or M1 (Fig. 6F). (Fig. 6I). Mutational activation of Wnt signalling aberrantly We detected no further significant changes in the total transactivates downstream TCF4/beta-catenin target genes, expression levels of TCF4 in MESP2-knockdown cells (Fig. 6H). It further contributing to malignant transformation in human is well known that the ability of the transcription factor to perform cancers. In addition, we examined that MESP2 overexpression its role always relies on transport between the cytoplasm and drastically decreased direct binding between beta-catenin and nucleus, which is controlled by specific sequences. Impressively, TCF4, on the basis that MESP2 did not bind to beta-catenin the bHLH domain of TCF4 contains specific sequences i.e. a (Fig. 6J). Luciferase reporter assay showed that TCF4 activity nuclear localization signal that overlaps two nuclear export signals decreased after overexpression of MESP2 (Fig. 6K). Altogether, [23]. Consequently, we found that the amount of TCF4 in the these observations support the proposal that MESP2 competes Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 5 MESP2 regulates p27 ubiquitination and degradation. A, B protein and mRNA levels of p27 in MESP2-knockdown cells were examined via western blotting and qRT-PCR. C. Western blots of cells overexpressing GFP or MESP2 after treatment with CHX (100 μg/ml) for a specific period. **P < 0.01. D GC cells with shGFP or shMESP2 were dealt with vehicle (−) or MG-132 (50 μg/ml) about 6 h prior to being immunoblotted for the protein expression levels of p27. E Effect of MESP2 knockdown on p27 ubiquitination in 293FT cells. with beta-catenin for binding to TCF4 to inhibit activation of the nucleotides and region are important in determining SKP2 TCF4/beta-catenin transcriptional complex. transcription, bioinformatics analyses using the promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/promoter/), MESP2 overexpression lessened the occupancy of TCF4/beta- JASPAR (http://jaspar.genereg.net/) were performed to identify catenin on SKP2 promoter, leading to subsequent the DNA bindings motifs of TCF4 (Fig. 7B). Further analyses of accumulation of p27 TCF4 binding sites revealed that the promoter of SKP2 contains four To narrow down potential MESP2 migratory targets that are binding motifs in a relatively concentrated and overlapped region relevant across GC, we analyzed the intersection between MESP2- (Fig. 7C). Then, we sought to identify whether MESP2 could affect the KD DEGs and migratory sample DEGs (GSE15459 and GSE34942). association of TCF4 with the SKP2 promoter. As expected, TCF4 could We found 30 overlapping genes. GO function enrichment analysis bind to the SKP2 promoter. Importantly, MESP2 overexpression for DEGs were performed using the DAVID (Fig. 7A). The results of significantly inhibited the association of TCF4 with the SKP2 GO function enrichment analysis for DEGs indicated that DEGs promoter (Fig. 7D). Next, luciferase reporter assay showed that were mainly enriched in post−translational protein modification transfection of TCF4 increased the luciferase activity of the SKP2 and extracellular matrix (ECM). Interestingly, for the cell compo- reporter in comparison with that in the control group (Fig. 7E). nent, the DEGs were enriched in SCF ubiquitin ligase complex Impressively, the luciferase activity of the SKP2 reporter was notably (Supplementary Fig. 2B). inhibited after co-transfection of MESP2 and TCF4 (Fig. 7E).Finally,we Previous studies have demonstrated that SKP2 is a major SCF found that knockdown of SKP2 and MESP2 suppressed the ubiquitin ligase that regulates p27 ubiquitination [17]. Thus, we proliferation and migration of GC cells (Figs. 7F–I), which further speculate that the accumulation of p27 by overexpressing ascertained that MESP2 regulated p27 ubiquitination through SKP2. MESP2 may be due to the inhibition of SKP2 transcription Consistently, these related protein expression levels were partially mediated by the TCF4/beta-catenin complex. To identify which inhibited (Fig. 7I). Similarly, there was a similar trend in the patient Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 6 MESP2 binds competitively to TCF4 to inhibit activation of the TCF4/beta-catenin transcriptional complex. A A plot of the TCF- dependent signalling in response to the Wnt pathway from gene set enrichment analysis (GSEA) of genes negatively correlated with MESP2 expression. B Schematic diagrams of the bHLH domain organization for human MESP2 and ITF2. C A prediction of the interaction between the MESP2 and TCF4 was made using the STRING website. D Immunofluorescence co-localization staining of Flag-MESP2 and TCF4 in HGC-27 and MKN-45 cells. Scale bar, 5 μm. E Interaction of endogenous MESP2 with endogenous TCF4. F Mapping of TCF4-binding domains on MESP2. HA-MESP2 (aa 1–397), HA-Mb (aa 79–137), HA-MΔ (aa 1–79,137–397), Flag-TCF4 (aa 1–619) Flag-T1 (aa 1–55) and Flag-TΔ (aa 56–619) were co- transfected in pairs into 293FT cells. G Proximity ligation assay indicating the interaction of MESP2 and TCF4 in HGC-27 and MKN-45 cells (red: PLA positive signal; blue: DAPI, scale bar = 5 μm). H TCF4 levels in MESP2-knockdown HGC-27 and MKN-45 cells. **P < 0.01. I Subcellular fractionation assays display TCF4 levels. Cytoplasmic α-tubulin and nuclear Histone H3 were used as reference standard. J Co-IP assays were used to examine TCF4 binding to beta-catenin after MESP2 overexpression. K Wnt-reporter luciferase activity in 293FT cells with or without MESP2 depletion and TCF4 overexpression. ***P < 0.001. Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 7 The depletion of MESP2 enhances the occupancy of TCF4/beta-catenin on the SKP2 promoter. A Venn diagram indicating 30 differential genes identified in all three cohorts. B JASPAR analysis of the DNA-binding motifs of TCF4. C Identification of TCF4 binding site in the SKP2 promoter. D Five primer sets were designed within the human SKP2 promoter. A Chromatin immunoprecipitation assay was carried out using Flag antibody. IgG was the negative control. E Luciferase activity was examined 48 h post-transfection. The pGL3-basic vector was used as the negative control. F MTT assays showing the proliferation ability of GC cells with knockdown of both MESP2 and SKP2. G Representative fluorescent images and quantification for BrdU staining of GC cells expressing shMESP2/shGFP or shMESP2/shSKP2. Scale bar, 20 μm. H Transwell assays showing the migration ability of GC cells with knockdown of both MESP2 and SKP2. Scale bar, 20 μm. I Western blots showing the levels of proliferation and transfer-associated proteins mentioned above. Data shown are means ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. Cell Death Discovery (2023) 9:79 L. Ge et al. database, although it did not show a significant difference compelling need for further research, as it remains to be (Supplementary Fig. 2C). determined whether the anti-GC activity of MESP2 depends on its abilities to inhibit the TCF4/beta-catenin complex formation in the cytoplasm or on dissociation in the nucleus. DISCUSSION In summary, we report that MESP2 functions as a completely Although investigators have achieved some success in treating new tumor suppressor in GC. Mechanistically, MESP2 competes patients with GC, there are currently strict criteria for determining with beta-catenin to bind to TCF4, and blocks TCF4/beta-catenin the prognosis of patients with GC [24]. Hence, targeting tumor complex transcriptional activity on the SKP2 promoter, which in suppressors in GC is a promising therapeutic strategy. Herein, we turn, enhances p27 stability and expression levels through detected for the first time, a decline in MESP2 expression in gastric SKP2 suppression (Fig. 8). Accordingly, this study reveals MESP2 tumor tissues that were simultaneously negatively correlated with as a novel player in TCF4/beta-catenin signalling. We believe that GC grade. Consistent with this result, MESP2 upregulation was this is the first comprehensive characterization of MESP2 role and intensively correlated with better prognosis, suggesting that mechanism in GC cells, implying that MESP2 is a prospective MESP2 may play a role in the inhibition of GC progression. It is therapeutic target for patients with GC. now becoming firmly established that MESP2 levels show signs of a periodic pattern that is necessary for the formation of segmental borders [25]. Other studies have demonstrated heritable muta- MATERIALS AND METHODS tions of MESP2 in human patients with heritable axial skeleton Cell lines and cultures All cell lines (HGC-27, MKN-45, SGC-7901, BGC-823, GES-1 and 293FT) were growth disorder [26]. Nevertheless, the function of MESP2 in derived from American Type Culture Collection (ATCC, Beijing, China) and human cancers have not been characterized till date. were cultured as described [31]. All cell lines tested negative for mycoplasma. Next, we found that a reduction in MESP2 expression levels increased proliferative and metastatic behaviors, and upregulation of MESP2 decreased relevant behaviors of GC cells. Indeed, MESP2 Protein extraction, western blotting, and overexpression strikingly inhibited GC cell proliferation via a G1 co-immunoprecipitation (co-IP) assays RIPA buffer, supplemented with protease and phosphatase inhibitors, was phase arrest, the cell cycle was regulated by cyclin-dependent utilized in cell lysis. Then sediments were removed by centrifugation at kinases, and the inhibitor p27 suppressed cell proliferation by 12,000 rpm and 4 °C for 10 min, and the supernatants were separated for hampering the cell cycle [27]. Accumulated evidence shows that western blot experiments. The Nuclear and Cytoplasmic Protein Extraction elevated p27 expression eventually causes growth inhibition in Kit (Beyotime, Shanghai, China) was utilized to analyze protein distribution. various human cancers [28]. We noticed a decrease in p27 levels, 30 µg of protein were used in equal amounts for western blot experiments. but p27 mRNA levels did not change after knockdown of MESP2. For co-IP assays, cell lysates with specific antibodies were incubated on a Reduced p27 expression is correlated with high rates of recurrence turn table at 4°C overnight. Then, Protein A/G Agarose (Santa Cruz, Dallas, and metastasis in a variety of malignancies. As documented, the Texas, USA) was added for antibody attachment and the immunopreci- p27 expression status is a key independent prognostic element in pitants were boiled for SDS-PAGE. Details about the antibodies used in this study are described in Supplementary Table 1. patients with GC [29]. Given that the impact of p27 can be disrupted in human cancers by excess proteolysis, C-terminal phosphorylation, or reduced translation, we found that deletion of immunohistochemical (IHC) staining MESP2 leading to p27 downregulation was due to SKP2-mediated Embedded tumor tissue sections in paraffin were probed with MESP2 ubiquitination through the proteasome pathway. Meanwhile, primary antibody (1:200, #bs-18796R, BIOSS, Beijing, China) or Ki67 primary antibody (1:200, #27309-1-AP, Proteintech, Wuhan, China), then tested as knockdown of SKP2 partially suppressed the growth and described [32, 33]. metastasis of GC cells enhanced via downregulation of MESP2. Furthermore, we provided evidence for the potential anti- cancer application of MESP2 as a marker for targeted therapy RNA extraction and qRT-PCR involving TCF4/beta-catenin signalling. After knockdown of Total RNA was extracted using TRIzoL as previously described [34]. NanoDrop ND-2000 instrument (Yeasen, Shanghai, China) was used for MESP2, we found that the activity of theTCF4/beta-catenin RNA concentration measurement. GoScript™ Reverse Transcription System transcription complex via the Wnt pathway and the expression Kit (Promega, Beijing, China) was used for complementary DNA synthesis. of its downstream target genes (MMPs) increased in GC cells. The qRT-PCR was used to examine cell mRNA levels. Details about the primers Wnt pathway dominates cell proliferation, cell differentiation, and used in qRT-PCR are described in Supplementary Table 2. EMT, which play increasingly crucial roles in human cancers [30]. Based on these results, we present a new hypothesis that TCF4/ shRNAs and Plasmids or lentiviruses for transfection or beta-catenin signalling is controlled inhibited by MESP2, but infection becomes significantly activated in the carcinoma stage due to shMESP2, shSKP2, and shGFP were purchased from Sangon and cloned MESP2 loss. Specifically, MESP2 suppressed formation of the TCF4/ into the pLKO.1 vector. Sequences of all the shRNAs and plasmids used are beta-catenin complex by competing with beta-catenin for binding provided in Supplementary Table 3. Lentiviruses were used for transfection to TCF4, which resulted in the inhibition of SKP2 transcriptional or infection as described [35]. activation and subsequent p27 accumulation. GSEA further confirmed that the action of MESP2 as a suppressor of TCF4/ Cell proliferation, migration, and invasion assays beta-catenin activity further regulated SKP2 /p27 signalling in GC. As previously mentioned, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazo- Interestingly, aside from MESP2, another bHLH transcription lium bromide (MTT) assays and bromodeoxyuridine (BrdU) staining were factor, ITF2, was also ascertained to inhibit activation of the TCF4/ used to assess cell proliferation [34]. Migration, invasion, and wound beta-catenin transcriptional complex. Different from MESP2, ITF2 healing assays were used for assessing the migratory and invasive directly interacted with beta-catenin, thereby reducing the binding behaviors of cells as described [35]. between TCF4 and beta-catenin [13]. These findings indicated that the different locations of the bHLH domains of MESP2 and ITF2 led Patient data analysis and patient tumor tissues to the differences in their biomolecular mechanisms. Of note, Patient data and gene expression data sets were obtained from R2: MESP2 was not co-precipitated with the TCF4/beta-catenin microarray analysis and visualization platform (http://hgserver1.amc.nl/ complex, which signified that TCF4, beta-catenin, and MESP2 did cgibin/r2/main.cgi). Kaplan–Meier analysis and survival curves were carried not form a trimeric complex. Considering the observation of MESP2 out by using GraphPad Prism (version 6.0, GraphPad Software, San Diego, in the cytoplasm and nucleus of GC cells, we have a particularly CA, USA). All cutoff values for separating high and low expression groups Cell Death Discovery (2023) 9:79 L. Ge et al. Fig. 8 Hypothesis diagram illustrates the mechanisms of MESP2 roles in GC. MESP2 and beta-catenin competitively bind to TCF4, which suppresses GC progression by regulating the SKP2/p27 axis. were determined by the online R2 database algorithm. Primary tumor were freely available online, and this study did not involve any experiment on specimens and prior approval were gained from the Ethics Committee of humans or animals performed by any of the authors. Daping Hospital (Chongqing, China). Tissue analysis was approved by the Ethics Committee of the Southwest University of China. All the patients Proximity ligation assay technique provided written informed consent to participate. A proximity ligation assay was performed using Duolink Proximity Ligation Assay reagents according to the supplier’s guidelines (Sigma- Animal studies Aldrich, Shanghai, China). Overexpred Flag-Mesp2 cells were cultured on All animal experiments were permitted by the Animal Care and Use the sliver. The cells were washed twice with 1 ml of PBS and fixed with 4% Committee of Southwest University and carried out in accordance with the paraformaldehyde. Then perforated with 0.3% Triton solution. After sealed Animal Care and Use Guidelines (Ministry of Science and Technology, with 10% goat serum (purchased by Beyotime, Shanghai, China), Paired Beijing, China). Five-week-old female nude mice were purchased and were combinations of mouse (anti-Flag,Proteintech, 66008-3-Ig, 1:50) and rabbit raised in SPF room for a week to adapt to the new environment. (anti-TCF4, Proteintech, 13838-1-AP, 1:50) antibodies were incubated with Subcutaneous xenotransplantation. Human GC lines (HGC-27) cells (1 × the samples for overnight at 4 °C temperature. Proximity ligation assay 10 cells) were stably transfected with shGFP or shMESP2, grown in 100 μl minus and PLA plus probes (containing the secondary antibodies medium (mixed with Matrigel at a 1:1 ratio), and injected hypodermically conjugated with oligonucleotides) were added and incubated 2 h at 4 °C. into the mice using a 1 ml Hamilton microliter syringe. Six female nude Afterwards, further oligonucleotides are added, allowed to hybridise to the mice were used in each group. After euthanizing the mice, the tumors PLA probes, and ligase joins the two hybridised oligonucleotides to a were removed, photographed, and weighed. closed circle. The DNA is then amplified, and detection of the amplicons In vivo metastasis assay. Five million HGC-27 cells that stably expressing was carried out using the Duolink® In Situ Red detection kit (including shGFP or shMESP2 were injected into the lateral tail vein of 5-week-old Hoechst 33342 dye nuclear staining were mounted with Vectashield female nude mice. Six female nude mice were used in each group. All mice mounting media), resulting in red fluorescence signals. were raised 25 days in SPF room and finally killed by cervical dislocation. All lung samples were collected. Chromatin immunoprecipitation GC cells were cultured in 10 cm culture dish followed by 1% formaldehyde Ubiquitination assay treatment for 10 min. Cells were pelletized and a supersonic device was The 293FT cells were co-transfected with relevant plasmid for 48 h. Then used for shearing DNA into fragments between 200 and 500 bps. After cells were dealt with MG-132 (50 μg/mL) for 6 h. Western blot and co-IP chromatin sonication and centrifugation, 2 µg of Flag antibody (#8146, assays were performed after cell lysis. CST, Shanghai, China) or normal IgG were added overnight. The precipitated DNA was analyzed via qRT-PCR and the primers used are listed in Supplementary Table 4. Expression microarray profiling Total RNAs were extracted from shGFP- and shMESP2-transfected HGC-27 cells using TRIzoL reagent. All samples were analyzed by Sangon Luciferase reporter assay (Shanghai, China) according to mRNA expression microarray protocols. The SKP2 promoter and TCF-reporter plasmid (TOP Flash and FOP Flash) were subcloned into pGL-basical vector (Promega), and 293FT cells were co-transfected with luciferase reporter and pGL-TK reporter for 48 h. The GSEA and GO analysis luciferase activity was measured using a Dual-Luciferase Reporter Assay kit GSEA analysis was performed by folding change data from differential (YEASEN, Shanghai, China) according to the manufacturer’s instructions at expression analysis into GSEA software (Broad Institute). GO analysis was 24 h post-transfection. performed with marker genes from each subcluster by tools from DAVID. The gene expression datasets (GSE15459 and GSE34942) analyzed in this study were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/ Statistical analysis geo/). GSE15459 and GSE34942 was based on platform GPL570 ([HG- All experiments were carried out with three technical and biological replicates. U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array). 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Skp2 over- Supplementary information The online version contains supplementary material expression is highly representative of intrinsic biological aggressiveness and available at https://doi.org/10.1038/s41420-023-01367-4. independently associated with poor prognosis in primary localized myxofi- brosarcomas. Clin Cancer Res: Off J Am Assoc Cancer Res. 2006;12:487–98. Correspondence and requests for materials should be addressed to Xiaoxue Ke or 20. Razavipour SF, Harikumar KB, Slingerland JM. p27 as a transcriptional regulator: Hongjuan Cui. new roles in development and cancer. Cancer Res. 2020;80:3451–8. 21. Jeannot P, Nowosad A, Perchey RT, Callot C, Bennana E, Katsube T, et al. p27 Reprints and permission information is available at http://www.nature.com/ promotes invadopodia turnover and invasion through the regulation of the reprints PAK1/Cortactin pathway. eLife. 2017;6:e22207. 22. Bencivenga D, Caldarelli I, Stampone E, Mancini FP, Balestrieri ML, Della Ragione Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims F, et al. p27 and human cancers: A reappraisal of a still enigmatic protein. Cancer in published maps and institutional affiliations. Lett. 2017;403:354–65. Cell Death Discovery (2023) 9:79 L. Ge et al. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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