The γ-secretase catalytic subunit PSEN1, regulated by YY1, promotes lung adenocarcinoma progression via the Notch1/EGFR pathway

doi:10.21203/rs.3.rs-4811218/v1

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The γ-secretase catalytic subunit PSEN1, regulated by YY1, promotes lung adenocarcinoma progression via the Notch1/EGFR pathway

https://doi.org/10.21203/rs.3.rs-4811218/v1

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Backgroud: Serving as a pivotal catalytic component of the γ-secretase, PSEN1 is recognized for its significant influence on the initiation and advancement of tumors. Nevertheless, the specific function and the fundamental mechanisms by which PSEN1 contributes to lung adenocarcinoma (LUAD) are yet to be elucidated. This study aimed to elucidate the role and regulation mechanism of PSEN1 in LUAD.

Methods: The expression of PSEN1 in LUAD tissues and cells was determined using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). The functional experiments in vitro and in vivo were used to investigate the effects of PSEN1 on LUAD progression. Bioinformatics analysis were used to initially identify the upstream transcription factors of PSEN1. Chromatin immunoprecipitation (ChIP) was used to validate the potential transcription factors of PSEN1 in LUAD.

Results: This study revealed distinct differential expression patterns of PSEN1 in different human tissues as well as in pan-cancer. PSEN1 is genetically altered in human cancers at a low frequency of about 1.2% and the genomic alteration is dominated by Missense Mutation, with the types varying according to the cancer type. And this genomic alteration does not affect the survival of cancer patients. Additionally, we found that PSEN1 was over-expressed in LUAD tumor tissues and associated with poor prognosis. PSEN1 promoted the malignant behaviors of LUAD both in vitro and in vivo. PSEN1 activated the Notch1/EGFR pathway and promoted the epithelial-mesenchymal transition (EMT) process in LUAD. Moreover, YY1 can directly bind to the promoter of the PSEN1 to promote its transcription to accelerate the progression of LUAD.

Conclusions: Our findings clarify that the YY1/PSEN1/Notch1/EGFR axis is associated with LUAD progression, and may provide new promising therapeutic targets for LUAD therapy.

γ-secretase

PSEN1

lung adenocarcinoma(LUAD).

Lung adenocarcinoma (LUAD), a predominant subtype of non-small cell lung carcinoma (NSCLC), is characterized by its high mortality, and complex, as well as its intricate and varied etiological factors (1). With the rapid development of medicine, LUAD prognosis has improved well, but still a considerable number of patients have not received significant clinical benefit. Further understanding of the mechanisms underlying the development of LUAD is still needed to explore more alternative therapeutic strategies.

Mutations in presenilin (PS), a key constituent of the γ-secretase complex, are significantly associated with the development of early-onset familial hereditary Alzheimer's disease (AD). The genes PSEN1 and PSEN2 are responsible for encoding presenilin proteins PS1 and PS2, respectively. Among Alzheimer's disease (AD) patients, PSEN1 is frequently mutated, accounting for approximately 70 to 80 percent of cases. In contrast, alterations in PSEN2 are less common (2). PSEN1, which shares a high degree of similarity with PSEN2, has been implicated in multiple cancer-related pathways (3–5). PSEN1 can cut the Notch family proteins to convert them into the activated Notch intracellular domain (NICD), which subsequently modulates the transcriptional activity of an array of specific genes (6). It also interacts with several oncogenic pathways such as Wnt/β-catenin, PI3K/AKT/mTOR and RAS/RAF/MEK (7–9).

The molecular biology of PSEN1 has not been characterized in human pan-cancer, and its expression pattern, molecular mechanism and clinical significance in LUAD are also currently unknown. In this study, we systematically characterized the differential expression model, genetic variation landscape and subcellular localization of PSEN1 in human pan-cancer, deeply explored the differential expression of PSEN1 in LUAD tumors and normal tissues and its correlation with prognosis, and revealed the potential mechanism of the regulation of LUAD progression, so as to provide a new field for exploring the potential therapeutic strategies for LUAD.

2.1 Clinical tissue specimens

Human LUAD tissues and paired adjacent normal tissues were collected from the Renmin Hospital of Wuhan University. All the patients were pathologically diagnosed as LUAD. Specimens were frozen in liquid nitrogen immediately after in vitro and stored at -80 ℃. All patients provided written informed consent, and the study was approved by the Ethics Committee of the Renmin Hospital of Wuhan University.

2.2 Public data preprocessing and bioinformatics analysis

The RNA-seq data for LUAD was downloaded from the Cancer Genome Atlas (TCGA) GDC (https://portal.gdc.cancer.gov/). As verification and supplement, three chip datasets (GSE10072, GSE30219 and GSE68465) for lung adenocarcinoma were downloaded from the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/). GSE10072 (10) included RNA data from 58 tumor tissues and 49 non-tumor tissues. GSE30219 (11) included RNA data from 85 tumor tissues and 14 non-tumor tissues. While GSE68465 (12) included RNA data from 443 tumor tissues and 19 non-tumor tissues. In addition, several large databases, including UALCAN(http://ualcan.path.uab.edu/analysis-prot.html), HPA(https://www.proteinatlas.org), cBioPortal(https://www.cbioportal.org/), SangerBox(http://sangerbox.com), LinkedOmics(http://www.linkedomics.orglogin.php), Xiantaoxueshu(https://www.xiantaozi.com/), String(https://string-db.org/), JASPAR(http://jaspar.genereg.net/ ), and ALGGEN-PROMO(http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/ promoinit.cgi?dirDB = TF_8.3), were also used for the online analysis.

2.3 Cell culture and treatment

A549 (CL-0016) and PC9 (CL-0668) cell lines were purchased from Wuhan Pricella Biotechnology Co., Ltd. A549 cells were cultured in F-12K medium (Gibco, USA) based complete medium and PC9 cells in RPMI-1640 medium (Gibco, USA) based complete medium. The complete medium contained 10% FBS and 100 µg/ml of penicillin-streptomycin. Cells were placed in a 37 C, 5% CO₂ environment. Cells were transfected with the siRNA / plasmid to interfere with gene expression. The specific siRNAs of PSEN1 and YY1 were purchased from Beijing Qingke Biological Co., Ltd. The human PSEN1 overexpression pCMV3-PSEN1 vector was obtained from Obio Technologies (China). Cells were infected with lentivirus (Genechem, Shanghai) and subsequently screened for puromycin (2.0 µg/ml) for two weeks to construct stable transfer cell lines.

2.4 qRT-PCR and Western Blot

Total RNA was extracted from cells using RNA extraction reagent (Servicebio, Wuhan) and reverse transcribed to cDNA using Servicebio®RT Enzyme Mix (Servicebio, Wuhan) following the manufacturer's protocol. The quantitative real time-polymerase chain reaction (qRT-PCR) was performed using 2×SYBR Green qPCR Master Mix (Servicebio, Wuhan) and GAPDH was used as endogenous control. The specifc primers used are listed in Table S1. The process for Western Blot includes protein “extraction of the samples”, “SDS-PAGE gel electrophoresis separation”, “transfer to PVDF or nitrocellulose membrane”, “sealing”, “antibody incubation” and “signal detection”. The details of the antibodies utilized in this research are presented in Table S2.

2.5 CCK-8 and EdU assays

CCK-8 and EdU assays were used to assess cell proliferation. After cells from each group were cultured for 0h, 24h, 48h, 72h and 96h, the cell plates were removed and CCK-8 assays were performed according to the manufacturer's protocol. For EdU assay, cells were cultured for 24 h in 6-well plates and incubated with EdU for 2 h at 37℃. Then, cells were stained with DAPI and cells were visualized under a fluorescence microscope.

2.6 Transwell assay

The transwell assay was performed with reference to the previous description (13). Differently, in transwell assay, we also calculated the migration and invasion rates while calculating the number of migration / invasion cells.

2.7 Immunofluorescence and immunohistochemistry

Immunofluorescence (IF) and immunohistochemistry (IHC) were performed as described previously (13, 14).

2.8 Molecular docking

The structures for molecular docking were predicted using native AlphaFold/2.3.1 and the predicted structures were visualized using PyMOL software.

2.9 ChIP-qPCR

Chromatin immunoprecipitation assay (ChIP) were performed using chromatin immunoprecipitation kits (Wanleibio, Lot: 15122020) according to the manufacturer's instructions. The data analysis of Q-PCR adopts the “ΔΔ Ct” method as follows: ΔCt [normalized ChIP] = (Ct [ChIP] - (Ct [Input] -Log2 (Input Dilution Factor)), Input Dilution Factor(IDF)= (fraction of the inputchromatin saved) ⁻¹, ΔΔCt [ChIP/NIS] = ΔCt [normalized ChIP] - ΔCt [ IgG ], %Input = 2 ^{^} (CtInput-CtChIP)×Fd×100%, Fold Enrichment = 2^ (-ΔΔCt [ChIP/NIS]).

2.10 Animal study

All procedures involving animals were carried out following the the guidelines of the Renmin Hospital of Wuhan University's Laboratory Animal Ethics Committee. Sixteen male BALB/c nude mice (5 weeks old, 15-20g) were randomly divided into four groups (A549-NC, A549-KD, PC9-NC, and PC9-KD). The resuspended cells (5×10⁶/ml, 100ul PBS) were injected subcutaneously into the left armpit of the mice, and the tumor growth was followed up every 5 days to calculate the volume of each tumor. After 30 days, the mice were killed and their tumor weight was examined.

2.11 Statistical analysis

All the experimental results were repeated no less than three times. Differences between groups were compared by t-test, chi-square test and one-way ANOVA. A P < 0.05 indicates that the differences were statistically significant. The statistical analysis of this study was done using the Graphpad Prism 9 and R 3.6.1 software.

3.1 The expression of PSEN1 in different human tissues and its differential expression pattern in pan-cancer

Based on the HPA data, we analyzed the expression of PSEN1 in various tissues and organs of the human body (Fig. 1A). PSEN1 expression is relatively high in gastrointestinal tissues and organs, especially in the jejunum and small intestine. And its expression is relatively low in skeletal muscle and epididymis. Furthermore, PSEN1 presents a differential expression pattern in the vast majority of human cancer types (Fig. 1B). For example, PSEN1 is highly expressed in tumor tissues of multiforming glioma (GBM), endometrial carcinoma (UCEC), breast invasive carcinoma (BRCA), cervical squamous carcinoma and adenocarcinoma (CESC), lung adenocarcinoma (LUAD), esophageal carcinoma (ESCA), gastric and esophageal carcinoma (STES), mixed renal carcinoma (KIRAN), colorectal adenocarcinoma (COADERAD), prostate cancer (PRAD), gastric adenocarcinoma (STAD), head and neck squamous cell carcinoma (HNSC), clear cell carcinoma, renal clear cell carcinoma (KIRC), lung squamous carcinoma (LUSC), hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (B LCA), thyroid cancer (THCA), uterine sarcoma (UCS), pheochromocytomas, and paragangliomas (PCPG). Protein expression data from the CPTAC database also showed that PSEN1 shows high expression status in tumor tissues of many cancer types, such as endometrial cancer, lung cancer, liver cancer, etc. (Fig. 1C).

Moreover, we also tried to further reveal the expression status of PSEN1 in the cells of each tissue from the single-cell level. We found that PSEN1 was significantly enriched in oligodendrocytes and monocytes (Figure S1A). From the SINGLE CELL TISSUES OVERVIEW (Figure S1B), we found the level of single-cell RNA enrichment of PSEN1 in each tissue/organ varies by tissue cell specificity. For example, the brain and ocular tissues mainly include neuronal cells and glial cells, and PSEN1 is predominantly enriched in oligodendrocytes. Lung tissue included specialized epithelial cells, blood & immune cells, endothelial cells, mesenchymal cells, glandular epithelial cells, and muscle cells, while PSEN1 was mainly enriched in macrophages in blood & immune cells (Figure S1C).

3.2 Genomic variation landscape and its subcellular localization of PSEN1 in human cancers

Data from cBioPortal demonstrate the frequency and type of genomic variation of the PSEN1 in human cancers. We found that the frequency of PSEN1 gene alteration in human pan-cancer was not high, at about 1.2%, and it was dominated by missense mutation, followed by CNV amplification and deep deletion (Figure S2A). We also further explored the specific proportion of each mutation type of the PSEN1 in each cancer type by SangerBox, as shown in Figure S2B. Most genomic alterations of PSEN1 are dominated by missense mutation. The amino acid range of PSEN 1 is 467AA. Furthermore, we explored the genetic alteration status of the PSEN1 in different types of cancer in the TCGA cohort. As shown in Figure S2C, PSEN1 was most frequently altered in patients with mature B cell tumors with “mutation” and “amplification” as the major changes, over 6%. However, in patients with NSCLC, genomic alterations in PSEN1 are dominated by “mutation”, “amplification” and “deep deletion”. Figure S2D also shows the 3 D structure of PSEN1 and its specific mutation types and sites in cancer. Overall, PSEN1 is genetically altered with low frequency in human cancers, and the types of its genomic alterations vary by cancer type. This genomic alteration did not affect the survival of cancer patients (Figure S3).

Next, we also investigated the expression of PSEN1 in various cell lines (Fig. 2A). PSEN1 showed relatively high expression in PC-3 and HaCaT as well as THP-1. Among them, THP-1 is a monocyte, which is consistent with the previous single-cell RNA analysis results. The function of genes is closely related to their protein subcellular localization. Subsequently, we also investigated the protein localization of PSEN1 in the three human cancer cells based on the immunofluorescence data from HPA (Fig. 2B). We found that PSEN1 was mainly localized to golgi apparatus, nucleoplasm, and cell Junctions. Figure 2C shows this situation more intuitively.

3.3 PSEN1 was highly expressed in LUAD tissues and associated with poor prognosis

In the previous part, we systematically investigated the expression pattern and gene variation landscape of PSEN1 in pan-cancer. Next, we focused on the exploration of the role of PSEN1 in a specific cancer (LUAD). Based on the TCGA paired and unpaired LUAD samples, we observed a higher expression of PSEN1 in the tumor tissues (Fig. 3A-B). We also observed similar conditions in the three GEO datasets (Fig. 3C-D). Figure S4 illustrates the protein expression levels based on PSEN1 in the CPTAC dataset. Figure S5 also validated the above results at the IHC level. Subsequently, to validate the above results, we assessed the levels of PSEN1 protein expression in LUAD samples and adjacent normal tissues samples from the Renmin Hospital of Wuhan University. IHC showed that PSEN1 was overexpressed in LUAD tumor tissues compared with adjacent normal lung tissue (Fig. 3F), which confirms our preliminary observations from external datasets. To further confirm this expression pattern, we randomly detected PSEN1 protein expression in LUAD tumor tissues and paired adjacent normal tissues by Western blot (Fig. 3G), and the results also indicated that PSEN1 showed high expression status in tumor tissues.

Given that the potential role of PSEN1 in LUAD has not been fully elucidated, we also performed a Kaplan-Meier survival analysis based on the information of 535 LUAD samples from the TCGA-LUAD dataset. We found that high expression of PSEN1 was associated with worse overall survival (Fig. 3H). The same phenomenon was observed in subsequent subgroup analyses (Figure S6). In addition, we also performed survival analysis validation based on the information of 399 LUAD samples from the GSE72094 dataset. The result, as we expected, was that patients expressing high PSEN1 had a shorter overall survival (Fig. 3I).

3.4 PSEN1 promotes the malignant phenotype of LUAD cells in vitro

We further explored the effect of PSEN1 on the malignant biological behavior of LUAD cells. We transfected cells with the siRNA-PSEN1 and PSEN1 overexpression plasmids. The qRT-PCR results showed that the RNA expression level of PSEN1 in the si-PSEN1-#1 and si-PSEN1-#2 groups was significantly lower than that in the si-NC group (Fig. 4A), while the RNA expression level of PSEN1 in the OE-PSEN1 group was significantly higher than that in the control group (Vector group) (Fig. 4B). Subsequent Western blot results also confirmed the success of the PSEN 1 knockdown / overexpression treatment. In the CCK-8 assay, the proliferation rates in the si-PSEN1-#1 and si-PSEN1-#2 groups were markedly reduced compared to the si-NC group (Fig. 4C). Conversely, the cell proliferation rate was significantly higher in the OE-PSEN1 group than that in the Vector group (Fig. 4D). In the EdU assay, the number of EdU-positive cells in the si-PSEN1-#1 and si-PSEN1-#2 groups was significantly lower than that in the si-NC group (Fig. 4E-F). In contrast, a substantial increase in the proportion of EdU-positive cells was observed in the OE-PSEN1 group compared to the Vector group (Fig. 4E-F). These results suggest that PSEN1 can promote the proliferation of LUAD cells in vitro.

We subsequently investigated the effect of PSEN1 on the metastasis of LUAD cells. In the transwell migration assay, a significant reduction in the number of cells that migrated was noted in the si-PSEN1-#1 and si-PSEN1-#2 groups when compared to the si-NC group, as shown in Fig. 5A. Conversely, the migration of cells in the OE-PSEN1 group was considerably higher than that of the Vector group, illustrated in Fig. 5B. This result indicates that PSEN1 can promote the cell migration of both A549 and PC9 in vitro.

In the Transwell invasion assay, it was noted that the count of cells that successfully invaded through the membrane was considerably reduced in the si-PSEN1-#1 and si-PSEN1-#2 groups compared to the si-NC group, as shown in Fig. 5C. In contrast, the count of invasive cells in the OE-PSEN1 group was significantly elevated compared to the Vector group, as indicated in Fig. 5D. This result indicates that PSEN 1 promoted the cell invasion of A549 and PC9 in vitro.

Epithelial-mesenchymal transition (EMT) plays an important role in the development of LUAD. Therefore, we investigated the association of PSEN1 expression with EMT in LUAD cells. The expression levels of EMT-related proteins in LUAD cells (A549 and PC9) were determined by Western blot for 48 h after siRNA-based treatment. The results showed that the expression level of E-cadherin in si-PSEN1-#1 and si-PSEN1-#2 groups was significantly higher than that in si-NC group, while the expression level of N-cadherin and Vimentin was significantly lower than that in si-NC group (Fig. 5E-F). This initially indicates that knockdown of PSEN1 can inhibit EMT progression in human LUAD cells. Subsequently, we assessed the fluorescence intensity of proteins associated with EMT across various cell groups using immunofluorescence. The results showed that the fluorescence intensity of E-cadherin in si-PSEN1-#1 and si-PSEN1-#2 groups was significantly higher than that of si-NC group, conversely, the fluorescence intensity for N-cadherin and Vimentin was notably reduced compared to the si-NC group (Fig. 5G). This further suggested that knockdown of PSEN1 could inhibit EMT progression in human LUAD cells in vitro.

3.5 PSEN1 promotes the malignant phenotype of LUAD cells associated with the Notch1/EGFR pathway

To further investigate the mechanism of PSEN1 in LUAD, we compared differential genes between the PSEN1 high and low expression groups. Among them, 12780 genes were up-regulated in the PSEN1 high expression group, while 8064 genes were down-regulated (Fig. 6A). We also used the LinkedOmics database to conduct a pilot study on the genes coexpressed with PSEN1 in LUAD from the TCGA dataset. Figure 6B shows the genes showing a positive and negative correlation of expression with PSEN1. Subsequently, we crossed the differentially expressed genes in LUAD and the PSEN1-related genes, and finally obtained 3251 key genes (Fig. 6C). The pathway enrichment analysis revealed that the implicated genes predominantly participated in pathways such as the Notch signaling, ErbB signaling, and Ras signaling pathways (Fig. 6D). Considering the relevance of these pathways to cancer development and PSEN1, we speculate that the oncogenic role played by PSEN1 might also be relevant in the aforementioned pathways.

As the central catalytic component of the γ-secretase, variations and expression levels of PSEN1 can obviously directly influence the function and operational dynamics of the γ-secretase. Through the String analysis, we searched for proteins with existing interactions with PSEN1. We found that besides the other five subunits of γ-secretase, the proteins that directly interact with PSEN1 include NOTCH1, APP, CDH 1, TRAF6, CTNNB1, and CTNND2 (Fig. 7A). Notch, serving as a key transmembrane receptor, is among the primary substrates for the enzymatic action of γ-secretase. The Notch signaling pathway is also widely proven to be involved in cancer development. The functional enrichment analysis in this study also indicated that the oncogenic role of PSEN1 in LUAD may be associated with the Notch signaling pathway (Fig. 6D). To further clarify the interaction between NOTCH1 and PSEN1, we performed molecular docking on this topic. The results suggest that there is a high confidence interaction between NOTCH1 and PSEN 1 (Fig. 7B). In Western Blot, the expression level of Notch1 (NICD) in the si-PSEN1-#1 and si-PSEN1-#2 groups was significantly lower than that in the si-NC group (Fig. 7C-D). These results suggest that Notch1 is a direct downstream regulator of PSEN1. We also observed that the expression level of EGFR in the si-PSEN1-#1 and si-PSEN1-#2 groups was significantly lower than that in the si-NC group (Fig. 7C-D). Furthermore, at the transcriptional level, a pronounced positive association was observed between the expression levels of EGFR and PSEN1 (R = 0.393, p < 0.001) (Fig. 7E). The close relationship between Notch1 and EGFR was also fully demonstrated in several previous studies (15, 16). Therefore, we propose that PSEN1 can exert an oncogenic effect through regulation of the Notch1/EGFR pathway in LUAD.

3.6 YY1 enhances the malignant behavior of LUAD cells through the regulation of PSEN1 expression

Given that the transcription factors (TFs) play important roles in cancer, we therefore went on to explore the transcription factors that may regulate PSEN1 expression. First, we obtained the location of PSEN1 in the genome through the UCSC database, which is located in Chr 14:73136507–73223691. Their corresponding promoter sequences were also obtained. In the JASPAR database, by entering this promoter sequence and setting relative profile score threshold to 99.9%, we finally obtained 156 putative sites for a total of 66 TFs. The association of these 66 TFs with PSEN1 in LUAD is shown in Figure S7A. At the same time, considering the transcription factor results of PSEN1 predicted by ALGGEN-PROMO database (Figure S7B), we finally locked the transcription factors regulating PSEN1 to YY1, and Figure S7C shows the motif of YY1 binding to PSEN1promoter. We further confirmed the binding of YY1 to the PSEN1 promoter region by ChIP-qPCR (Fig. 7F). Subsequently, we performed YY1 knockdown in A549 cells, and the results of qRT-PCR and Western blot showed that the expression of PSEN1 was decreased after YY1 knockdown (Fig. 7G-H). In addition, based on the mRNA correlation analysis of the TCGA database, we also identified a distinct correlation linking YY1 with PSEN1 (R = 0.694, p < 0.001) (Fig. 7I). The above results indicate that YY1 is an upstream transcription factor of PSEN1 and can bind the PSEN1 promoter region to regulate the expression of PSEN1.

Previous studies (17, 18) showed that YY1 plays a crucial role in lung cancer, being overexpressed in tumor tissues and promoting the malignant cell phenotype. We next explored whether PSEN1 participates in the function of YY1 in LUAD cells. We noted that the suppression of YY1 substantially hindered the proliferation, migration, and invasion of LUAD cells. Conversely, elevating PSEN1 levels notably counteracted these inhibitory effects caused by YY1 knockdown (Fig. 8A-D). Since knockdown of PSEN1 inhibited EMT progression, we subsequently explored whether this process is regulated by YY1. We observed that the knockdown of YY1 enhanced the expression of E-cadherin and downregulated the expression of mesenchymal markers N-cadherin and Vimentin, and the overexpression of PSEN1 could largely reverse the EMT progression in LUAD cells caused by YY1 knockdown (Fig. 8E-F). Overall, these results indicate that PSEN1 is an important target of YY1 to promote LUAD progression.

3.7 PSEN1 promotes LUAD proliferation and EMT in vivo

To further investigate the role of PSEN1 in vivo, we injected the stable cell line PC9 as well as A549 cells into BALB/c nude mice to observe subcutaneous tumorigenesis in mice. Our findings indicated that the dimensions and mass of tumors in the PSEN1 knockdown group (KD) were considerably smaller than those in the control group (NC) (Fig. 9A-D). In addition, we analyzed the expression of EMT markers in tumor samples by IHC, and the results showed that the reduction in PSEN1 expression notably upregulated E-cadherin levels and concurrently reduced the levels of mesenchymal markers such as N-cadherin and Vimentin (Fig. 9E-G). These results suggest that PSEN1 can promote LUAD cell proliferation and EMT progression in vivo.

Current treatments have significantly improved the prognosis of LUAD patients, but there is still a large room for improvement. Further exploration of the mechanism of LUAD development is still needed to find potential molecular targets to improve prognosis. PSEN1 is the core catalytic subunit of the γ -secretase complex, which cleaves Notch family proteins to generate the activated Notch intracellular domain (NICD) and control the transcriptional activity of multiple target genes.It also interacts with several oncogenic pathways such as Wnt/ β -catenin, PI3K/AKT/ mTOR and RAS/RAF/MEK to participate in the development of malignant tumors (7–9). PSEN1 can exhibit either promoting or exhibiting cancer suppression functions in different tumor-specific genetic insults (3–5), which could potentially be associated with the distinct tissue-specific microenvironments present in various types of cancers. Therefore, it is necessary to investigate the pan-cancer biology of PSEN1. By integrating multiple online large data resources, we found that PSEN1 presents differential expression patterns in the vast majority of human cancer types. In this study, we discovered that PSEN1 levels were elevated in lung adenocarcinoma (LUAD) and linked to an unfavorable outcome. Prior investigations have reported PSEN1 overexpression in head and neck squamous cell carcinoma (HNSCC) (19), hepatocellular carcinoma (20)and esophageal carcinoma (21), with elevated levels correlating to more severe prognosis (20, 21).

The function of genes is closely related to their protein subcellular localization. PSEN1 protein was reported mainly localized to the endoplasmic reticulum, Golgi apparatus, and plasma membrane(22). We investigated the protein localization of PSEN1 in three human cancer cells based on HPA immunofluorescence data, and found that PSEN1 was mainly localized at the Golgi apparatus, nucleoplasm, and cell junctions. This suggests that the subcellular localization of PSEN1 has not exactly the same results in different studies.

In addition to investigating the expression pattern of PSEN1, we further explored the role and potential mechanism of PSEN1 in LUAD. Our findings indicated that PSEN1 knockdown inhibited LUAD proliferation and metastasis, whereas up-regulation of PSEN1 caused the opposite effect. EMT is an important link in the carcinogenesis process in most cancers (23). Several studies (24, 25) have implicated EMT with LUAD progression. This is in a line with our finding. Increasing evidence indicates that multiple signaling pathways play key roles in many cancers, such as PI3K/AKT (26), Notch (27), Wnt/β-catenin(28) signaling pathway, etc. The Notch signaling pathway, a pathway conserved across species, has been associated with the development of numerous types of human cancer due to its dysregulation. Given the substantial body of evidence highlighting Notch's significant contribution to cancer progression, targeting this pathway has emerged as a crucial strategy in oncology. It has been reported that Notch is a primary substrate for the γ-secretase. Upon cleavage by γ-secretase, the NICD is released and translocates to the nucleus, leading to the activation of downstream signaling pathways (29, 30). PSEN1, a crucial player in the development of multiple cancers, may have its influence modulated either by or independent of γ-secretase activity. Our study found that knockdown of PSEN1 decreased Notch1 (NICD) expression. Even, the molecular docking approach identified PSEN1 to interact directly with Notch1. Therefore, we propose that PSEN1 can play an oncogenic role through Notch1 in LUAD.

EGFR signaling has been demonstrated to play important roles in multiple cancers including lung cancer (31, 32). PSEN1 has been reported to activate EGFR signaling in colorectal cancer in a Notch1-signaling-dependent manner (33). We also explored whether PSEN1 affects the EGFR signaling pathway in LUAD. Our results indicated that the total protein level of EGFR decreased significantly after the knockdown of PSEN1. Meanwhile, bioinformatics analysis also found a pronounced positive association between EGFR and PSEN1 expression levels, which indicated that PSEN1 could promote the progression of LUAD by EGFR pathway. Bioinformatics studies have also revealed a pronounced positive association between EGFR and PSEN1 expression levels.

YY1 is a widely distributed intracellular transcription factor that belongs to the GLI-Kruppel class of zinc-finger proteins (34). In this study, with the help of JASPAR and ALGGEN-PROMO databases, we predicted the presence of YY1 binding sites in the promoter region of PSEN1, and bioinformatics analysis also revealed a pronounced positive association between the expression of the two. This was also confirmed by ChIP-qPCR. YY1 is reported to be overexpressed in various cancers and promotes cancer cell proliferation and metastasis, clearly associated with poor prognosis in tumor patients (35, 36). In lung cancer, YY1 was also identified as an oncogene, over-expressed in tumor tissues and promoting the malignant cell phenotype (17, 18). In our study, siRNA-YY1 significantly inhibited the proliferation, migration, and invasion of LUAD cells, whereas the over-expression of PSEN1 could partially rescue the effects caused by YY1 inhibition. Meanwhile, YY1 can promote EMT progression, which is consistent with previous studies (37, 38). Taken together these findings, we propose that YY1 can accelerate LUAD progression by regulating the transcriptional activity of PSEN1.

In conclusion, our results indicate that PSEN1 plays a crucial role in LUAD progression. Mechanistically, PSEN1, regulated by YY1, can promotes LUAD progression ability by activating the Notch1/ EGFR pathway and promoting EMT process. This study is the first to clarify that the YY1 / PSEN1 / Notch / EGFR axis is associated with LUAD progression and may provide new promising therapeutic targets.

Author contributions

CKS, MLG, YL and RYH made significant contributions to the study design, and article writing. CKS,MLG, NL, and QG designed the experiments. CKS, MLG and YL carried out the experiments and acquired the data. BYL, TLF, and WJW participated in drafting the article critically, and were responsible for clinical sample collection. QG and other co-authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the the Fundamental Research Funds for the Central Universities (2042023kf0011), National Natural Science Foundation of China (No.82300103, No.82102269).

Availability of data and materials

The datasets generated during and/or analysed during the current study are available in UALCAN, HPA, cBioPortal, SangerBox, LinkedOmics, Xiantaoxueshu, String, JASPAR, ALGGEN-PROMO, GEO and TCGA databases.

Ethics approval and consent to participate

All animal experiments were approved by the Laboratory Animal Ethics Committee of Renmin Hospital of Wuhan University. All procedures involving human participants in this study were approved by the Ethics Committee of the Renmin Hospital of Wuhan University. All patients included in the present study signed informed consent.

Consent for publication

All authors provided their consent to publish the study.

Competing interests

The authors declared that they had no competing interests.

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No competing interests reported.

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The γ-secretase catalytic subunit PSEN1, regulated by YY1, promotes lung adenocarcinoma progression via the Notch1/EGFR pathway

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Clinical tissue specimens

2.2 Public data preprocessing and bioinformatics analysis

2.3 Cell culture and treatment

2.4 qRT-PCR and Western Blot

2.5 CCK-8 and EdU assays

2.6 Transwell assay

2.7 Immunofluorescence and immunohistochemistry

2.8 Molecular docking

2.9 ChIP-qPCR

2.10 Animal study

2.11 Statistical analysis

3 Results

3.1 The expression of PSEN1 in different human tissues and its differential expression pattern in pan-cancer

3.2 Genomic variation landscape and its subcellular localization of PSEN1 in human cancers

3.3 PSEN1 was highly expressed in LUAD tissues and associated with poor prognosis

3.4 PSEN1 promotes the malignant phenotype of LUAD cells in vitro

3.5 PSEN1 promotes the malignant phenotype of LUAD cells associated with the Notch1/EGFR pathway

3.6 YY1 enhances the malignant behavior of LUAD cells through the regulation of PSEN1 expression

3.7 PSEN1 promotes LUAD proliferation and EMT in vivo

4 Discussion

Declarations

References

Additional Declarations

Supplementary Files

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