The PLOD2 promotes tumor progression in colorectal cancer through the PI3K-AKT pathway

doi:10.21203/rs.3.rs-5266104/v1

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The PLOD2 promotes tumor progression in colorectal cancer through the PI3K-AKT pathway

https://doi.org/10.21203/rs.3.rs-5266104/v1

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Based on reports, Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) constitutes an oncogenic gene that undergoes upregulation in multiple malignancies, encompassing cervical cancer, endometrial cancer, and lung cancer. PLOD2 is competent to stiffen the extracellular matrix and modify the ECM architecture. Despite the fact that PLOD2 has been demonstrated to be associated with an unfavorable prognosis in colorectal cancer (CRC), the regulatory mechanism and functionality of PLOD2 in human colorectal cancer remain enigmatic. In this investigation, we validated the elevated expression of PLOD2 in human CRC tissue and its correlation with tumor pathological staging. Concurrently, we discerned through public databases that the expression of PLOD2 is interrelated with patient prognosis. Furthermore, functional experiments have manifested that PLOD2 facilitates CRC cell metastasis and proliferation via the PI3K-AKT pathway and predicts the potential binding of compounds to its protein.

PLOD2

colorectal cancer

PI3K-AKT pathway

Colorectal cancer (CRC), characterized by its high incidence and mortality rates, ranks third and second, respectively, among all cancers worldwide[1]. D Owing to the advancement of sophisticated treatment modalities, the survival rate of patients with malignant tumors has been enhanced. Regrettably, tumor metastasis persists as one of the principal causes of death in patients with malignant tumors. Hence, this study is purposed to delineate the genetic alterations associated with the development and progression of CRC.

Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) assumes a pivotal role in the hydroxylation of lysine residues, facilitating the cross-linking and subsequent deposition of collagen precursors[2–4]. Emerging investigations suggest that PLOD2 can modify the cell shape and movement capacity under hypoxia, stiffening the extracellular matrix (ECM)[5, 6]. This modification facilitated the invasion and metastasis of breast cancer cells[7]. In actuality, collagen deposition and cross-linking occur frequently in malignant tumors, escalating the risk of tumor occurrence and invasion[8]. In addition to its high expression in breast cancer, recent studies have demonstrated that PLOD2 is also up-regulated in cervical cancer, endometrial cancer, and colorectal cancer[2, 9]. Further research is requisite to elucidate how PLOD2 regulates the progression of CRC.

The PI3K/AKT/mTOR signaling pathway is a highly conserved signaling network in eukaryotic cells, renowned for its critical roles in cell survival, growth, and regulation of cell cycle progression[10, 11]. The PI3K/AKT/mTOR signaling pathway demonstrates comprehensive interaction with numerous other pathways, leading to a complex balance governed by a diversity of molecular events[12, 13]. Hence, research on the upstream and downstream regulation of the PLOD2 gene on the PAM signaling axis can illuminate the mechanism of occurrence and development of CRC.

In this study, we demonstrated that PLOD2 can promote CRC metastasis by enhancing migration through the PI3K/AKT signaling pathway. Our research findings indicate that PLOD2 is an important factor in the progression of CRC and provide a potential target for the treatment of CRC.

1. Colon cancer cell lines and clinical samples

The human colon cancer cell line LoVo and the 293T cell line were obtained from authenticated sources; LoVo from the Fudan University Cell Bank, and 293T from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and maintained in a humidified incubator at 37°C with 5% CO2. Clinical samples were collected from colorectal cancer patients following pathological confirmation, under protocols approved by the Ethics Committee of Jiaxing First Hospital (Approval No:2024-KY-377), ensuring compliance with the ethical guidelines outlined in the Declaration of Helsinki. Informed consent was obtained from all participants.

2. RNA extraction and qRT PCR

Total RNA was extracted from clinical specimens using Trizol reagent according to the manufacturer's protocol, followed by measurement of RNA concentration and quality assessment via agarose gel electrophoresis. Reverse transcription was carried out using the ReverTra Ace qPCR RT Kit (Toyobo) to synthesize cDNA from 1 µg of total RNA. Subsequently, RT-qPCR was performed using SYBR Green Master Mix. The primer sequences were as follows: PLOD2-F 5'-TCCAAAAGGCAAACCACAAAG-3', PLOD2-R 5'-AGATAGCGTTTCCCAATGTGC-3'; 18S-F 5'-GGCCCTGTAATTGGAATGAGTC-3', 18S-R 5'-CCAAGATCCAACTACGAGTT-3'. Data were analyzed using the comparative Ct method (∆∆Ct) to determine relative gene expression, normalized to the endogenous control 18S rRNA.

3. Western blotting

Protein was extracted using RIPA lysate containing a mixture of protease and phosphatase inhibitors. Protein quantification was performed using a BCA protein quantification kit. Gel electrophoresis was carried out with a 10% gel, after which proteins were transferred onto a PVDF membrane and blocked with a quick-blocking solution for 20 minutes. Primary antibodies (β-Actin 1:50000, mTOR 1:10000, PI3K 1:500, PLOD2 1:2000, p-AKT 1:10000, AKT 1:10000) (Proteintech) were incubated overnight at 4°C. Secondary antibodies (HRP-conjugated goat anti-mouse and anti-rabbit 1:50,000) (Affinity Biosciences) were incubated at room temperature for 1 hour, followed by development with ECL luminescent reagent.

4. Cell transfection

After 293T cells were prepared, lentiviral liquid containing PLOD2 knockout gene, purinomycin resistance gene and luciferase gene was prepared using lipofectamine 3000 kit. Colon cancer cell line LoVo was selected and added with chronic disease venom. After 8h, puromycin was screened by 2µL/ml (Sigma, USA). The transfection efficiency was detected by immunofluorescence microscopy and RT-qPCR.

5. Cell biological function

Transwell Invasion Assays: A 3 × 104 cell suspension in serum-free DMEM was added to the upper chamber of each Transwell plate (300 µL/well), and 600 µL of DMEM with 20% FBS was added to the lower chamber. After incubation, cells in the upper chamber were washed, dried, and photographed; Cell Migration Assay: Cells were seeded to near-confluence (about 100%) in a hole plate. A scratch perpendicular to the well's horizontal axis was made using a 100 µL pipette tip. The wound closure rates were observed and photographed under a microscope at 24, 48, 72, and 96 hours after scratching; Cell Proliferation Assay: Cells were plated in 96-well plates with four replicates per condition. CCK-8 reagent was added to each well on days 1, 2, 3, and 4 after 24-hour culturing to assess cell proliferation.

6. Molecular docking

PyMol 2.4 software was used to add hydrogen atoms to LH2 protein (PLOD2) and delete water molecules and redundant ligands. AutoDock Vina 1.1.2 software was used to perform semi-flexible docking between small molecular compounds and PLOD2 to form stable complexes. The docking conformation with the lowest binding energy and the highest clustering frequency is considered to be the most potential binding mode between ligands and proteins. PLIP and Pymol v.2.4 software was used to visualize the docking results.

7. Bioinformatics analysis

Clinical information data and RNA-Seq data were acquired from TCGA database (https://portal.gdc.cancer.gov/), and Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/). The survival difference between the subgroups was tested by the Kaplan–Meier (KM) and log‐rank methods with the functions Survfit and Survdiff in the survival package for R (v 3.1.12). GSEA was performed on RNA measurements for samples using the clusterProfiler package (v4.10.1) with R.

8. statistical analysis

Image J, PLIP and Pymol v.2.4 software was used to process and analyze the images. R4.0.0 software was used for statistical analysis of the data. The quantitative data between the two groups was compared by independent t test, χ2 to compare the correlation between PLOD2 expression and clinicopathologic features of colorectal cancer patients. The statistical data were represented by percentage (%), and P < 0.05 indicated that the difference was statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.

1.High expression of PLOD2 is an important predictive factor for poor prognosis in tumor patients

We quantified the mRNA expression of PLOD2 in 42 primary colon cancer tissues and corresponding normal tissues collected from our hospital. We discovered that PLOD2 was highly expressed in tumor tissues (P < 0.001; Fig. 1.A). Subsequently, we gathered clinical and pathological information from these patients and ascertained that the expression level of PLOD2 in tumor tissues of CRC patients (N = 42) rose along with the increase of clinical staging (Fig. 1.B). For further exploration, we searched multiple datasets in the GEO database (GSE17536; GSE29621, P < 0.05) for comparison and identified that high expression of PLOD2 frequently indicates a poor prognosis in patients ((Fig. 1.C-D). In the TCGA colorectal cancer patient cohorts, we found that patients with high expression of PLOD2 had a poorer prognosis in MSS type tumors, but no significant difference was observed in MSI type patients (Fig. 1.E; S1.A). This might imply the distinctive role of PLOD2 in MSS type tumors, which demands further investigation. We analyzed the relationship between the six major immune cells and PLOD2 in colorectal cancer using the Tumor Immune Estimation Resource (TIMER) database. PLOD2 was positively correlated with immune cells in colorectal cancer (Fig. 1.F). We found that PLOD2 is predominantly associated with macrophages in colorectal cancer, suggesting the role of macrophages in ECM remodeling. We sought the expression of PLOD2 in colorectal cancer on the HPA website (https://www.proteinatlas.org/), and based on the intensity of IHC staining, we selected slices with three intensities for display (Fig. 1.G). In conclusion, our findings imply that PLOD2 holds potential as a prospective therapeutic target and diagnostic biomarker for CRC.

2.Construction of PLOD2 knockout cell lines

To further elucidate the role of PLOD2 in CRC, we selected the LoVo cell line with higher levels of PLOD2 expression as our knockdown PLOD2 transfection targets (LoVo sh-PLOD2), while the empty lentiviral vector was employed as the negative control (LoVo Vector). Meanwhile, we also observed the transfection efficiency of plasmids in a fluorescent environment, and the transfection efficiency was satisfactory (Fig. 2.A). The efficiency of transfection was verified by WB and qPCR results (Fig. 2.B-C). The CCK8 results demonstrated that PLOD2 knockdown inhibited the proliferation of tumor cells (Fig. 2.D). Cell scratch experiments also indicated the migration ability of cells subsequent to PLOD2 Knockdown. Transwell and scratch experiments attested that silencing PLOD2 could inhibit the migration ability of tumors (Fig. 2.E-F).

3. PLOD2 affects tumor progression through the PI3K-AKT signaling pathway

Simultaneously, we conducted KEGG pathway analysis on the three groups and generated heatmaps of recurrent signaling pathways, as depicted in Fig. 3A. The signaling pathways that are commonly upregulated by the three encompass various signaling pathways, such as the TGF-beta signaling pathway and the PI3K-AKT signaling pathway (Fig. 3B). The PI3K-AKT signaling pathway influences multiple biological processes within cells and is impacted by multiple signaling pathways. Consequently, we will select and present the key nodes of the signaling pathway separately (Fig. 3C). We hypothesize that PLOD2 is regulated by the PI3K-AKT signaling pathway and affects tumor progression. To validate our notion, the expression of PLOD2 and PI3K-AKT signaling pathway-related proteins was determined by protein blotting (Fig. 3D). The results indicated that in sh-PLOD2 cells, the silencing of PLOD2 suppressed p-AKT expression in LoVo cells, but had no effect on total AKT and mTOR (Fig. 3E). These findings imply that PLOD2 might promote the proliferation and migration of CRC cells by activating the PI3K-AKT signaling pathway.

4. Drug prediction for PLOD2

In this study, we adopted a semi-flexible docking approach to identify a stable complex. Specifically, datasets GSE17536, GSE29621, and TCGA-MSS were incorporated into the Genomics of Drug Sensitivity in Cancer (GDSC) database for further analysis to screen for target drugs associated with high PLOD2 expression. The GDSC database is currently the most extensive public resource for tumor cell drug sensitivity, encompassing 621 anti-cancer compounds across more than 1000 cancer cell lines, with a wide range of activities involving 24 pathways[14]. Based on the IC50 values, 19 small molecule compounds correlated with high PLOD2 expression were identified (Table S1). The top 5 small molecule drugs with potential therapeutic effects are: Doramapimod, NU7441, RVX-208, BMS-754807, and RO-3306, with binding energies of -8.489, -7.756, -7.688, -7.655, and − 7.614 kcal/mol, respectively (Fig. 4.A-J). Generally, smaller binding energies indicate stronger binding affinities. Therefore, Doramapimod is regarded as the most promising compound for highly stable binding to PLOD2.

The PLOD family of proteins catalyzes the post-translational modification of collagen through converting lysine to hydroxylysine, thereby facilitating stable interactions and deposition of collagen[15, 16]. The transformation of lysine from collagen end peptide to hydroxylysine is contingent upon the PLOD family of proteins, among which LH2 encoded by PLOD2 is the sole member capable of hydroxylating collagen end peptide lysine. Thus, research on PLOD2 could be more substantive[3, 17]. Studies have manifested that PLOD2 relies on protein inhibition to stabilize the expression of USP15, thereby influencing the development of CRC[18]. There are also studies indicating that PLOD2 is induced by L1CAM and promotes CRC progression via ezrin signaling and the SMAD2/3 pathway[19]. Therefore, these studies suggest that PLOD2, as a promising therapeutic target, merits further exploration in the direction of precise tumor treatment.

The intervention in ECM changes and regulatory factors within tumors has been verified to be an important therapeutic direction in clinical practice[6, 20], yet the role of PLOD2 in the progression of CRC remains ambiguous. In this study, we initially confirmed the high expression of PLOD2 in human CRC tissue and its association with tumor pathological staging. Concurrently, we discovered in public databases that the expression of PLOD2 is related to patient prognosis. Additionally, knocking out PLOD2 significantly inhibits the proliferation and metastasis of CRC and promotes the activation of the PI3K-AKT signaling. Specifically, we found that PLOD2 is one of the crucial genes influencing the development of CRC.

Nevertheless, PLOD2 might be a promising target for combating these diseases, but an effective inhibitor is still lacking. Scholars have demonstrated that enol compounds in the form of enols can establish stronger interactions with Fe (II) in the active site, enhancing the branching of enol compounds with functional groups such as phenyl and pyridine groups and strengthening their interactions with various residues around the active site[21, 22]. Although this provides a direction for the study of PLOD2 inhibitors, a considerable amount of work remains to be done.

In summary, our research suggests that PLOD2 regulates proliferation and invasion mediated progression of colorectal cancer through PI3K-AKT, and may be used for precision treatment in clinical practice.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

DezhenChen, Yuan Zhou, Mili Zhang,Yuxuan Zhou designed the research. DezhenChen, Haiqiang Zhu, Mili Zhang conducted interpretation and analysis of the data. DezhenChen, Yuan Zhou, Zhongquan Deng, Mili Zhang,Yuxuan Zhou,Ouxuan Li performed the experiments. Mili Zhang, Yuan Zhou, Dezhen Chen wrote, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by 2023 Jiaxing city and provinces to build medical key disciplines-Oncology (2023-SSGJ-001) and Jiaxing Science and Technology plan Project(2024AD30034).

Acknowledgments

We thank everyone who has contributed to this article.

Data Availability Statement

All datasets presented in this study are included in the article.

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No competing interests reported.

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The PLOD2 promotes tumor progression in colorectal cancer through the PI3K-AKT pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

1. Colon cancer cell lines and clinical samples

2. RNA extraction and qRT PCR

3. Western blotting

4. Cell transfection

5. Cell biological function

6. Molecular docking

7. Bioinformatics analysis

Results

1.High expression of PLOD2 is an important predictive factor for poor prognosis in tumor patients

2.Construction of PLOD2 knockout cell lines

3. PLOD2 affects tumor progression through the PI3K-AKT signaling pathway

4. Drug prediction for PLOD2

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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