PAK5 Promotes Esophageal Squamous Cell Carcinoma Progression Revealed by Transcriptomic Profiling

Summary

This study presents a method to assess PAK5 function in ESCC, showing that its overexpression predicts poor prognosis and promotes tumor progression through the PAK5-GAREM1 signaling axis.

Abstract

Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related mortality globally, particularly in Asia. Due to the lack of early symptoms, most patients are diagnosed at advanced stages, limiting treatment efficacy and worsening prognosis. Understanding the mechanisms underlying ESCC and identifying biomarkers or therapeutic targets are crucial for improving patient outcomes. P21-activated protein kinase 5 (PAK5), a member of the mitogen-activated protein kinase family, has been implicated in various malignancies by regulating cell cycle, migration, and invasion. However, its role in ESCC remains unclear. This study assessed PAK5 expression in ESCC tissues and adjacent normal tissues using immunohistochemistry and performed Kaplan-Meier survival analysis to evaluate the association between PAK5 and prognosis. ESCC cell models with PAK5 overexpression and knockdown were established, and functional assays, including CCK-8, colony formation, and Transwell assays, were conducted. Furthermore, mRNA sequencing was performed to identify downstream targets and signaling pathways regulated by PAK5. These results showed that PAK5 expression was significantly elevated in ESCC tissues compared with normal tissues and was associated with poor prognosis. Functional assays revealed that PAK5 promoted ESCC cell proliferation, colony formation, migration, and invasion, while transcriptomic analysis highlighted GAREM1 as a key downstream effector. These findings indicate that PAK5 contributes to ESCC progression and may serve as a prognostic biomarker and therapeutic target.

Introduction

Globally, esophageal cancer ranks among the most lethal malignancies. In China, esophageal squamous cell carcinoma (ESCC) represents the predominant histological subtype. Although therapeutic advances have been achieved, the prognosis for ESCC patients continues to be dismal, manifesting as a mere 10% to 25% 5-year survival rate¹. This is largely due to the challenges of early detection and the limited availability of effective molecularly targeted therapies². Unlike other solid tumors where targeted therapies have shown significant promise, such approaches in ESCC are still in their infancy, hampered by a scarcity of actionable molecular targets.

PAK5, a group II PAK, integrates cytoskeletal dynamics and pro-survival signaling and has been connected to chemoresistance in several malignancies^{3,4,5,6,7,8,9,10,11}. Given the prominent role of MAPK signaling in ESCC biology and therapy response^1,2,12, it was hypothesized that PAK5 is upregulated in ESCC and promotes malignant phenotypes through modulation of MAPK signaling, potentially involving GAREM1 as an adaptor/regulator^13,14,15,16. Quantitative IHC, functional genetic perturbation, and transcriptomic profiling were employed to test this hypothesis.

The management of ESCC typically involves a combination of esophagectomy, chemotherapy, and radiotherapy. While esophagectomy can be curative, it carries a high risk of morbidity^4,8. Additionally, both chemotherapy and radiotherapy often fail to eliminate all cancer cells, resulting in resistance, recurrence, and metastasis^8,9. Immunotherapy has emerged as a prominent focus in oncology recently, demonstrating effectiveness in treating advanced cancers. This growing interest highlights the urgent need for new biomarkers that can aid in early diagnosis and treatment planning for ESCC. Previous studies have shown that other PAK family members, such as PAK1 and PAK4, promote proliferation, invasion, and therapy resistance in ESCC, underscoring the importance of this kinase family in ESCC biology. However, the role of PAK5, a less studied group II PAK, remains poorly defined, motivating the current study^17,18.

P21-activated kinase 5 (PAK5), a member of the PAK II subfamily, was first identified as a brain-specific kinase in 2002. Located on chromosome 20p12, PAK5 encodes a protein of approximately 80 kDa and is primarily found in the mitochondria and nucleus, where it plays diverse roles in cellular regulation^10,11,13. Its functions in the mitochondria include energy metabolism and regulation, while its nuclear presence is linked to gene transcription and cell cycle control. Although PAK5 is less studied than its counterparts, it is believed to be crucial for neurodevelopment, cellular survival, and cancer progression¹⁰. Notably, PAK5 has been implicated in cytoskeletal regulation, anti-apoptotic mechanisms, and cellular proliferation¹². Its upregulation in neoplastic cells has been linked to emergent drug resistance, resonating with findings that associate PAK5 overexpression with heightened tumor cell resistance to chemotherapy^10,11,13,14. Yet, the PAK5's precise function in ESCC progression remains elusive. This study probes PAK5's influence on the ESCC transcriptomic milieu and its functional ramifications. Transcriptomic profiling was performed to identify gene expression alterations associated with PAK5 activity in ESCC cells. Additionally, an ESCC cell model with controlled PAK5 modulation was generated to elucidate its role in tumor growth and metastatic potential. Unlike conventional biomarker discovery strategies in ESCC that rely primarily on Western blotting or single-modality transcript measurements, the present workflow integrates quantitative immunohistochemistry, transcriptomic profiling, and functional assays. This combined approach enables simultaneous molecular and phenotypic validation, providing mechanistic insight while improving biological relevance. In addition, the protocol emphasizes experimental reproducibility, utilizing paired tumor and adjacent tissue samples, standardized immunostaining procedures, and high-quality RNA inputs (RNA integrity number ≥ 7) for sequencing. These features enhance assay robustness and applicability across laboratories, particularly for studies seeking to define clinically meaningful kinase-driven regulatory networks in ESCC.

Protocol

All procedures were approved by the Ethics Committee of the Second Affiliated Hospital of Hainan Medical University (Approval No. LW2022602), and informed consent was obtained from each participant in accordance with the Declaration of Helsinki. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Patient and tissue sample collection

A total of 40 patients with pathologically confirmed ESCC were enrolled, all with normal hepatic and renal function prior to surgery. Tumor specimens (Group T) and adjacent normal tissues (Group N; ≥2 cm from the tumor margin) were collected immediately after surgical resection. Tissues were fixed in 4% paraformaldehyde (PFA) in PBS at room temperature for 7 days before paraffin embedding.

2. Immunohistochemical staining

Paraffin-embedded blocks were sectioned at 5 µm and baked at 60 °C for 1 h. Sections were deparaffinized with xylene substitute, rehydrated through graded ethanol, and subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 95-98 °C for 20 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 5% normal serum for 30 min at room temperature. Sections were incubated overnight (12-16 h) at 4 °C with anti-PAK5 primary antibody (1:100 dilution, see Table of Materials). After PBS washes, slides were incubated with horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature, developed with DAB until color appeared (1-5 min, monitored under microscope to avoid over-staining), and counterstained with hematoxylin for 30-60 s. Sections were dehydrated, mounted, and imaged under a brightfield microscope. Staining intensity and proportion of positive cells were quantified using image analysis software with at least five random fields per section (200× magnification). The quantification method adopted H-score calculation, with the formula: H-score = ∑ (staining intensity grade × percentage of positive cells) (intensity graded as 0 = negative, 1 = weak, 2 = moderate, 3 = strong; percentage categorized as 0-25%, 26-50%, 51-75%, 76-100%). To ensure reliability, two independent pathologists performed blind scoring, with an inter-observer agreement κ value ≥ 0.75. Quality control criteria were strictly implemented: Positive controls (known PAK5-positive ESCC tissue) and negative controls (primary antibody replaced with PBS) must pass validation; background staining intensity must be below a pre-set threshold (e.g., H-score < 50); coefficient of variation (CV) of staining results across duplicate batches must be <20% to exclude experimental variability.

3. Cell lines and culture

KYSE-150 (human ESCC) and HEK-293 (viral packaging) cells were authenticated by short tandem repeat (STR) profiling and confirmed to be mycoplasma-free. KYSE-150 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin). HEK-293 cells were maintained in DMEM supplemented with the same additives. Cells were subcultured using 0.25% trypsin-EDTA when they reached 70%-80% confluence, and were seeded to appropriate densities 24 h before each experiment.

4. Lentiviral vector construction and infection

Lentiviral vectors were designed to either overexpress PAK5 (pLV-EF1α-PAK5-PGK-Puro) or silence PAK5 (pLV-U6-shRNA-PAK5-PGK-Puro). HEK-293 cells were seeded in 10 cm dishes and transfected at 70%-80% confluence with the transfer plasmid, packaging plasmids (gag/pol and rev), and envelope plasmid (VSV-G) at a 4:3:1 ratio using a lipid-based transfection reagent, with a total DNA amount of ~20 µg per 10 cm dish. The medium was replaced 6-8 h after transfection. Viral supernatants were collected at 48 h and 72 h, centrifuged at 500 × g for 10 min to remove debris, filtered through a 0.45 µm filter, and either concentrated or stored at −80 °C. For transduction, KYSE-150 cells were seeded one day prior to infection and exposed to viral supernatant at a multiplicity of infection (MOI) of 5-10 in the presence of 8 µg/mL polybrene. MOI was defined as the ratio of infectious units to target cell number. A pre-experiment was conducted to estimate MOI using GFP counting or qPCR, and an MOI of 5-10 was selected. Medium was replaced after 12-16 h, and a puromycin kill-curve was established by treating non-transduced cells with puromycin concentrations ranging from 0-4 µg/mL for 72-96 h to determine the minimal lethal concentration. The selected concentration for selection was 1-2 µg/mL, and the maintenance concentration was 0.5-1 µg/mL. Cells were selected with puromycin (1-2 µg/mL) beginning 48 h later. PAK5 overexpression or silencing was verified by RT-qPCR using specific primers for PAK5 (forward 5′-CCAAAGCCTATGGTGGGACCC-3′, reverse 5′-AGGCCGTTGATGGAGGTTTC-3′) and GAPDH (forward 5′-GTGGACATCCGCAAAGAC-3′, reverse 5′-AAAGGGTGTAACGCAACTA-3′). The relative expression of PAK5 was calculated using the 2^−ΔΔCt method. All experiments were performed with at least three biological replicates.

5. CCK-8 cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8), which is based on the reduction of the WST-8 reagent by cellular dehydrogenases. Logarithmically growing KYSE-150 cells were seeded at 2,000 cells per well in 96-well plates in 100 µL complete medium. At 0 h, 24 h, 48 h, 72 h, and 96 h, 10 µL of CCK-8 reagent (containing WST-8) was added to each well. After incubation at 37 °C for 2 h, absorbance at 450 nm (A450) was measured with a microplate reader. Blank wells containing medium only were included. Each experimental group contained at least three biological replicates, and each measurement was performed with five technical replicates.

6. Colony formation assay

Single-cell suspensions of KYSE-150 were seeded at 1,000 cells per well in 6-well plates and cultured for 14 days, with medium replaced every 2-3 days. Colonies were fixed with 4% PFA for 15 min, stained with 0.1% crystal violet in 20% methanol for 15 min, rinsed with water, and air-dried. Colonies containing more than 50 cells were counted using image analysis software, and the colony formation rate was calculated.

7. Transwell invasion assay

Inserts with 8 µm pores were pre-coated with basement membrane matrix (50 µL per insert,1:8 dilution) and incubated at 37 °C for 1 h; quality control was performed before seeding cells: blank inserts (without cells) and negative control inserts (without chemoattractant) were set up to verify assay validity, and the uniformity of the basement membrane matrix layer was confirmed by visual inspection (or weight measurement for stricter control). Cells were serum-starved in 1% FBS medium for 6-12 h, and 2 × 10⁵ cells/mL (200 µL per insert) were seeded into the upper chamber. The lower chamber contained 600 µL medium with 20% FBS as a chemoattractant. After 24 h incubation, non-invaded cells on the upper surface were removed, and invaded cells on the lower surface were fixed with methanol for 10 min and stained with 0.1% crystal violet for 15 min. Stained cells were imaged under a brightfield microscope and counted in at least five random microscopic fields per insert (200× magnification); counting was performed using a blind method (counting personnel were unaware of group assignments) to avoid subjective bias.

8. Flow cytometric detection of apoptosis

Cells were harvested (including floating cells), washed twice with PBS, and resuspended in binding buffer at 1 × 10⁶ cells/mL. Annexin V-FITC (5 µL) and propidium iodide (5 µL) were added to 100 µL of the suspension, followed by incubation at room temperature for 15 min in the dark;after incubation, 400 µL binding buffer was added to each sample. Prior to flow cytometric analysis, compensation adjustment was performed using single-stained controls (Annexin V-FITC single-stained cells and PI single-stained cells) to eliminate fluorescence spillover, and the FSC/SSC gate was set to select single cells (excluding cell debris and aggregates). Samples were analyzed within 1 h on a flow cytometer equipped with a 488 nm laser, with at least 10,000 events acquired per sample. Apoptotic indices were quantified using flow cytometry analysis software: a uniform gating threshold was applied across all samples to ensure consistency, and the proportions of early apoptotic cells (Annexin V⁺/PI⁻) and late apoptotic cells (Annexin V⁺/PI⁺) were separately reported to characterize the apoptotic profile of each group.

9. RNA sequencing and bioinformatics analysis

Total RNA was extracted and assessed for purity (A₂₆₀/₂₈₀ between 1.8 and 2.1) and integrity (RIN ≥7 as a quality control threshold). mRNA was purified using oligo(dT) beads, fragmented at 94 °C for 5-7 min, and reverse transcribed into cDNA. Libraries were prepared by end repair, adaptor ligation, and PCR amplification (8-12 cycles), and sequenced on an Illumina platform to generate paired-end 150 bp reads at a depth of 30-50 million read pairs per sample. Reads were trimmed and aligned to the human reference genome using STAR or HISAT2. Gene counts were obtained with featureCounts, and differential expression analysis was performed with DESeq2, applying thresholds of FDR < 0.05 and |log₂ fold change| ≥ 1. Functional enrichment analysis of differentially expressed genes was conducted using clusterProfiler for Gene Ontology and KEGG pathways (with Benjamini-Hochberg (BH) corrected FDR < 0.05), and protein-protein interaction networks were constructed using the STRING database.

10. Statistical analysis

Continuous data were expressed as mean ± SD from at least three independent experiments. Comparisons between two groups were performed with two-tailed unpaired t-tests, and comparisons among three or more groups were performed with one-way ANOVA followed by Tukey's post-hoc test. Non-parametric tests were applied where appropriate. Survival distributions were estimated by Kaplan-Meier analysis with log-rank tests. For RNA-seq data, multiple testing correction was performed using the Benjamini-Hochberg method. Exact p-values are reported in figure legends; statistical significance was considered at p < 0.05, with* indicating p < 0.05 and **indicating p < 0.01.

Results

PAK5 is highly expressed in ESCC and is associated with poor prognosis
Figure 1 illustrates the overall workflow, from the clinical validation of PAK5 to functional and transcriptomic analysis, which identifies GAREM1 as a downstream target. To test the hypothesis that PAK5 promotes ESCC progression by modulating the MAPK pathway via GAREM1, the expression patterns of PAK5 in ESCC tissues and their association with clinical prognosis were examined. Firstly, the ...

Discussion

This study provides an in-depth examination of the role of PAK5 in the progression of esophageal squamous cell carcinoma (ESCC) by combining clinical samples, functional assays, and transcriptomic analysis. These findings broaden the understanding of PAK5's involvement in ESCC, highlighting its potential as both a prognostic marker and a therapeutic target.

The marked overexpression of PAK5 in ESCC tissues and its association with poorer patient survival is consistent with findings from ot...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V-FITC apoptosis detection kit	Beyotime Biotechnology	C1062S	Apoptosis detection staining kit
Anti-PAK5 antibody	Abcam	ab110069	Primary antibody for IHC, dilution 1:100
Automated electrophoresis system	Agilent	2100 Bioanalyzer	RNA integrity assessment
cDNA synthesis kit	Invitrogen	1896649	Reverse transcription kit
Crystal violet solution	Sigma-Aldrich	C3886	Staining dye for colonies and invasion
DMEM Medium	Gibco (Thermo Fisher)	11965092	Cell culture medium
Fetal Bovine Serum (FBS)	Gibco (Thermo Fisher)	16000044	Cell culture supplement
Flow cytometer	BD Biosciences	FACSCanto II	Instrument for cell analysis
GraphPad Prism	GraphPad Software	Version 8	Statistical analysis and graphing software
H&E Staining Kit	Solarbio	G1120	Hematoxylin and eosin staining solution
HRP-conjugated Anti-Rat IgG	Abcam	ab150165	Secondary antibody for IHC
Illumina NovaSeq sequencer	Illumina	NovaSeq 6000	High-throughput sequencing platform
KYSE-150 cell line	RIKEN Cell Bank	RCB2057	Human esophageal squamous carcinoma cell line
Lipofectamine 3000	ThermoFisher	L3000008	Transfection reagent
Microplate reader	Bio-Tek Instruments	ELx808	Absorbance measurement instrument
DynabeadTM Oligo(dT)25s	Thermo Fisher Scientific	61005	For mRNA purification
Penicillin-Streptomycin	Gibco (Thermo Fisher)	15140122	Antibiotics for cell culture
Propidium iodide (PI)	Sigma-Aldrich	P4864	DNA staining dye
RPMI-1640 Medium	Gibco (Thermo Fisher)	11875093	Cell culture medium
Spectrophotometer	Thermo Fisher	NanoDrop ND-1000	RNA concentration and purity measurement
SPSS software	IBM	Version 23	Statistical analysis software
Transwell inserts	Corning	3422	Cell invasion assay inserts