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 ac....

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....

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. Firstl.......

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