Heat Shock Transcription Factor 4 Promotes Malignant Biological Behavior in Colorectal Cancer Through Stiffness Rather Than Collagen Content.

Background Colorectal cancer (CRC), a malignancy with high incidence and mortality rates, presents particularly severe prognoses at the stages of invasion and metastasis despite significant advancements in diagnostic and therapeutic technologies. The tumour microenvironment, especially the extracellular matrix (ECM) stiffness, has garnered considerable attention for its impact on CRC progression and metastasis. However, the precise relationship and molecular mechanisms between ECM stiffness and CRC prognosis remain unclear. Methods This study included 107 CRC patients. Tumor stiffness was assessed using magnetic resonance elastography (MRE), and collagen content was analysed with Masson staining. CRC cell lines SW480 and HCT116 were cultured on matrices of varying stiffness, followed by transcriptome sequencing to identify stiffness-related differential genes and investigate their functions and signalling pathways. Additionally, an HSF4 knockout CRC cell model was constructed to evaluate the effects of HSF4 on cell proliferation, migration, and invasion through both in vitro and in vivo experiments, exploring its role in tumour growth and metastasis. Results The study found that CRC tumour stiffness was significantly higher than normal tissue and positively correlated with collagen content and TNM staging. Transcriptome analysis revealed that high-stiffness matrices significantly regulated cell functions and signalling pathways. High expression of HSF4 was strongly associated with tumour stiffness and poor prognosis. HSF4 expression increased with higher TNM stages in CRC tissues, and its knockout significantly inhibited cell proliferation, migration, and invasion, particularly on high-stiffness matrices. In vivo experiments further confirmed that HSF4 promoted tumour growth and metastasis, independent of collagen protein increase. Conclusion This study reveals a close relationship between tumour stiffness and CRC prognosis, identifying HSF4 as a critical player in CRC progression. HSF4 promotes tumour proliferation and metastasis by regulating EMT-related signalling pathways, with its high expression closely linked to tumour stiffness. Although LOXL1 increased collagen content and a-SMA expression, HSF4's role in promoting tumour growth and metastasis was independent of collagen protein. HSF4 holds potential as a target for CRC prognosis evaluation and treatment. Future research should further explore the regulatory mechanisms of HSF4 and its potential for clinical application. SW480 cells were cultured on 6-well plates coated with 2 kPa or 25 kPa hydrogels (MATRIGEN, USA). Plates were washed with PBS, and 2 ml of cell suspension at 1×10^6 cells/ml was added to each well. Each stiffness condition was tested in triplicate, and cells were incubated at 37°C in a CO2-free incubator for 72 hours. Cells were harvested by scraping, and the reaction was stopped with a complete medium. The cell suspension was centrifuged, washed with PBS, and treated with TRIzol reagent (Life Technologies) for RNA extraction. The RNA was sent to BGI Genomics, where it was reverse-transcribed into cDNA, and libraries were constructed for deep sequencing at a depth of 200×. RNA was quantified using a NanoDrop and Agilent 2100 bioanalyser (Thermo Fisher Scientific). Libraries were prepared from 1 μg of total RNA per sample, and small RNA (18-30 nt) was purified via PAGE gel electrophoresis, reverse-transcribed, and amplified by PCR. Enriched cDNA fragments were selected, purified, and assessed for quality and quantity using an Agilent 2100 bioanalyser and real-time qPCR with TaqMan probes. Sequencing was performed on the Illumina HiSeq platform (BGI, Shenzhen).