Introduction

Circular RNAs (circRNAs) are a class of singlestranded, covalently closed RNA molecules that are formed by precursor mRNA back-splicing or skipping events of thousands of genes in eukaryotes. CircRNAs play an essential role in the initiation and progression of human cancers by participating in the regulation of one or several cancer hallmarks.

Aims & Methods

In our study, we aimed to reveal the functions and mechanisms of circRNAs in colorectal cancer (CRC) progression. High-throughput RNA sequencing (RNA-seq) was performed to assess RNA expression profiles in 10 paires of CRC tumor tissues and matched adjacent peritumor tissues, then the expression level of top five dysregulated circRNAs were tested in CRC cancer cell lines. The circLRBA with concordant alteration in RNA-seq data and cell lines was validated in tumor and corresponding adjacent tissues (85 pairs) from a CRC cohort by qRT-PCR analysis followed by correlation and prognosis analysis between circLRBA and clinical characteristics. Sanger sequencing, RNase R and Actinomycin D assays were used to verify the ring structure of circLRBA. Nuclear-cytoplasmic separation experiment and the fluorescence in situ hybridization (FISH) were performed to show the cellular location of circLRBA. Functional experiments, including CCK8 assay, colony formation assay, transwell assay, 3D multicellular tumor spheroid invasion assay and orthotopic-injected CRC cell line-derived xenograft (CDX) mouse models, were performed by using circLRBA overexpression and knockdown cell lines to investigate the effects of circLRBA on CRC progression in vitro and in vivo. Mechanistically, RNA-sequencing was used to reveal the dysregulated genes followed by circLRBA overexpression. Bioinformatic analysis was used to predict the possible downstream pathways. Furthermore, Biotin‑labeled RNA pulldown and RNA immunoprecipitation (RIP) assays were carried out to identify the proteins directly interacting with circLRBA.

Results

Totally 853 differentially expressed circRNAs were identified, including 778 down-regulated and 75 up-regulated circRNAs. We identified a circular RNA generated from the LRBA gene, designated circLRBA, that was significantly down-regulated in CRC tissues and associated with unfavorable clinicopathological parameters. CircLRBA was dominantly distributed in the cytoplasm in several CRC cell lines. Gain‑ and loss‑of‑function experiments suggested that circLRBA could inhibit CRC cell proliferation, metastasis and apoptosis in vitro and in vivo. RNA-seq of circLRBA-overexpressing cells and the controls revealed that these dysregulated genes were significantly enriched in pathways of malignant neoplasm of colon stage IV (DisGeNET database), protein localization to endoplastic reticulum and lysosomal lumen (Gene Ontology analyse). Furthermore, RNA pulldown and RIP assays validated that circLRBA directly binds to the HSPA8 protein, which is an essential chaperone for mediating cell autophagy by translocating proteins containing the KFERQ-like motif in their amino acid sequence to undergo lysosomal degradation.

Conclusion

Our study uncovered a novel LRBA gene-generated circRNA with a tumor suppression function. CircLRBA probably inhibited CRC progression by chaperone-mediated autophagy (CMA) through interacting with HSPA8. However, further exploration is needed to reveal the substrate protein of HSPA8 and its effects on CRC. CircLRBA is expected to be a new therapeutic target for CRC patients in the future.

Disclosure

No conflict of interest.

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide^1,2. Liquid–liquid phase separation (LLPS) describes a reversible and dynamic process in which intracellular biomacromolecules condense into liquid-like biomolecular assemblies through multivalent interactions³. Emerging evidence indicates that dysregulated phase separation may play a pivotal role in cancer initiation and progression^4-6. However, the specific role and regulatory mechanisms of LLPS in the progression and metastasis of CRC remain largely unexplored.

Aims & Methods

Magnifying endoscopy combined with narrow-band imaging (ME-NBI) was used to perform targeted biopsies of distinct colorectal tissue subpopulations. Adjacent normal tissues, non-invasive cancer, and invasive colorectal cancer samples were separately collected for ATAC-seq and RNA-seq to identify core transcription factor (TF) involved in the progression of CRC. Then, the TF was purified in vitro and over-expressed in vivo separately to observe the phase separation phenomenon. Besides, phase separation-deficient mutants were identified and characterized to determine their biophysical properties. Finally, we evaluated its effects on CRC invasion and metastasis both in vitro and in vivo by using transwell migration and invasion assays, wound healing assays, and a lung metastasis model in nude mice.

Results

Multi-omics analysis identified ETV4 as a key driver of CRC progression. qRT-PCR and Western blot revealed that ETV4 were significantly elevated in CRC tissues compared to adjacent normal tissues. Correlation analysis based on data from The Cancer Genome Atlas (TCGA) further demonstrated that high ETV4 expression was positively associated with distant metastasis and advanced tumor stage in CRC. Meanwhile, the purified ETV4 readily formed droplet-like condensates in vitro. Transfection of HEK-293T and CRC cell lines with ETV4-GFP plasmids similarly resulted in the formation of distinct intracellular droplet-like condensates. Besides, fluorescence recovery after photobleaching (FRAP) assays using confocal laser microscopy demonstrated that fluorescence signals in the bleached regions of ETV4 condensates gradually recovered over time, indicating that these condensates exhibit dynamic liquid-like properties.
Based on the intrinsically disordered regions (IDRs) of ETV4, a series of truncated mutants (ΔIDR1, ΔIDR2, and ΔIDR3) were constructed. When these mutant plasmids were transfected into HEK-293 cells, only deletion of the IDR1 region (ΔIDR1) abrogated the formation of droplet-like condensates. Notably, expression of the IDR1 region alone was sufficient to restore condensate formation within cells, indicating that IDR1 is essential and sufficient for ETV4-mediated phase separation. Furthermore, in vitro experiments demonstrated that ETV4 overexpression significantly enhanced the migratory and invasive capacities of colon cancer cells, whereas ETV4 knockdown markedly suppressed these malignant phenotypes compared to control groups. Additionally, mice injected with ETV4 wild-type (ETV4-FL) cells developed significantly more lung metastases compared to controls, whereas those injected with the phase separation-deficient mutant (ETV4-ΔIDR1) failed to enhance metastatic dissemination.

Conclusion

This study identifies ETV4 as a pivotal transcriptional regulator that drives CRC progression and metastasis. Besides, ETV4 undergoes LLPS both in vitro and in vivo, with this process being specifically mediated by its first IDR1. Importantly, the pro-metastatic function of ETV4 is mechanistically dependent on its LLPS capability.

References

• 1. Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74, 229–263 (2024).
• 2. Biller, L. H. & Schrag, D. Diagnosis and Treatment of Metastatic Colorectal Cancer: A Review. JAMA 325, 669–685 (2021).
• 3. Mehta, S. & Zhang, J. Liquid-liquid phase separation drives cellular function and dysfunction in cancer. Nat Rev Cancer 22, 239–252 (2022).
• 4. Zong, Z. et al. Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis. Cell 187, 2375-2392.e33 (2024).
• 5. Liu, Q. et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell 184, 5559-5576.e19 (2021).
• 6. Ahn, J. H. et al. Phase separation drives aberrant chromatin looping and cancer development. Nature 595, 591–595 (2021).

Introduction

Colorectal cancer (CRC) is one of the most common malignancies worldwide, and early detection significantly improves patient outcomes. Long non-coding RNAs (lncRNAs) are >200 nucleotide transcripts that regulate gene expression and are frequently dysregulated in cancer. Notably, certain lncRNAs are overexpressed during colorectal tumorigenesis and detectable in circulation, making them promising non-invasive biomarkers. This study aimed to identify circulating lncRNAs with progressive expression changes along the adenoma-carcinoma sequence and evaluate their potential as early diagnostic markers for CRC.

Aims & Methods

We performed bioinformatic screening of three public gene expression datasets (GSE117606, GSE289499, GSE154548) containing colorectal normal, adenoma, and carcinoma tissues. Twelve lncRNA candidates showing linear differential expression across the adenoma-to-carcinoma sequence were identified. The five most significantly dysregulated lncRNAs (ZFAS1, FOXP4-AS1, CRNDE, UCA1, SNHG17) were selected for further validation. Plasma samples were collected from healthy controls (n=21), patients with advanced adenomas (precancerous lesions, n=51), early-stage CRC (Tis-T1, n=24), and advanced CRC (T2-T4, n=53). Levels of the five lncRNAs in plasma were measured by quantitative PCR and normalized to 5S rRNA using the 2^-ΔCt method. Expression trends across the four groups were analyzed. Receiver operating characteristic (ROC) curves (area under the curve, AUC) were generated to assess the ability of individual lncRNAs to distinguish early CRC from controls. A logistic regression model was used to combine multiple lncRNAs into a panel and evaluate its diagnostic performance (sensitivity and specificity).

Results

Bioinformatic analysis identified 12 lncRNAs with stepwise expression changes from adenoma to carcinoma, and the top five candidates were successfully measured in plasma. Among these, UCA1 and SNHG17 showed progressively increasing plasma levels from healthy controls to adenoma to early CRC, with the highest levels in advanced CRC (trend P<0.05). In contrast, CRNDE levels remained unchanged between healthy and early CRC groups but rose significantly in advanced CRC, suggesting its upregulation is associated with tumor progression. The other two lncRNAs (ZFAS1, FOXP4-AS1) exhibited less pronounced changes. In diagnostic tests, UCA1 alone distinguished early-stage CRC from controls with an AUC of 0.82, whereas SNHG17 achieved an AUC of 0.73. Notably, a combined three-lncRNA panel (UCA1 + SNHG17 + CRNDE) improved early CRC detection, achieving an AUC of 0.85 with 87.5% sensitivity and 61.9% specificity for differentiating early CRC from healthy individuals. This multi-marker panel outperformed any single lncRNA biomarker in identifying early CRC.

Conclusion

A GEO dataset-guided approach coupled with plasma validation provided robust evidence that circulating lncRNAs have significant diagnostic potential for CRC. UCA1 and SNHG17 emerged as promising blood-based markers for early detection of colorectal neoplasia, while CRNDE may serve as a complementary marker indicative of advanced disease progression. Moreover, a panel combining these lncRNAs demonstrated superior accuracy over individual markers, underscoring the value of a multi-lncRNA approach. These findings support further development of non-invasive, lncRNA-based blood tests for early colorectal cancer screening.

Introduction

Liver metastasis is one of the main reasons for colorectal cancer (CRC)-related deaths due to the lack of effective therapeutic. CRC cell line-derived xenograft (CDX) mouse models developed by orthotopic injection in the cecum, ectopic injection in the spleen or the portal vein are essential invivo tools for exploring the mechanism of liver metastasis and testing potential chemotherapeutic agents. We did this study to evaluate and optimize the establishment of these models, and emphasize characteristics of liver metastatic foci derived from different CRC cell lines.

Aims & Methods

Luciferin-expressing HCT116 cells, SW480 cells and SW620 cells were used to induce mouse models of CRC with liver metastases by the cecum injection, intrasplenic injection and intraportal injection. The metastatic characteristics of tumors were confirmed and evaluated by bioluminescence imaging, macroscopic and microscopic appearance. Furthermore, we improved and evaluated the procedures of these CRC CDX mouse models, and comprehensively summarized their performances in the aspect of liver metastasis with three popular cell lines.

Results

Models established by cecal injection and intrasplenic injection displayed similarly in the operation difficulty, cell number to need and frequency of metastasis. The bioluminescence imaging usually had a strong and concentrated signal in cecum, but a weak or moderate signal in the spleen respectively, and accompanied with a diffuse signal or no signal in the liver. The model for HCT116-induced liver metastasis could be well established by intrasplenic injection, showing granular lesions with tumor pleomorphic variation, invasion, erosion and necrosis. SW480 and SW620-derived models by cecal or intrasplenic injection characterized macroscopically with nodular, white grey metastatic foci throughout the liver tissue, and identified with scattered, sub-rotund, uniform-sized and infiltrative pathological changes. However, the metastatic lesions were more and larger in the interior of liver tissues in the cecum injection model within 6 weeks, suggesting its superiority. Intraportal injection had technique difficulty in the discovery and puncture of the portal vein, probably leading to post-operated hemostasis and mouse deaths. The metastatic foci developed from intraportal injection were hardly displayed obvious findings in macroscopic appearance, and few or small-sized sporadic lesions under microscopy could be detected.

TABLE 1: The opitimization and evaluation of the establishment of three CDX models.

Conclusion

The study provides not only useful information for the establishment of CRC CDX models for liver metastasis, but also novel insights for identifying characteristics of metastatic foci induced by different cell lines.

Disclosure

No conflict of interest

Introduction

Colorectal cancer (CRC) is a leading global malignancy and a major cause of cancer-related mortality. Exosomes—small extracellular vesicles—mediate intercellular communication by transferring microRNAs, which regulate gene expression and influence tumor behavior. Elevated levels of miR-1290 have been observed in CRC patient blood and tumor tissues. However, the biological function of exosomal miR-1290 in CRC progression remains poorly understood.

Aims & Methods

Plasma and tissue samples from CRC patients and healthy individuals were collected with ethical approval. Exosomes were isolated from patient plasma and CRC cell line-conditioned media using ultracentrifugation and membrane affinity methods. In vitro, CRC cells were transfected with miR-1290 mimics or inhibitors to assess effects on proliferation (CCK-8, EdU assays), clonogenicity (colony formation), apoptosis (flow cytometry), and migration (transwell assay). Candidate miR-1290 targets were predicted bioinformatically, and CBFA2T3 was validated by dual-luciferase reporter assay. Stable CBFA2T3-overexpressing CRC cell lines were established to evaluate phenotypic rescue. In vivo, CRC xenografts in nude mice were treated with exosomes derived from miR-1290–overexpressing or control cells. Tumor growth and expression of Ki-67 and PCNA were analyzed.

Results

Plasma exosomal miR-1290 levels were lowest in healthy controls and progressively increased across advanced adenoma, early-stage CRC, and advanced CRC groups. Notably, miR-1290 distinguished advanced CRC from controls with an AUC of 0.77 and moderately distinguished early CRC (AUC = 0.69). Consistent with clinical findings, miR-1290 expression was significantly elevated in CRC tissues versus adjacent normal mucosa and in CRC cell lines compared to normal colonic epithelial cells. CRC cells also secreted exosomes enriched in miR-1290. In vitro, miR-1290 mimics enhanced CRC cell proliferation, DNA synthesis, and clonogenic potential, without significantly affecting apoptosis or migration. Bioinformatic analysis identified CBFA2T3 as a key target of miR-1290. Dual-luciferase assays confirmed direct binding of miR-1290 to the 3′-UTR of CBFA2T3, resulting in its downregulation. In CRC cells, miR-1290 upregulation—via mimics or exosomal transfer—decreased CBFA2T3 mRNA and protein levels. Restoration of CBFA2T3 expression significantly counteracted miR-1290-induced proliferation and colony formation. In vivo, nude mice injected with CRC cells and treated with miR-1290–enriched exosomes developed significantly larger tumors compared to controls. Tumors exhibited reduced CBFA2T3 levels and elevated expression of proliferation markers Ki-67 and PCNA, supporting the tumor-promoting role of exosomal miR-1290.

Conclusion

Exosomal miR-1290 correlates with CRC progression and shows promise as a non-invasive diagnostic biomarker. Functionally, it promotes CRC proliferation and tumor growth by targeting and downregulating the tumor suppressor CBFA2T3, highlighting its potential as a therapeutic target.

Introduction

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death worldwide. While early CRC has a five-year survival rate of 91%, this drops to 14% for advanced CRC. Although endoscopic resection effectively detects and treats precancerous lesions and early CRC (Tis/T1), its invasiveness, cost, and reliance on operator expertise limit its use in large-scale screening. Noninvasive tests such as FIT, stool DNA, and methylated SEPT9—despite FDA approval—show limited sensitivity for advanced adenomas (AA). Plasma proteomics has emerged as a promising alternative, with Proximity Extension Assay (PEA) enabling highly sensitive, multiplex protein detection from minimal samples. In this study, we used PEA to profile plasma proteins in patients with advanced adenomas and early CRC, developed predictive models, and validated their diagnostic potential—offering a promising noninvasive strategy for early detection.

Aims & Methods

We collected plasma samples from patients with AA, early CRC, and healthy controls, and analyzed them using the Olink Explore panel with PEA technology in a discovery (n=80) and validation cohort (n=69). Protein biomarkers were identified in the discovery cohort using multivariate logistic regression adjusted for clinical confounders. Four machine learning algorithms—LASSO, Random Forest, SVM, and XGBoost—were applied to develop diagnostic models for colorectal neoplasia, assessing their performance in detecting early CRC and AA.

Results

Plasma proteomic profiles of AA and early CRC patients were analyzed using PEA technology. In the discovery cohort, multivariate logistic regression identified 15 protein biomarkers that exhibited significant differential expression between AA, ECRC, and control groups. Machine learning models based on these biomarkers demonstrated strong diagnostic performance. Specifically, the LASSO model selected five proteins with an AUC of 0.958, while the Support Vector Machine model achieved an AUC of 0.983 with seven proteins. Random Forest and XGBoost models also showed robust performance, with AUCs of 0.983 and 0.974, respectively. These models demonstrated excellent diagnostic performance for AA and ECRC, with AUCs consistently above 0.90 in the discovery cohort and ranging from 0.908 to 0.923 in the external validation cohort. MMP7 and GDF15 were confirmed as key biomarkers, with ELISA assays showing significant differences (MMP7: R = 0.894, GDF15: R = 0.723). A combined prediction model using MMP7 and clinical factors (age, gender, family history, BMI) achieved an AUC of 0.927, outperforming the clinical model (AUC = 0.863). This study underscores the potential of protein biomarkers and machine learning models for noninvasive early CRC detection.

Conclusion

In summary, to enable early intervention and improve CRC survival, we utilized PEA technology to analyze protein profiles and develop machine learning models for diagnosing early colorectal neoplasia (AA and early CRC). These multi-target models demonstrated superior diagnostic performance in both discovery and validation cohorts compared to previous studies on early lesions, highlighting their potential for early CRC detection and timely endoscopic intervention.

Disclosure

Nothing to disclose.