SEC22B inhibition attenuates colorectal cancer aggressiveness and autophagic flux under unfavorable environment.

Autophagy has bidirectional functions in cancer by facilitating cell survival and death in a context-dependent manner. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a large family of proteins essential for numerous biological processes, including autophagy; nevertheless, their potential function in cancer malignancy remains unclear. Here, we explored the gene expression patterns of SNAREs in tissues of patients with colorectal cancer (CRC) and discovered that SEC22B expression, a vesicle SNARE, was higher in tumor tissues than in normal tissues, with a more significant increase in metastatic tissues. Interestingly, SEC22B knockdown dramatically decreased CRC cell survival and growth, especially under stressful conditions, such as hypoxia and serum starvation, and decreased the number of stress-induced autophagic vacuoles. Moreover, SEC22B knockdown successfully attenuated liver metastasis in a CRC cell xenograft mouse model, with histological signs of decreased autophagic flux and proliferation within cancer cells. Together, this study posits that SEC22B plays a crucial role in enhancing the aggressiveness of CRC cells, suggesting that SEC22B might be an attractive therapeutic target for CRC.

The Small-Molecule Wnt Inhibitor ICG-001 Efficiently Inhibits Colorectal Cancer Stemness and Metastasis by Suppressing MEIS1 Expression.

Recurrence and metastasis remain major obstacles in colorectal cancer (CRC) treatment. Recent studies suggest that a small subpopulation of cells with a self-renewal ability, called cancer stem-like cells (CSCs), promotes recurrence and metastasis in CRC. Unfortunately, no CSC inhibitor has been demonstrated to be more effective than existing chemotherapeutic drugs, resulting in a significant unmet need for effective CRC therapies. In this study, transcriptomic profiling of metastatic tumors from CRC patients revealed significant upregulation in the Wnt pathway and stemness genes. Thus, we examined the therapeutic effect of the small-molecule Wnt inhibitor ICG-001 on cancer stemness and metastasis. The ICG-001 treatment efficiently attenuated self-renewal activity and metastatic potential. Mechanistically, myeloid ecotropic viral insertion site 1 (MEIS1) was identified as a target gene of ICG-001 that is transcriptionally regulated by Wnt signaling. A series of functional analyses revealed that MEIS1 enhanced the CSC behavior and metastatic potential of the CRC cells. Collectively, our findings suggest that ICG-001 efficiently inhibits CRC stemness and metastasis by suppressing MEIS1 expression. These results provide a basis for the further clinical investigation of ICG-001 as a targeted therapy for CSCs, opening a new avenue for the development of novel Wnt inhibitors for the treatment of CRC metastasis.

Roles of Wnt Target Genes in the Journey of Cancer Stem Cells.

The importance of Wnt/ß-catenin signaling in cancer stem cells (CSCs) has been acknowledged; however, the mechanism through which it regulates the biological function of CSCs and promotes cancer progression remains elusive. Hence, to understand the intricate mechanism by which Wnt controls stemness, the specific downstream target genes of Wnt were established by analyzing the genetic signatures of multiple types of metastatic cancers based on gene set enrichment. By focusing on the molecular function of Wnt target genes, the biological roles of Wnt were interpreted in terms of CSC dynamics from initiation to metastasis. Wnt signaling participates in cancer initiation by generating CSCs from normal stem cells or non-CSCs and augmenting persistent growth at the primary region, which is resistant to anti-cancer therapy. Moreover, it assists CSCs in invading nearby tissues and in entering the blood stream, during which the negative feedback of the Wnt signaling pathway maintains CSCs in a dormant state that is suitable for survival. When CSCs arrive at distant organs, another burst of Wnt signaling induces CSCs to succeed in re-initiation and colonization. This comprehensive understanding of Wnt target genes provides a plausible explanation for how Wnt allows CSCs variation during cancer progression.

The dysadherin/FAK axis promotes individual cell migration in colon cancer.

Dysregulation of cancer cell motility is a key driver of invasion and metastasis. High dysadherin expression in cancer cells is correlated with invasion and metastasis. Here, we found the molecular mechanism by which dysadherin regulates the migration and invasion of colon cancer (CC). Comprehensive analysis using single-cell RNA sequencing data from CC patients revealed that high dysadherin expression in cells is linked to cell migration-related gene signatures. We confirmed that the deletion of dysadherin in tumor cells hindered local invasion and distant migration using in vivo tumor models. In this context, by performing cell morphological analysis, we found that aberrant cell migration resulted from impaired actin dynamics, focal adhesion turnover and protrusive structure formation upon dysadherin expression. Mechanistically, the activation of focal adhesion kinase (FAK) was observed in dysadherin-enriched cells. The dysadherin/FAK axis enhanced cell migration and invasion by activating the FAK downstream cascade, which includes the Rho family of small GTPases. Overall, this study illuminates the role of dysadherin in modulating cancer cell migration by forcing actin dynamics and protrusive structure formation via FAK signaling, indicating that targeting dysadherin may be a potential therapeutic strategy for CC patients.

Dysadherin awakens mechanical forces and promotes colorectal cancer progression.

Rationale: Dysadherin is a tumor-associated, membrane-embedded antigen found in multiple types of cancer cells, and associated with malignant behavior of cancer cells; however, the fundamental molecular mechanism by which dysadherin drives aggressive phenotypes of cancer is not yet fully determined. Methods: To get a mechanistic insight, we explored the physiological relevance of dysadherin on intestinal tumorigenesis using dysadherin knockout mice and investigated its impact on clinicopathological features in patients with advanced colorectal cancer (CRC). Next, to discover the downstream signaling pathways of dysadherin, we applied bioinformatic analysis using gene expression data of CRC patient tumors and dysadherin knockout cancer cells. Additionally, comprehensive proteomic and molecular analyses were performed to identify dysadherin-interacting proteins and their functions. Results: Dysadherin deficiency suppressed intestinal tumorigenesis in both genetic and chemical mouse models. Moreover, increased dysadherin expression in cancer cells accounted for shorter survival in CRC patients. Comprehensive bioinformatics analyses suggested that the effect of dysadherin deletion is linked to a reduction in the extracellular matrix receptor signaling pathway. Mechanistically, the extracellular domain of dysadherin bound fibronectin and enhanced cancer cell adhesion to fibronectin, facilitating the activation of integrin-mediated mechanotransduction and leading to yes-associated protein 1 activation. Dysadherin-fibronectin interaction promoted cancer cell growth, survival, migration, and invasion, effects collectively mediated the protumor activity of dysadherin. Conclusion: Our results highlight a novel function of dysadherin as a driver of mechanotransduction that stimulates CRC progression, providing a potential therapy strategy for CRC.

DCLK1 promotes colorectal cancer stemness and aggressiveness via the XRCC5/COX2 axis.

Rationale: Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that selectively marks cancer stem-like cells (CSCs) and promotes malignant progression in colorectal cancer (CRC). However, the exact molecular mechanism by which DCLK1 drives the aggressive phenotype of cancer cells is incompletely determined. Methods: Here, we performed comprehensive genomics and proteomics analyses to identify binding proteins of DCLK1 and discovered X-ray repair cross-complementing 5 (XRCC5). Thus, we explored the biological role and downstream events of the DCLK1/XRCC5 axis in human CRC cells and CRC mouse models. Results: The results of comprehensive bioinformatics analyses suggested that DCLK1-driven CRC aggressiveness is linked to inflammation. Mechanistically, DCLK1 bound and phosphorylated XRCC5, which in turn transcriptionally activated cyclooxygenase-2 expression and enhanced prostaglandin E2 production; these events collectively generated the inflammatory tumor microenvironment and enhanced the aggressive behavior of CRC cells. Consistent with the discovered mechanism, inhibition of DCLK1 kinase activity strongly impaired the tumor seeding and growth capabilities in CRC mouse models. Conclusion: Our study illuminates a novel mechanism that mediates the pro-inflammatory function of CSCs in driving the aggressive phenotype of CRC, broadening the biological function of DCLK1 in CRC.

Lipid raft-disrupting miltefosine preferentially induces the death of colorectal cancer stem-like cells.

BACKGROUND: Lipid rafts (LRs), cholesterol-enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem-like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear. METHODS: This study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR-disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses. RESULTS: Miltefosine suppresses CSC populations and their self-renewal activities in CRC cells, a CSC-targeting effect leading to irreversible disruption of tumour-initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44high CSCs, we found that CSCs exhibited stronger therapy resistance than non-CSC counterparts by preventing cell death through CHEK1-mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death. CONCLUSION: Our findings underscore the therapeutic potential of LR-targeting APLs for CRC treatment that overcomes the therapy-resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.

Aberrant activation of the CD45-Wnt signaling axis promotes stemness and therapy resistance in colorectal cancer cells.

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes. Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models. Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the ß-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype. Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.

The JAK2/STAT3/CCND2 Axis promotes colorectal Cancer stem cell persistence and radioresistance.

BACKGROUND: Radiotherapy (RT) is a highly effective multimodal nonsurgical treatment that is essential for patients with advanced colorectal cancer (CRC). Nevertheless, cell subpopulations displaying intrinsic radioresistance survive after RT. The reactivation of their proliferation and successful colonization at local or distant sites may increase the risk of poor clinical outcomes. Recently, radioresistant cancer cells surviving RT were reported to exhibit a more aggressive phenotype than parental cells, although the underlying mechanisms remain unclear. METHODS: By investigating public databases containing CRC patient data, we explored potential radioresistance-associated signaling pathways. Then, their mechanistic roles in radioresistance were investigated through multiple validation steps using patient-derived primary CRC cells, human CRC cell lines, and CRC xenografts. RESULTS: Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling was activated in radioresistant CRC tissues in correlation with local and distant metastases. JAK2 was preferentially overexpressed in the CRC stem cell subpopulation, which was accompanied by the phosphorylation of STAT proteins, especially STAT3. JAK2/STAT3 signaling played an essential role in promoting tumor initiation and radioresistance by limiting apoptosis and enhancing clonogenic potential. Mechanistically, the direct binding of STAT3 to the cyclin D2 (CCND2) promoter increased CCND2 transcription. CCND2 expression was required for persistent cancer stem cell (CSC) growth via the maintenance of an intact cell cycle and proliferation with low levels of DNA damage accumulation. CONCLUSION: Herein, we first identified JAK2/STAT3/CCND2 signaling as a resistance mechanism for the persistent growth of CSCs after RT, suggesting potential biomarkers and regimens for improving outcomes among CRC patients.

Inhibition of LEF1-Mediated DCLK1 by Niclosamide Attenuates Colorectal Cancer Stemness.

PURPOSE: Niclosamide, an FDA-approved anthelmintic drug, has been characterized as a potent Wnt inhibitor that can suppress tumor growth and cancer stem-like cell (CSC) populations. However, the underlying molecular mechanisms remain poorly understood. This study aimed to examine how Wnt inhibition by niclosamide preferentially targets CSCs. EXPERIMENTAL DESIGN: The mechanistic role of niclosamide in CSC inhibition was examined in public databases, human colorectal cancer cells, colorectal cancer xenografts, and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colorectal cancer model. RESULTS: Niclosamide suppresses CSC populations and their self-renewal activities in colorectal cancer cells, and this CSC-targeting effect leads to irreversible disruption of tumor-initiating potential in vivo. Mechanistically, niclosamide downregulates multiple signaling components of the Wnt pathway, specifically lymphoid enhancer-binding factor 1 (LEF1) expression, which is critical for regulating stemness. Subsequently, we identified that the doublecortin-like kinase 1 (DCLK1)-B is a target of LEF1 and upregulates cancer stemness in colorectal cancer cells. We first documented that niclosamide blocks the transcription of DCLK1-B by interrupting the binding of LEF1 to DCLK1-B promoter. DCLK1-B depletion impairs cancer stemness resulting in reduced survival potential and increased apoptosis, thus sensitizing colorectal cancer to chemoradiation. CONCLUSIONS: Disruption of the LEF1/DCLK1-B axis by niclosamide eradicates cancer stemness and elicits therapeutic effects on colorectal cancer initiation, progression, and resistance. These findings provide a preclinical rationale to broaden the clinical evaluation of niclosamide for the treatment of colorectal cancer.