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.

BackgroundLipid 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.MethodsThis 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.ResultsMiltefosine 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.ConclusionOur 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.

Background: Cancer-specific ligands have been of great interest as pharmaceutical carriers due to the potential for site-specific delivery. In particular, cancer-specific peptides have many advantages over nanoparticles and antibodies, including high biocompatibility, low immunogenicity, and the formation of nontoxic metabolites. The goal of the present study was the development of a novel cancer-specific ligand. Methods: Cancer-specific peptide ligands were screened using a one-bead-one-compound (OBOC) combinatorial method combined with a multiple-antigen-peptide (MAP) synthesis method. The specificity of the peptide ligands toward cancer cells was tested in vitro using a whole-cell binding assay, flow cytometry, and fluorescence confocal microscopy. The tissue distribution profile and therapeutic efficacy of a paclitaxel (PTX)-conjugated peptide ligand was assessed in vivo using xenograft mouse models. Results: We discovered that AGM-330 specifically bound to cancer cells in vitro and in vivo . Treatment with PTX-conjugated AGM-330 dramatically inhibited cancer cell growth in vitro and in vivo compared to treatment with PTX alone. The results of pull-down assay and LC-MS/MS analyses showed that membrane nucleolin (NCL) was the target protein of AGM-330. Although NCL is known as a nuclear protein, we observed that it was overexpressed on the membranes of cancer cells. In particular, membrane NCL neutralization inhibited growth in cancer cells in vitro . Conclusions: In summary, our findings indicated that NCL-targeting AGM-330 has great potential for use in cancer diagnosis and targeted drug delivery in cancer therapy.

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 β-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.

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.