Characterization of Anoikis-Resistant Cells in Mouse Colonic Epithelium

Kawasaki, Hideyoshi; Yoshida, Tetsuya; Horiguchi, Kotaro; Sato, Koichi

doi:10.1292/jvms.13-0005

Cited by 8 publications

(7 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…During the experiments, we noticed that MCF10A-DCLK1 cells did not attach tightly to plates. It was previously reported that anoikis-resistant mouse colon epithelial cells express DCLK1 [23] . To confirm this observation in MCF10A cells, we performed a soft-agar colony formation assay.…”

Section: Resultsmentioning

confidence: 96%

RETRACTED: Doublecortin-like kinase 1 compromises DNA repair and induces chromosomal instability

Maruyama

Kuwata³

et al. 2018

Biochemistry and Biophysics Reports

View full text Add to dashboard Cite

Doublecortin-like kinase 1 (DCLK1) is a serine/threonine-kinase with two doublecortin (DCX) domains. DCLK1 is associated with microtubules via DCX domains and regulates microtubule polymerization. DCLK1 is known to be expressed in cancer stem cells and provides cancer cells with tumor-initiating capacity. Accumulating clinical evidence supports that DCLK1 is associated with tumor aggressiveness and is an important prognostic marker in various human cancers. However, the mechanism, by which DCLK1 causes oncogenesis, is not yet elucidated. In this study, we showed that DCLK1 empowers human mammary epithelial MCF10A cells to form spheres under floating condition in serum-free medium, which are reminiscent of mammospheres formed by mammary epithelial stem cells. We demonstrated that DCLK1 causes chromatin instability in MCF10A cells. DCLK1 impairs DNA repairs in human colon cancer HCT116 and lung cancer H1299 cells. The kinase-negative DCLK1 mutant and the mutant that is not associated with microtubules compromise DNA repair. In conclusion, DCLK1 interferes with DNA repair and induces tumorigenesis through genomic instability and this function is independent of the kinase activity and the regulation of microtubules.

show abstract

Section: Resultsmentioning

confidence: 96%

RETRACTED: Doublecortin-like kinase 1 compromises DNA repair and induces chromosomal instability

Maruyama

Kuwata³

et al. 2018

Biochemistry and Biophysics Reports

View full text Add to dashboard Cite

show abstract

“…DCLK1 might be correlated with apoptosis pathway since knockdown of DCLK1 gene expression induced apoptosis of neuroblastoma cells [33] and anoikis-resistant mouse colonic epithelial cells expressed increased amount of DCLK1 [34]. Our RNA-Seq data indicated that DCLK1 over expression modulates gene expression of multiple key caspases, specifically casp-4 and 10 (Figure 2A), which might due to antibiotics G418 selection.…”

Section: Dclk1 Suppresses Gene Expression Of Multiple Critical Caspasmentioning

confidence: 90%

“…We are not the first one to find that DCLK1 was involved in the apoptosis pathway. It was identified that DCLK1 is essential for the survival of neuroblastoma cells since knockdown of DCLK1 gene expression induced apoptosis of the cells [33] and in the anoikis-resistant mouse colonic epithelial cells, DCLK1 was upregulated [34]. However, it is the first time that DCLK1 was demonstrated to increase chemoresistance of CRC cells via anti-apoptosis pathway.…”

Section: However How Dclk1 Enhances Chemoresistance Of Crc Cells Is mentioning

confidence: 99%

See 1 more Smart Citation

Doublecotin-like kinase 1 increases chemoresistance of colorectal cancer cells through the anti-apoptosis pathway

Jones

Mei

2019

Preprint

View full text Add to dashboard Cite

Colorectal cancer (CRC) is the third most common cancer diagnosed and the second leading cause of cancer-related deaths in the United States. About 50% of CRC patients relapsed after surgical resection and ultimately died of metastatic disease. Cancer stem cells (CSCs) are believed to be the primary reason for the recurrence of CRC. Specific stem cell marker, doublecortin-like kinase 1 (DCLK1) plays critical roles in initiating tumorigenesis, facilitating tumor progression, and promoting metastasis of CRC. It is up-regulated in CRC and upregulation of DCLK1 indicates poor prognosis. Whether DCLK1 is correlated with enhanced chemoresistance of CRC cells is unclear. Our research aims to reveal association of DCLK1 with chemoresistance of CRC cells and the underlying molecular mechanisms. In order to achieve our goal, we established stable DCLK1 over-expression cells (DCLK1+) using the HCT116 cells (WT). DCLK1+ and WT cells were treated with 5-Fluorouracil (5-Fu) at different doses for 24 or 48 hours. MTT assay was used to evaluate cell viability and IC 50 of 5-Fu was determined. Quantitative real time PCR was applied to determine gene expression of caspase-3 (casp-3), caspase-4 (casp-4), and caspase-10 (casp-10). Cleaved casp-3 expression was investigated using Western blot and immunofluorescence. Our results demonstrated that IC 50 of 5-Fu for the DCLK1+ cells was significantly higher than that of the WT cells for both 24 and 48hour treatment (P=0.002 and 0.048 respectively), indicating increased chemoresistance of the DCLK1+ cells. Gene expression of casp-3, casp-4, and casp-10 were significantly inhibited in the DCLK1+ cells after 5-Fu treatment compared to the WT cells (P=7.616e-08, 1.575e-05 and 5.307e-08, respectively). Cleaved casp-3 amount and casp-3 positive cells were significantly decreased in the DCLK1+ cells after 5-Fu treatment compared to the WT cells (P=0.015). In conclusion, our results demonstrated that DCLK1 overexpression enhanced the chemoresistance of CRC cells to 5-Fu treatment by suppressing gene expression of key caspases in the apoptosis pathway and activation of apoptosis pathway. DCLK1 can be an intriguing therapeutic target for the effective treatment of CRC patients.

show abstract

“…Spheroid cultures (reviewed by Fennema et al [ 14 ]) use cells grown in small aggregates that are thought to allow more natural cell-cell and cell-matrix contacts to develop. The anchorage-independent cultures were used to test for antibodies interfering with anoikis resistance–the ability of cells to avoid apoptosis in the absence of normal cell-cell contacts, an important pre-requisite for metastasis [ 16 ]. We generated a panel of antibodies in two molecular formats by performing phage-display selections against the primary NSCLC cells; a designed ankyrin repeat protein (DARPin) antibody mimetic library was used, in addition to a scFv library.…”

Section: Introductionmentioning

confidence: 99%

Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling

Sandercock¹,

Rust²,

Guillard³

et al. 2015

Mol Cancer

View full text Add to dashboard Cite

BackgroundMonolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets.MethodsScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin.ResultsTwo primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model.ConclusionsPhenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0415-0) contains supplementary material, which is available to authorized users.

show abstract

Characterization of Anoikis-Resistant Cells in Mouse Colonic Epithelium

Cited by 8 publications

References 34 publications

RETRACTED: Doublecortin-like kinase 1 compromises DNA repair and induces chromosomal instability

RETRACTED: Doublecortin-like kinase 1 compromises DNA repair and induces chromosomal instability

Doublecotin-like kinase 1 increases chemoresistance of colorectal cancer cells through the anti-apoptosis pathway

Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling

Contact Info

Product

Resources

About