Subpopulation Of Colon Cancer Cells Research Articles

Identification of a subpopulation of long-term tumor-initiating cells in colon cancer

Long-term tumor-initiating cells (LT-TICs) are viewed as a quantifiable target for colon cancer therapy owing to their extensive self-renewal and tumorigenic and metastatic capacities. However, it is unknown which subpopulation of colon cancer cells contains LT-TICs. Here, based on the methods for isolating and identifying cancer stem cells (CSCs) and the functional features of LT-TICs, we aimed to identify a subpopulation of LT-TICs. Among the six cell lines assessed, our results showed that CD133 and CD44 coexpression was only detected in HCT116 and HT29 cell lines. In HCT116 and HT29 cells, CD133+CD44+ cells not only shared the extensive tumorigenic potential of LT-TICs but also functionally reproduced the behaviors of LT-TICs that drive tumor metastasis (TM) formation, suggesting that CD133+CD44+ cells are a typical representation of LT-TICs in colon cancer. Mechanistically, the enhanced capacity of CD133+CD44+ cells to drive metastasis involves the up-regulated expression of Wnt-, epithelial–mesenchymal transition (EMT)-, and metastasis-related genes in these cells. Additionally, CD133+CD44+ cells presented significant chemoresistance compared with corresponding nontumorigenic CD133−CD44− cells following exposure to oxaliplatin (OXLP) or 5-fluorouracil (5-FU). Accordingly, CD133+CD44+ cells contained lower reactive oxygen species (ROS) levels than CD1133−CD44− cells, and the low ROS levels in CD133+CD44+ cells were related to the enhancement of antioxidant defense systems. More importantly, CD133+CD44+ cells developed less DNA damage after exposure to chemotherapeutics than CD133−CD44− cells. In conclusion, we identified a subpopulation of LT-TICs in colon cancer.

Different Camptothecin Sensitivities in Subpopulations of Colon Cancer Cells Correlate with Expression of Different Phospho-Isoforms of Topoisomerase I with Different Activities.

The heterogeneity of tumor cells and the potential existence of rare cells with reduced chemotherapeutic response is expected to play a pivotal role in the development of drug resistant cancers. Herein, we utilized the colon cancer cell lines, Caco2 and DLD1, to investigate heterogeneity of topoisomerase 1 (TOP1) activity in different cell subpopulations, and the consequences for the chemotherapeutic response towards the TOP1 targeting drug, camptothecin. The cell lines consisted of two subpopulations: one (the stem-cell-like cells) divided asymmetrically, was camptothecin resistant, had a differently phosphorylated TOP1 and a lower Casein Kinase II (CKII) activity than the camptothecin sensitive non-stem-cell-like cells. The tumor suppressor p14ARF had a different effect in the two cell subpopulations. In the stem-cell-like cells, p14ARF suppressed TOP1 activity and downregulation of this factor increased the sensitivity towards camptothecin. It had the opposite effect in non-stem-cell-like cells. Since it is only the stem-cell-like cells that have tumorigenic activity our results point towards new considerations for future cancer therapy. Moreover, the data underscore the importance of considering cell-to-cell variations in the analysis of molecular processes in cell lines.

MiR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-β/Smad Pathway.

Quiescent caner stem cells are identified as a subpopulation of colon cancer cells in dormant state and possess strong stem-cell like characteristics. Previously, we have identified this subpopulation in colorectal cancer (quiescent colon cancer stem cells, QCCSCs), and find QCCSCs are sensitive to the apoptotic effect of IFN-γ, which is attributed to their high IFN-γR expression levels. Microarray and bioinformatic analysis indicate miR-4666-3p is low expressed in QCCSCs and target IFN-γR1/2, which is proved by luciferase assay and western-blot. Furthermore, we find miR-4666-3p could also target TGF-βR1 to block the activation of TGF-β1/Smad pathway, therefore function as a tumor suppressor gene to inhibit the stemness of colon cancer cells. Besides, compared with QCCSCs, we find the TGF-β1 expression also decreased with the weakening of stemness properties. In terms of mechanism, our result reveal TGF-β1 is the target gene of miR-329, which is also high expressed in non-QCCSCs. Thereafter, we perform gain- and loss- function experiments to confirm the synergistic effect between miR-4666-3p and miR-329 in blocking the activation of TGF-β/Smad pathway. Finally, we evaluate the expression of both miR-4666-3p and miR-329 in 73 tumor specimens and paired normal tissue, and find both two miRNAs are related to unfavorable prognosis and advanced tumor stage in colorectal cancer. Our study revealed a novel epigenetic regulation mechanism in colon cancer stem cells, which could be exploited as a novel therapeutic strategy for cancer treatment.

MiR-139-5p reverses stemness maintenance and metastasis of colon cancer stem-like cells by targeting E2-2.

Colon cancer stem cells (CCSCs) stand for a critical subpopulation of colon cancer cells that possess self-renewal and multilineage differentiation potentials and drive tumorigenicity. Due to their impact on treatment tolerance, CCSCs have been a hot research topic in the past few years. We have previously reported that miR-139-5p is a vital tumor repressive noncoding RNA whose level decreases in the clinical colon cancer samples with the increase of tumor malignancy. This research discovered that miR-139-5p targets the Wnt/β-catenin/TCF7L2 downstream effector E2-2 in CCSCs. E2-2 is a pivot molecule in the negative feedback loop of miR-139-5p/Wnt/β-catenin/TCF7L2. Its small interfering RNA reverses the stemness maintenance and epithelial-mesenchymal transition of colon cancer CSCs. This study provides a theoretical foundation for the clinical diagnosis and medical treatment of recurrent or metastatic colon cancer with miR-139-5p and its target E2-2.

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.

Abstract 5212: Loss of G-protein coupled calcium sensing receptor augments malignancy and bestows cancer stem cell-like properties in human colon carcinoma cells

Abstract The G-protein coupled calcium sensing receptor (CaSR) is a robust promoter of differentiation in colonic epithelial cells. Loss of CaSR expression allows cancer cells to escape from CaSR control. Loss of CaSR expression augments the malignant phenotype in vitro and is associated with undifferentiated and invasive colon carcinomas in vivo (Cancer Res. 63: 67, 2003; 65: 493, 2005; Int. J. Cancer 121: 1455, 2007;126: 631, 2010; Mol. Carcinogenesis 48: 202, 2009). Interestingly, human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). Here, we report the isolation and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. We isolated CaSR null cells by magnetic cell sorting using monoclonal anti-CaSR antibody. CaSR null cells were cultured and maintained in stem cell culture medium. Biologic characterization of CaSR null cells showed that these cells were non-adherent and grew as three-dimensional spherical clusters with an increase in propensity for anchorage independent growth. CaSR null cells were highly invasive in matrigel invasion assays and resistant to treatment with cytotoxic drugs. Western analysis showed that CaSR null cells expressed a high level of thymidylate synthase and survivin which have been reported to be an underlying basis of drug resistance in colon cancer. Western analysis also showed that CaSR null cells expressed a high level of the previously reported cancer stem cell markers CD133, CD44 and Nanog. Molecular profiling by quantitative RT-PCR revealed a significant increase in the expression of epithelial-mesenchymal transition associated mRNAs in CaSR null cells. These mRNAs were N-cadherin, β-catenin, vimentin, fibronectin, FSP1, Snail1, Snail2, Twist, FOXC2 and SIP1. Interestingly, CaSR null cells re-expressed CaSR, over time, when placed in culture medium used to propagate the parental CBS and HCT116 cells. Re-expression of CaSR in CaSR null cells attenuated the expression of markers associated with the malignant phenotype. We conclude that CaSR null cells represent a highly malignant subpopulation of colon cancer cells and that these cells may possess cancer stem cell-like properties. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5212. doi:10.1158/1538-7445.AM2011-5212

Abstract 5205: Selectively high Wnt pathway activity in subpopulations of colon cancer cells and its significance for tumor initiation

Abstract Several cell surface markers that are reported to enrich for tumor-initiating colon cancer cells are presumed targets of the canonical Wnt/beta-catenin pathway. Furthermore, colon cancers typically express the Wnt-effector beta-catenin in a heterogeneous pattern, with strong nuclear expression detected only in a small fraction of tumor cells. We therefore hypothesized that high beta-catenin expression and Wnt pathway activity mark a tumorigenic subpopulation. To label viable tumor cells according to the level of Wnt pathway activity, we constructed lentiviral vectors that express GFP under control of an optimal, beta-catenin-responsive TCF/LEF1 promoter (TOP-GFP). We transduced primary colon cancer xenografts and two well characterized colon cancer cell lines with these vectors, used flow cytometry to isolate cell subpopulations with differential GFP expression, and examined these subpopulations for tumorigenicity and gene expression. High GFP expression accurately marked tumor cells with nuclear beta-catenin immunostaining in Caco2 but not SW1222 cells and in a fraction of primary colon cancer xenografts. Gene expression analysis in the concordant cases was consistent with increased Wnt-pathway activity within the GFP-high cell population, including high expression of the putative stem cell markers CD133, CD44 and LGR5. In SW1222 cells and some primary tumor xenografts, the GFP-high fraction correlated poorly with nuclear beta-catenin localization and GFP flow cytometry did not separate cells with a gene expression profile suggesting Wnt pathway activity. Among these various sources, only Caco2 cells showed increased tumorigenicity of GFP-high cells in immunodeficient mouse xenografts. The utility of TOP-GFP and related reporters to identify tumor-initiating colon cancer cells with high Wnt activity thus varies among primary human colorectal tumors and established cell lines. Furthermore, Wnt pathway activity might fluctuate substantially within individual tumor cells. We conclude that high Wnt activity among tumor cell subpopulations does not necessarily signify tumor-initiating potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2011-5205

Abstract A100: STAT3 as a novel cancer prevention target in colorectal cancer stem cells

Abstract Constitutive activation of signal transducers and activators of transcription 3 (STAT3) signaling is frequently detected in human cancer including colon cancer, and has emerged as an attractive molecular target for cancer prevention. Recent experimental evidence suggests that the existence of a small population of tumorigenic stem/progenitor cells may be responsible for tumor initiation, chemotherapy and radiation resistance, invasion, recurrence, and metastasis. An increasing body of evidence suggests that the cancer stem cell concept is also relevant to colorectal cancer. To date, however, whether STAT3 is activated in colon cancer stem cells or cancer-initiating cells and what the role of STAT3 signaling may play in these cancer stem cells is still unknown. If STAT3 is activated in colorectal cancer stem cells, inhibiting STAT3 may offer a promising opportunity to target colorectal cancer stem cells and prevent the recurrence of cancer. We utilized the colon cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive (ALDH+) and CD133-positive (CD133+) subpopulation. We demonstrated that ALDH+/CD133+ cells have capacity than ALDH-/CD133- cells to form more tumorspheres and to exhibit more potent tumor-initiating ability in mice. We then examined the STAT3 activation in these colon cancer cells. Interestingly, we observed that the ALDH+/CD133+ subpopulation of colon cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to the ALDH-/CD133- subpopulation and un-separated colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. We demonstrated that dietary agent, curcumin and a new curcumin analog, GO-Y030 can inhibit STAT3 phosphorylation, cell viability, and induced apoptosis in colon cancer stem cells. The inhibition of STAT3 was confirmed using a novel STAT3-selecitve curcumin analog, FLLL32, STAT3 ShRNA, as well as STAT3 inhibitors, LLL12 and Stattic. All these STAT3 inhibitors, inhibited STAT3 phosphorylation, cell viability, and tumorsphere growth in colon cancer stem cells. Furthermore, both GO-Y030 and LLL12 inhibited tumor growth of colon cancer stem cells in NOD/SCID mouse model in vivo. In Summary, this is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in colon cancer stem cells. Our study is also the first attempt to target STAT3 in colon cancer stem cells and we demonstrated for the first time that colon cancer stem cells are indeed sensitive to the inhibition by small molecular STAT3 inhibitors, FLLL32, LLL12, Stattic, STAT3 shRNA, and curcumin analog, GO-Y030. Our results suggest that STAT3 is a novel prevention target in colon cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer an effective preventive approach for colorectal carcinoma. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A100.

Expression of CD133 correlates with differentiation of human colon cancer cells.

CD133 has been identified as a cancer stem cell marker in colon and several other cancers, but its function is still unknown. We examined the CD133 expression in 44 human cancer cell lines, and found five of the 8 positive lines were from colon cancer. The CD133 positive subpopulation of colon cancer cells showed more vigorous growth and lower differentiation. Induction of differentiation reduced the CD133-positive population. Knockdown of CD133 expression in colon cancer cells could not induce cellular differentiation. Care must be taken if CD133 is used as the only marker of cancer stem cells in colon cancer, especially in established cell lines. CD133 negatively correlates with cell differentiation, but it is not a regulator of differentiation.

An Epigenetic Switch Involving NF-κB, Lin28, Let-7 MicroRNA, and IL6 Links Inflammation to Cell Transformation

Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. We show that transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal.

Characterization of a subpopulation of colon cancer cells with stem cell‐like properties

The biology of the normal colonic mucosa suggests that colon cancer originates from normal colon stem cells. CD44 cancer stem cells have been identified in breast and prostate cancer, and we therefore examined whether CD44 similarly identified colon cancer stem cells. Initial assays found CD44(hi) colon tumor cells to have enhanced soft agar colony-forming ability. Subsequently, CD44(hi) cells isolated from 4 primary colon adenocarcinoma xenografts were found to be highly tumorigenic in immune deficient mice. CD44(hi) cells consistently formed tumors with 1,000 cells, and in multiple experiments, as few as 10 and 100 CD44(hi) cells formed tumors in 7/10 and 21/28 mice, respectively. In contrast, CD44(-) colon tumor cells were either nontumorigenic or 10-50-fold less tumorigenic. CD44(hi) cells could be serially passaged up to 4 times in vivo, suggesting self-renewal capacity, and formed tumors that recapitulated the heterogeneity of the original patient tumor. CD44(hi) cells were significantly enriched for nuclear activated beta-catenin, a key element in normal stem/progenitor cells and in early colon tumor progression. Bromodeoxyuridine (BrdU) labeling studies indicated that CD44(hi) cells divide slowly relative to the CD44(-) cells, suggesting their tumorigenicity is not simply due to faster proliferation. Aldehyde dehydrogenase (ALDH) sort further increased the tumorigenicity of CD44(hi) cells from 2/2 patient tumors, but CD133 tumor cells in our hands did not have increased tumorigenicity. Our observations indicate that CD44 is a marker of stem-like cells in colon cancer, and support the use of additional markers to further purify colon cancer stem cells.

Sulindac sulfide induces several subpopulations of colon cancer cells, defined by PCNA/Ki-67 and DNA strand breaks

We assessed the effect of sulindac sulfide (SS), a colon cancer chemopreventive agent, on the proliferation and apoptosis in the colon cancer cell lines HCT-15 and HT-29. We applied a triparameter flow cytometric analysis that simultaneously determined DNA content, expression of Ki-67 or proliferating cell nuclear antigen (PCNA), and extent of DNA strand breaks by TUNEL (TdT-mediated dUTP nick end labeling). HCT-15 and HT-29 cells were exposed to SS 200 μM and 175 μM, respectively, for up to 72 h. As expected, SS inhibited proliferation and induced apoptosis. SS also induced several subpopulations of cells defined by their expression of proliferation markers and DNA strand breaks. By 72 h the rapidly proliferating cells [PCNA/Ki-67(+)/TUNEL(−)] were reduced from >90% to about one third. Of the remaining cells, about one third were apoptotic [PCNA/Ki-67(−)/TUNEL(+)] and one third were quiescent [PCNA/Ki-67(−)/TUNEL(−)]. Another subpopulation was detected that was PCNA/Ki-67(+)/TUNEL(+), some had a dominant subdiploid peak and over half were in S or G 2/M phases by DNA content. Thus, a subpopulation of apoptotic cells strongly expressed PCNA and Ki-67, suggesting that their specificity as proliferation markers may need reassessment. Similar results were obtained with the HL-60 promyelocytic cell line.

Subpopulations of colon cancer cells survive freezing

Subpopulations of colon cancer cells survive freezing