Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemoresistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.Key words: IGF-1, cancer stem cell, colon cancer, figitumumab     

Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemo-resistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.     

Mitochondrial metabolism in cancer stem cells: a therapeutic target for colon cancer

It has been proposed that the selective elimination of cancer stem cells (CSCs) using targeted therapy could greatly reduce tumor growth, recurrence, and metastasis. To develop effective therapeutic targets for CSC elimination, we aimed to define the properties of CSC mitochondria, and identify CSC-mitochondria-specific targets in colon cancer. We found that colon CSCs utilize mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP. We also found that forkhead box protein 1 (FOXM1)-induced peroxiredoxin 3 (PRDX3) maintains the mitochondrial function, and the FOXM1/PRDX3 mitochondrial pathway maintains survival of colon CSCs. Furthermore, FOXM1 induces CD133 (PROM1/prominin 1) expression, which maintains the stemness of colon CSCs. Together, our findings indicate that FOXM1, PRDX3, and CD133 are potential therapeutic targets for the elimination of CSCs in colon cancer. [BMB Reports 2015; 48(10): 539-540]     

Cancer stem cells and angiogenesis

Cancer stem cells (CSCs) or tumor initiating cells were identified and characterized as a unique subpopulation with stem cell features in many types of cancer. Current CSC studies provide novel insights regarding tumor initiation, progression, angiogenesis, resistance to therapy and interplay with the tumor micro-environment. A cancer stem cell niche has been proposed based on these findings. The niche provides the soil for CSC self-renewal and maintenance, stimulating essential signaling pathways in CSCs and leading to secretion of factors that promote angiogenesis and long term growth of CSCs. We present evidence which has emerged over the past 5 years indicating interaction of CSCs with angiogenesis in the proposed "vascular niche". Based on these findings, targeting the "cancer stem cell niche" by combining an individualized anti-CSC approach with treatment of their microenvironment may represent a novel therapeutic strategy against solid tumor systems.     

Cancer stem cell, niche and EGFR decide tumor development and treatment response: A bio-computational simulation study

Recent research in cancer biology has suggested the hypothesis that tumors are initiated and driven by a small group of cancer stem cells (CSCs). Furthermore, cancer stem cell niches have been found to be essential in determining fates of CSCs, and several signaling pathways have been proven to play a crucial role in cellular behavior, which could be two important factors in cancer development. To better understand the progression, heterogeneity and treatment response of breast cancer, especially in the context of CSCs, we propose a mathematical model based on the cell compartment method. In this model, three compartments of cellular subpopulations are constructed: CSCs, progenitor cells (PCs), and terminal differentiated cells (TCs). Moreover, (1) the cancer stem cell niche is, considered by modeling its effect on division patterns (symmetric or asymmetric) of CSCs, and (2) the EGFR signaling pathway is integrated by modeling its role in cell proliferation, apoptosis. Our simulation results indicate that (1) a higher probability for symmetric division of CSC may result in a faster expansion of tumor population, and for a larger number of niches, the tumor grows at a slower rate, but the final tumor volume is larger; (2) higher EGFR expression correlates to tumors with larger volumes while a saturation function is observed, and (3) treatments that inhibit tyrosine kinase activity of EGFR may not only repress the tumor volume, but also decrease the CSCs percentages by shifting CSCs from symmetric divisions to asymmetric divisions. These findings suggest that therapies should be designed to effectively control or eliminate the symmetric division of CSCs and to reduce or destroy the CSC niches.     

Bladder cancer stem cells

Stem cells are undifferentiated cells that renew themselves while simultaneously producing differentiated tissue- or organspecific cells through asymmetric cell division. The appreciation of the importance of stem cells in normal tissue biology has prompted the idea that cancers may also develop from a progenitor pool (the "cancer stem cell (CSC) hypothesis"), and this idea is gaining increasing acceptance among scientists. CSCs are sub-populations of cancer cells responsible for tumor initiation, differentiation, recurrence, metastasis, and drug resistance. First identified in the hematopoietic system, CSCs have also been discovered in solid tumors of the breast, colon, pancreas, and brain. Recently, the tissue-specific stem cells of the normal urothelium have been proposed to reside in the basal layer, and investigators have isolated phenotypically similar populations of cells from urothelial cancer cell lines and primary tumors. Herein, we review the CSC hypothesis and apply it to explain the development of the two different types of bladder cancer: noninvasive ("superficial") carcinoma and invasive carcinoma. We also examine potential approaches to identify CSCs in bladder cancer as well as therapeutic applications of these findings. While exciting, the verification of the existence of CSCs in bladder cancer raises several new questions. Herein, we identify and answer some of these questions to help readers better understand bladder cancer development and identify reasonable therapeutic strategy for targeting stem cells.     

Cancer stem cell markers in common cancers - therapeutic implications

Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.     

Stem cells: The root of prostate cancer?

The cancer stem cell (CSC) model states that tumors contain a reservoir of self-renewing cells that maintain the heterogeneous cell population of the tumor. These cells appear to be resistant to therapy and can therefore survive to repopulate the tumor during progression to therapy resistant disease. The biology of CSCs is still not definitive since it is difficult to isolate them from solid tumors and analyze their characteristics in vitro. Another challenge is to correlate these characteristics with tumor development and progression in vivo. Using the prostate CSC as a model, this review presents the CSC hypothesis, reviews the origin, identification and functions of prostate CSCs, and discusses the clinical implications and therapeutic challenges CSCs have for cancer therapy.     

Combination of aging and dimethylhydrazine treatment causes an increase in cancer—stem cell population of rat colonic crypts

Aging is associated with increased incidence of colon cancers. It is also becoming evident that cancer stem cells (CSC) play a vital role in the pathogenesis and prognosis of colon cancer. Recently, we reported the presence of colon cancer stem-like cells in macroscopically normal mucosa in patients with adenomatous polyps and that they increase with aging, suggesting that aging may predispose the colon to carcinogenesis. In the current study we have examined the combined effects of aging and carcinogen exposure on the status of colon CSCs in an experimental model. We used young (4-6 months) and aged (22-24 months) rats and exposed them to the carcinogen, dimethylhydroxide (DMH). We investigated the expression of colon cancer stem cell markers, CD44, CD166, EpCam, and ALDH1 as well as EGFR expression in normal colonic crypt epithelium following carcinogen treatment. Our results demonstrate that aging per se or carcinogen treatment alone causes an increase in the number of colon cancer stems cells, as evidenced by increased immunoreactive-CSC-markers positive cells in the colonic mucosa. In aged rats, carcinogen exposure results in a more pronounced increase in colon cancer stem cells. Our study shows that in aging colon the effects of carcinogens are more pronounced, and an increase in colon CSCs is one of the earliest changes preceding tumor development. Moreover, the current investigation of the use of a panel of immunohistochemical markers of colon CSC can potentially serve as a prognostic marker during screening for colon cancer.     

Cancer stem cells: current status and evolving complexities

The cancer stem cell (CSC) model has been established as a cellular mechanism that contributes to phenotypic and functional heterogeneity in diverse cancer types. Recent observations, however, have highlighted many complexities and challenges: the CSC phenotype can vary substantially between patients, tumors may harbor multiple phenotypically or genetically distinct CSCs, metastatic CSCs can evolve from primary CSCs, and tumor cells may undergo reversible phenotypic changes. Although the CSC concept will have clinical relevance in specific cases, accumulating evidence suggests that it will be imperative to target all CSC subsets within the tumor to prevent relapse.     

Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors

Cancer stem cells (CSCs) are considered to be responsible for the dismal prognosis of cancer patients. However, little is known about the molecular mechanisms underlying the acquisition and maintenance of CSC properties in cancer cells because of their rarity in clinical samples. We herein induced CSC properties in cancer cells using defined factors. We retrovirally introduced a set of defined factors (OCT3/4, SOX2 and KLF4) into human colon cancer cells, followed by culture with conventional serum-containing medium, not human embryonic stem cell medium. We then evaluated the CSC properties in the cells. The colon cancer cells transduced with the three factors showed significantly enhanced CSC properties in terms of the marker gene expression, sphere formation, chemoresistance and tumorigenicity. We designated the cells with CSC properties induced by the factors, a subset of the transduced cells, as induced CSCs (iCSCs). Moreover, we established a novel technology to isolate and collect the iCSCs based on the differences in the degree of the dye-effluxing activity enhancement. The xenografts derived from our iCSCs were not teratomas. Notably, in contrast to the tumors from the parental cancer cells, the iCSC-based tumors mimicked actual human colon cancer tissues in terms of their immunohistological findings, which showed colonic lineage differentiation. In addition, we confirmed that the phenotypes of our iCSCs were reproducible in serial transplantation experiments. By introducing defined factors, we generated iCSCs with lineage specificity directly from cancer cells, not via an induced pluripotent stem cell state. The novel method enables us to obtain abundant materials of CSCs that not only have enhanced tumorigenicity, but also the ability to differentiate to recapitulate a specific type of cancer tissues. Our method can be of great value to fully understand CSCs and develop new therapies targeting CSCs.     

Cancer cells stemness: A doorstep to targeted therapy

Recent advances in research on cancer have led to understand the pathogenesis of cancer and development of new anticancer drugs. Despite of these advancements, many tumors have been found to recur, undergo metastasis and develop resistance to therapy. Accumulated evidences suggest that small population of cancer cells known as cancer stem cells (CSC) are responsible for reconstitution and propagation of the disease. CSCs possess the ability to self-renew, differentiate and proliferate like normal stem cells. CSCs also appear to have resistance to anti-cancer therapies and subsequent relapse. The underlying stemness properties of the CSCs are reliant on multiple molecular targets such as signaling pathways, cell surface molecules, tumor microenvironment, apoptotic pathways, microRNA, stem cell differentiation, and drug resistance markers. Thus an effective therapeutic strategy relies on targeting CSCs to overcome the possible tumor relapse and chemoresistance. The targeted inhibition of these stem cell biomarkers is one of the promising approaches to eliminate cancer stemness. This review article summarizes possible targets of cancer cell stemness for the complete treatment of cancer.     

Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells

Metastatic colorectal cancer remains a serious health concern with poor patient survival. Although 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) is the standard therapy for colorectal cancer, it has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategies that target CSCs for successful treatment of this malignancy is warranted. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary agent with pleiotropic effect) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs sub-population. Remnants of spontaneous adenomas from APC ^{Min +/-} mice treated with dasatinib and/or curcumin were analyzed for several cancer stem cell markers (ALDH, CD44, CD133 and CD166). Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant (referred to as CR) cells. The effectiveness of the combination therapy in inhibiting growth, invasive potential and stemness was examined in colon cancer CR cells. The residual tumors from APC ^{Min +/-} mice treated with dasatinib and/or curcumin showed 80-90% decrease in the expression of the CSC markers ALDH, CD44, CD133, CD166. The colon cancer CR cells showed a higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR HCT-116 and CR HT-29 cells, as determined by Calcusyn analysis. The combinatorial therapy inhibited cellular growth, invasion and colonosphere formation and also reduced CSC population as evidenced by the decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. Our data suggest that the combination therapy of dasatinib and curcumin may be a therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population.     

Overlapping Genes May Control Reprogramming of Mouse Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) and Breast Cancer Stem Cells

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.     

Three‐dimensional cell culture model utilization in cancer stem cell research

Three‐dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.     

Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.     

The Tumor Growth Paradox and Immune System-Mediated Selection for Cancer Stem Cells

Cancer stem cells (CSCs) drive tumor progression, metastases, treatment resistance, and recurrence. Understanding CSC kinetics and interaction with their nonstem counterparts (called tumor cells, TCs) is still sparse, and theoretical models may help elucidate their role in cancer progression. Here, we develop a mathematical model of a heterogeneous population of CSCs and TCs to investigate the proposed “tumor growth paradox”—accelerated tumor growth with increased cell death as, for example, can result from the immune response or from cytotoxic treatments. We show that if TCs compete with CSCs for space and resources they can prevent CSC division and drive tumors into dormancy. Conversely, if this competition is reduced by death of TCs, the result is a liberation of CSCs and their renewed proliferation, which ultimately results in larger tumor growth. Here, we present an analytical proof for this tumor growth paradox. We show how numerical results from the model also further our understanding of how the fraction of cancer stem cells in a solid tumor evolves. Using the immune system as an example, we show that induction of cell death can lead to selection of cancer stem cells from a minor subpopulation to become the dominant and asymptotically the entire cell type in tumors.     

Reprogramming strategies for the establishment of novel human cancer models

Cancer comprises heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. The emergence of the “cancer stem cell” (CSC) hypothesis that they are the cells responsible for resistance, metastasis and secondary tumor appearance identifies these populations as novel obligatory targets for the treatment of cancer. CSCs, like their normal tissue-specific stem cell counterparts, are multipotent, partially differentiated, self-sustaining, yet transformed cells. To date, most studies on CSC biology have relied on the use of murine models and primary human material. In spite of much progress, the use of primary material presents several limitations that limit our understanding of the mechanisms underlying CSC formation, the similarities between normal stem cells and CSCs and ultimately, the possibility for developing targeted therapies. Recently, different strategies for controlling cell fate have been applied to the modeling of human cancer initiation and for the generation of human CSC models. Here we will summarize recent developments in the establishment and application of reprogramming strategies for the modeling of human cancer initiation and CSC formation.     

Modulatory roles of microRNAs in the regulation of different signalling pathways in large bowel cancer stem cells

There are emerging data to suggest that microRNAs (miRNAs) have significant roles in regulating the function of normal cells and cancer stem cells (CSCs). This review aims to analyse the roles of miRNAs in the regulation of colon CSCs through their interaction with various signalling pathways. Studies showed a large number of miRNAs that are reported to be deregulated in colon CSCs. However, few of the studies available were able to outline the function of miRNAs in colon CSCs and uncover their signalling pathways. From those miRNAs, which are better described, miR‐21 followed by miR‐34, miR‐200 and miR‐215 are the most reported miRNAs to have roles in colon CSC regulation. In particular, miRNAs have been reported to regulate the stemness features of colon CSCs mainly via Wnt/B‐catenin and Notch signalling pathways. Additionally, miRNAs have been reported to act on processes involving CSCs through cell cycle regulation genes and epithelial–mesenchymal transition. The relative paucity of data available on the significance of miRNAs in CSCs means that new studies will be of great importance to determine their roles and to identify the signalling pathways through which they operate. Such studies may in future guide further research to target these genes for more effective cancer treatment. miRNAs were shown to regulate the function of cancer stem cells in large bowel cancer by targeting a few key signalling pathways in cells.     

EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis

Objectives

Cancer stem cells (CSCs) compose a subpopulation of cells within a tumour that can self‐renew and proliferate. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (b‐FGF) promote cancer stem cell proliferation in many solid tumours. This study assesses whether EGF, bFGF and IGF signalling pathways are essential for colon CSC proliferation and self‐renewal.

Material and methods

Colon CSCs were cultured in serum‐free medium (SFM) with one of the following growth factors: EGF, bFGF or IGF. Characteristics of CSC gene expression were evaluated by real time PCR. Tumourigenicity of CSCs was determined using a xenograft model in vivo. Effects of EGF receptor inhibitors, Gefitinib and PD153035, on CSC proliferation, apoptosis and signalling were evaluated using fluorescence‐activated cell sorting and western blotting.

Results

Colon cancer cell HCT116 transformed to CSCs in SFM. Compared to other growth factors, EGF was essential to support proliferation of CSCs that expressed higher levels of progenitor genes (Musashi‐1, LGR5) and lower levels of differential genes (CK20). CSCs promoted more rapid tumour growth than regular cancer cells in xenografts. EGFR inhibitors suppressed proliferation and induced apoptosis of CSCs by inhibiting autophosphorylation of EGFR and downstream signalling proteins, such as Akt kinase, extracellular signal‐regulated kinase 1/2 (ERK 1/2).

Conclusions

This study indicates that EGF signalling was essential for formation and maintenance of colon CSCs. Inhibition of the EGF signalling pathway may provide a useful strategy for treatment of colon cancer.