"Molecular Anatomy": a new multi-dimensional hierarchical scaffold analysis tool.

The scaffold representation is widely employed to classify bioactive compounds on the basis of common core structures or correlate compound classes with specific biological activities. In this paper, we present a novel approach called "Molecular Anatomy" as a flexible and unbiased molecular scaffold-based metrics to cluster large set of compounds. We introduce a set of nine molecular representations at different abstraction levels, combined with fragmentation rules, to define a multi-dimensional network of hierarchically interconnected molecular frameworks. We demonstrate that the introduction of a flexible scaffold definition and multiple pruning rules is an effective method to identify relevant chemical moieties. This approach allows to cluster together active molecules belonging to different molecular classes, capturing most of the structure activity information, in particular when libraries containing a huge number of singletons are analyzed. We also propose a procedure to derive a network visualization that allows a full graphical representation of compounds dataset, permitting an efficient navigation in the scaffold's space and significantly contributing to perform high quality SAR analysis. The protocol is freely available as a web interface at https://ma.exscalate.eu .

Binding Mode Exploration of B1 Receptor Antagonists' by the Use of Molecular Dynamics and Docking Simulation-How Different Target Engagement Can Determine Different Biological Effects.

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

Publisher Correction: Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

ADPredict: ADP-ribosylation site prediction based on physicochemical and structural descriptors.

Motivation: ADP-ribosylation is a post-translational modification (PTM) implicated in several crucial cellular processes, ranging from regulation of DNA repair and chromatin structure to cell metabolism and stress responses. To date, a complete understanding of ADP-ribosylation targets and their modification sites in different tissues and disease states is still lacking. Identification of ADP-ribosylation sites is required to discern the molecular mechanisms regulated by this modification. This motivated us to develop a computational tool for the prediction of ADP-ribosylated sites. Results: Here, we present ADPredict, the first dedicated computational tool for the prediction of ADP-ribosylated aspartic and glutamic acids. This predictive algorithm is based on (i) physicochemical properties, (ii) in-house designed secondary structure-related descriptors and (iii) three-dimensional features of a set of human ADP-ribosylated proteins that have been reported in the literature. ADPredict was developed using principal component analysis and machine learning techniques; its performance was evaluated both internally via intensive bootstrapping and in predicting two external experimental datasets. It outperformed the only other available ADP-ribosylation prediction tool, ModPred. Moreover, a novel secondary structure descriptor, HM-ratio, was introduced and successfully contributed to the model development, thus representing a promising tool for bioinformatics studies, such as PTM prediction. Availability and implementation: ADPredict is freely available at www.ADPredict.net. Supplementary information: Supplementary data are available at Bioinformatics online.

Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

Transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel, is the predominant mammalian cold temperature thermosensor and it is activated by cold temperatures and cooling compounds, such as menthol and icilin. Because of its role in cold allodynia, cold hyperalgesia and painful syndromes TRPM8 antagonists are currently being pursued as potential therapeutic agents for the treatment of pain hypersensitivity. Recently TRPM8 has been found in subsets of bladder sensory nerve fibres, providing an opportunity to understand and treat chronic hypersensitivity. However, most of the known TRPM8 inhibitors lack selectivity, and only three selective compounds have reached clinical trials to date. Here, we applied two virtual screening strategies to find new, clinics suitable, TRPM8 inhibitors. This strategy enabled us to identify naphthyl derivatives as a novel class of potent and selective TRPM8 inhibitors. Further characterization of the pharmacologic properties of the most potent compound identified, compound 1, confirmed that it is a selective, competitive antagonist inhibitor of TRPM8. Compound 1 also proved itself active in a overreactive bladder model in vivo. Thus, the novel naphthyl derivative compound identified here could be optimized for clinical treatment of pain hypersensitivity in bladder disorders but also in different other pathologies.

TRAP1 regulates stemness through Wnt/ß-catenin pathway in human colorectal carcinoma.

Colorectal carcinoma (CRC) is a common cause of cancer-related death worldwide. Indeed, treatment failures are triggered by cancer stem cells (CSCs) that give rise to tumor repopulation upon initial remission. Thus, the role of the heat shock protein TRAP1 in stemness was investigated in CRC cell lines and human specimens, based on its involvement in colorectal carcinogenesis, through regulation of apoptosis, protein homeostasis and bioenergetics. Strikingly, co-expression between TRAP1 and stem cell markers was observed in stem cells located at the bottom of intestinal crypts and in CSCs sorted from CRC cell lines. Noteworthy, TRAP1 knockdown reduced the expression of stem cell markers and impaired colony formation, being the CSC phenotype and the anchorage-independent growth conserved in TRAP1-rich cancer cells. Consistently, the gene expression profiling of HCT116 cells showed that TRAP1 silencing results in the loss of the stem-like signature with acquisition of a more-differentiated phenotype and the downregulation of genes encoding for activating ligands and target proteins of Wnt/ß-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/ß-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of ß-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of ß-catenin and several Wnt/ß-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of ß-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/ß-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics.

HMGA1 silencing reduces stemness and temozolomide resistance in glioblastoma stem cells.

OBJECTIVE: Glioblastoma multiforme (GBM) develops from a small subpopulation of stem-like cells, which are endowed with the ability to self-renew, proliferate and give rise to progeny of multiple neuroepithelial lineages. These cells are resistant to conventional chemo- and radiotherapy and are hence also responsible for tumor recurrence. HMGA1 overexpression has been shown to correlate with proliferation, invasion, and angiogenesis of GBMs and to affect self-renewal of cancer stem cells from colon cancer. The role of HMGA1 in GBM tumor stem cells is not completely understood. RESEARCH DESIGN AND METHODS: We have investigated the role of HMGA1 in brain tumor stem cell (BTSC) self-renewal, stemness and resistance to temozolomide by shRNA- mediated HMGA1 silencing. RESULTS: We first report that HMGA1 is overexpressed in a subset of BTSC lines from human GBMs. Then, we show that HMGA1 knockdown reduces self-renewal, sphere forming efficiency and stemness, and sensitizes BTSCs to temozolomide. Interestingly, HMGA1 silencing also leads to reduced tumor initiation ability in vivo. CONCLUSIONS: These results demonstrate a pivotal role of HMGA1 in cancer stem cell gliomagenesis and endorse HMGA1 as a suitable target for CSC-specific GBM therapy.

CD26-positive/CD326-negative circulating cancer cells as prognostic markers for colorectal cancer recurrence.

The present study evaluated the presence and clinical relevance of a cluster of differentiation (CD)26+/CD326- subset of circulating tumor cells (CTCs) in pre- and post-operative blood samples of colorectal cancer patients, who had undergone curative or palliative intervention, in order to find a novel prognostic factor for patient management and follow-up. In total, 80 colorectal cancer patients, along with 25 healthy volunteers were included. The easily transferable methodology of flow cytometry, along with multiparametric antibody staining were used to selectively evaluate CD26+/CD326- CTCs in the peripheral blood samples of colorectal cancer patients. The multiparametric selection allowed any enrichment methods to be avoided thus rendering the whole procedure suitable for clinical routine. The presence of CD26+/CD326- cells was higher in advanced Dukes' stages and was significantly associated with poor survival and high recurrence rates. Relapsing and non-surviving patients showed the highest number of CD26+/CD326- CTCs. High pre-operative levels of CD26+/CD326- CTCs correctly predicted tumor relapse in 44.4% of the cases, while 69% of post-operative CD26+/CD326- CTC-positive patients experienced cancer recurrence, with a test accuracy of 88.8%. By contrast, post-operative CD26+/CD326- CTC-negative patients showed an increase in the three-year progression-free survival rate of 86%, along with a reduced risk of tumor relapse of >90%. In conclusion, CD26+/CD326- CTCs are an independent prognostic factor for tumor recurrence rate in multivariate analysis, suggesting that their evaluation could be an additional factor for colorectal cancer recurrence risk evaluation in patient management.

miR-142-3p down-regulation contributes to thyroid follicular tumorigenesis by targeting ASH1L and MLL1.

CONTEXT: A previous micro-RNA expression profile of thyroid follicular adenomas identified miR-142 precursor among the miRNAs downregulated in the neoplastic tissues compared to normal thyroid gland. OBJECTIVE: The aim of this work has been to assess the expression of miR-142-3p in a large panel of follicular thyroid adenomas and carcinomas and evaluate its effect on thyroid cell proliferation and target expression. DESIGN: The expression of miR-142-3p was analyzed by qRT-PCR in thyroid follicular adenomas and carcinomas, compared to normal thyroids. MiR-142-3p expression was restored in WRO cells and the effects on cell proliferation and target expression were evaluated. RESULTS: Here we show that miR-142-3p is downregulated in FTAs, FTCs, and FVPTCs. MiR-142-3p was demonstrated to reduce the proliferation rate of WRO and FTC133 cells, supporting its tumor suppressor role in thyroid cancerogenesis. Moreover, this microRNA was able to downregulate the expression of ASH1L and MLL1, by direct and indirect mechanisms, respectively. Consistently, an inverse correlation between miR-142-3p expression and ASH1L and MLL1 proteins was found in thyroid follicular adenomas and carcinomas. ASH1L and MLL1, which belong to the Trithorax group (TrxG) proteins and are major regulators of Homeobox gene expression, maintain active target gene transcription by histone 3 lysine 4 methylation. Interestingly, we found that FTCs and FTC cell lines express tumor specific, shorter forms of the two proteins. The capability of miR-142-3p to modulate the levels of these tumor-associated forms and to reactivate thyroid-specific Hox gene expression, likely contributes to its tumor suppressive function. CONCLUSIONS: These data demonstrate that miR-142-3p downregulation has a role in thyroid tumorigenesis, by regulating ASH1L and MLL1.

HMGA1 silencing restores normal stem cell characteristics in colon cancer stem cells by increasing p53 levels.

High-mobility group A1 (HMGA1) proteins are architectural chromatinic proteins, abundantly expressed during embryogenesis and in most cancer tissues, but expressed at low levels or absent in normal adult tissues. Several studies have demonstrated that HMGA1 proteins play a causal role in neoplastic cell transformation. The aim of this study was to investigate the role of these proteins in the control of cancer stem cells (CSCs), which have emerged as a preferred target in cancer therapy, because of their role in cancer recurrence. We observed that HMGA1 is overexpressed in colon tumour stem cell (CTSC) lines compared to normal and colon cancer tissues. We demonstrated that HMGA1 silencing in CTSCs increases stem cell quiescence and reduces self-renewal and sphere-forming efficiency (SFE). The latter, together with the upregulation and asymmetric distribution of NUMB, is indicative of the recovery of an asymmetric division pattern, typical of normal stem cells. We further found that HMGA1 transcriptionally regulates p53, which is known to control the balance between symmetric and asymmetric divisions in CSCs. Therefore, our data indicate a critical role for HMGA1 in regulating both self-renewal and the symmetric/asymmetric division ratio in CSCs, suggesting that blocking HMGA1 function may be an effective anti-cancer therapy.

Novel pancreas organogenesis markers refine the pancreatic differentiation roadmap of embryonic stem cells.

The generation of pancreatic endocrine and exocrine functional precursors from embryonic stem cells (ESCs) is an intriguing opportunity to address cell therapy challenges. The main goal of cellular regeneration is to derive, in vitro, pancreatic progenitor cells (PPCs) that retain the capacity to differentiate following the in vivo developmental ontogeny. In our work, we aim to refine the pancreatic in vitro cellular transitions, through the identification of the intrinsic factors that mark the pancreas budding process at embryonic stage 10.5 (E10.5), in which pancreas precursor specification predominantly occurs. We identified a cohort of genes (Bex1, Nepn, Pcbd1, Prdxdd1, Rnf160, Slc2a1, and Tff3) that marked the pancreas budding genesis, and above all signaled ESC differentiation transitions during pancreatic lineage commitment. Noticeably, we demonstrated that the expression of Nepn marked a naïve pancreatic cellular state that resembled PPC-like specification. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

Hematopoietic stem/progenitor cells express myoglobin and neuroglobin: adaptation to hypoxia or prevention from oxidative stress?

Oxidative metabolism and redox signaling prove to play a decisional role in controlling adult hematopoietic stem/progenitor cells (HSPCs) biology. However, HSPCs reside in a hypoxic bone marrow microenvironment raising the question of how oxygen metabolism might be ensued. In this study, we provide for the first time novel functional and molecular evidences that human HSPCs express myoglobin (Mb) at level comparable with that of a muscle-derived cell line. Optical spectroscopy and oxymetry enabled to estimate an O2-sensitive heme-containing protein content of approximately 180 ng globin per 10(6) HSPC and a P50 of approximately 3 µM O2. Noticeably, expression of Mb mainly occurs through a HIF-1-induced alternative transcript (Mb-V/Mb-N = 35 ± 15, p < .01). A search for other Mb-related globins unveiled significant expression of neuroglobin (Ngb) but not of cytoglobin. Confocal microscopy immune detection of Mb in HSPCs strikingly revealed nuclear localization in cell subsets expressing high level of CD34 (nuclear/cytoplasmic Mb ratios 1.40 ± 0.02 vs. 0.85 ± 0.05, p < .01) whereas Ngb was homogeneously distributed in all the HSPC population. Dual-color fluorescence flow cytometry indicated that while the Mb content was homogeneously distributed in all the HSPC subsets that of Ngb was twofold higher in more immature HSPC. Moreover, we show that HSPCs exhibit a hypoxic nitrite reductase activity releasing NO consistent with described noncanonical functions of globins. Our finding extends the notion that Mb and Ngb can be expressed in nonmuscle and non-neural contexts, respectively, and is suggestive of a differential role of Mb in HSPC in controlling oxidative metabolism at different stages of commitment.

Surface proteomic analysis of differentiated versus stem-like osteosarcoma human cells.

Cancer stem cell characterization represents a breakthrough in cancer research. Despite evidence showing the existence and the role of cancer stem cells in osteosarcoma (OS) onset and progression, little is known about their specific surface phenotype. To address this issue, we carried out a cytometric analysis with an antibody-array comprising 245 membrane proteins comparing the stem and differentiated OS cells. As experimental model, we chose the stem-like cell line 3aminobenzamide-OS and its parental, differentiated, cell line MG63. We identified 50 differentially expressed, 23 homogeneously expressed, and 172 not expressed proteins in the two cell line models, thus defining a surface protein signature specific for each of them. Furthermore, we selected ERK1/2 (p44/42 mitogen-activated protein kinases) as a potential pathway correlated with processes that characterize tumorigenic potential and stemness of 3aminobenzamide-OS cells.

Postoperative detection of circulating tumor cells predicts tumor recurrence in colorectal cancer patients.

INTRODUCTION: Circulating tumor cells are thought to play a crucial role in the development of distant metastases. Their detection in the blood of colorectal cancer patients may be linked to poor outcome, but current evidence is controversial. MATERIALS AND METHODS: Pre- and postoperative flow cytometric analysis of blood samples was carried out in 76 colorectal cancer patients undergoing surgical resection. The EpCAM/CD326 epithelial surface antigen was used to identify circulating tumor cells. RESULTS: Fifty-four (71%) patients showed circulating tumor cells preoperatively, and all metastatic patients showed high levels of circulating tumor cells. Surgical resection resulted in a significant decrease in the levels of circulating tumor cells. Among 69 patients undergoing radical surgery, 16 had high postoperative levels of circulating tumor cells, and 12 (75%) experienced tumor recurrence. High postoperative level of circulating tumor cells was the only independent variable related to cancer relapse. In patients without circulating tumor cells, the progression-free survival rate increased from 16 to 86%, with a reduction in the risk of tumor relapse greater than 90%. CONCLUSIONS: High postoperative levels of circulating tumor cells accurately predicted tumor recurrence, suggesting that assessment of circulating tumor cells could optimize tailored management of colorectal cancer patients.

Cytometric profiling of CD133+ cells in human colon carcinoma cell lines identifies a common core phenotype and cell type-specific mosaics.

In colorectal cancer, CD133+ cells from fresh biopsies proved to be more tumorigenic than their CD133- counterparts. Nevertheless, the function of CD133 protein in tumorigenic cells seems only marginal. Moreover, CD133 expression alone is insufficient to isolate true cancer stem cells, since only 1 out of 262 CD133+ cells actually displays stem-cell capacity. Thus, new markers for colorectal cancer stem cells are needed. Here, we show the extensive characterization of CD133+ cells in 5 different colon carcinoma continuous cell lines (HT29, HCT116, Caco2, GEO and LS174T), each representing a different maturation level of colorectal cancer cells. Markers associated with stemness, tumorigenesis and metastatic potential were selected. We identified 6 molecules consistently present on CD133+ cells: CD9, CD29, CD49b, CD59, CD151, and CD326. By contrast, CD24, CD26, CD54, CD66c, CD81, CD90, CD99, CD112, CD164, CD166, and CD200 showed a discontinuous behavior, which led us to identify cell type-specific surface antigen mosaics. Finally, some antigens, e.g. CD227, indicated the possibility of classifying the CD133+ cells into 2 subsets likely exhibiting specific features. This study reports, for the first time, an extended characterization of the CD133+ cells in colon carcinoma cell lines and provides a "dictionary" of antigens to be used in colorectal cancer research.

CD66c is a novel marker for colorectal cancer stem cell isolation, and its silencing halts tumor growth in vivo.

BACKGROUND: Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell-directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen-related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell-directed treatment of colorectal cancer. METHODS: First, the authors characterized CD66c expression by flow cytometry and immunohistochemistry in colon cancer samples and in normal colon tissues. Then, the coexpression of CD66c and CD133 was evaluated on putative CCSCs. CD66c expression also was measured in stem cell-enriched colon spheres. Finally, the effects of small-interfering RNA-mediated CD66c silencing on the in vitro and in vivo growth of Caco2 colon cancer cells were evaluated. RESULTS: CD66c expression was significantly higher in colon cancers than in contiguous normal colon tissues and paralleled cancer stage. CD66c was absent in CD133-positive cells that were isolated from normal colon, whereas its expression was brightest (CD66c(bright) ) in CD133-positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66c(bright) population in a fashion comparable to the enrichment observed in fresh liver metastases. In vitro proliferation and clonogenic potential were hampered when CD66c was silenced in Caco2 cells. Finally, in vivo xenograft experiments demonstrated that CD66c silencing almost completely abrogated the tumorigenic potential of Caco2 cells. CONCLUSIONS: CD66c(bright) expression was associated with colon cancer stem cells and CD66c silencing blocked tumor growth, thereby opening the way to a potential new treatment for colon cancer.

Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity.

The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/ß-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.

Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63(+/L514F) ) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b(-/-) mice. Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome.

Combined CD133/CD44 expression as a prognostic indicator of disease-free survival in patients with colorectal cancer.

HYPOTHESIS: Because of some inconsistencies in the traditional model of human colorectal carcinogenesis, the cancer stem cell (CSC) model was recently proposed, in which tumor results from neoplastic transformation of stem cells, which become CSCs. Identification of CSCs by expression of surface antigens remains a critical issue because no biomarker has been shown to be completely reliable. CD133 and CD44 are commonly used as CSC markers, and correlation of their expression with colorectal cancer (CRC) clinicopathological features and outcomes may be useful. DESIGN: Pilot study. SETTING: University hospital. PATIENTS: Thirty-six consecutive patients with CRC. CD133 and CD44 expression (alone or combined) was determined in nontumor cells and in tumor cells by flow cytometry, which identified viable cells only. MAIN OUTCOME MEASURES: Correlation of CD133 and CD44 expression with each other, with other prognostic indicators, and with disease-free survival. RESULTS: CD133 and CD44 expression was significantly higher in tumor cells than in nontumor cells, and expression of one did not necessarily correlate with expression of the other. CD133 or CD44 expression alone was variable, while combined CD133/CD44 expression identified a small subset of cells positive for CRC. CD133 or CD44 overexpression was not associated with CRC recurrence; only high frequencies of CD133(+)/CD44(+) cells were a strong indicator of worse disease-free survival and an independent risk factor for CRC recurrence. CONCLUSION: Evaluation of combined CD133/CD44 expression could be useful to identify putative colorectal CSCs and tumors with a poor prognosis.